Competitive Strategies in Life Sciences [1st ed.] 9789811575891, 9789811575907

Table of contents :
Front Matter ....Pages i-xv
Large Biomolecules: An Overview (Basanta Kumara Behera, Ram Prasad, Shyambhavee Behera)....Pages 1-42
Management and Manufacturing Process of Biologics (Basanta Kumara Behera, Ram Prasad, Shyambhavee Behera)....Pages 43-104
Downstream Processes (Basanta Kumara Behera, Ram Prasad, Shyambhavee Behera)....Pages 105-136
Bioprinting (Basanta Kumara Behera, Ram Prasad, Shyambhavee Behera)....Pages 137-156
Biopharmaceuticals: New Frontier (Basanta Kumara Behera, Ram Prasad, Shyambhavee Behera)....Pages 157-183
Back Matter ....Pages 185-189

Citation preview

New Paradigms of Living Systems 1

Basanta Kumara Behera Ram Prasad Shyambhavee Behera

Competitive Strategies in Life Sciences

New Paradigms of Living Systems Volume 1

Series Editors Basanta Kumara Behera, Advanced Centre for Biotechnology, Maharshi Dayanand University, Rohtak, Haryana, India Ram Prasad, Department of Botany, School of Life Sciences, Mahatma Gandhi Central University, Motihari, Bihar, India

This book series aims at how traditional life sciences has developed its broad discipline as applied life science by interacting with the basic principle of physical sciences, engineering and medical sciences. Additionally, it also aims on how armed with deeper understanding of applied life sciences, the biotechnology companies have formed a growing number of formal and informal partnerships with researchers in government, academic to fulfil the significance of personalized health care products are to get the right application for the needy people through the use of molecular diagnostic tests and targeted therapies. The main objective of this book series is to display the main areas in: i) biologics a new challenge; ii) basic understanding of applied life sciences for product development, and manufacturing; iii) microbial platform for innovative biomolecule production iv) biologics from host cells of mammal, animal and plant cells; v) applied genetics vi) Biosimilar; vii) when quality meet confidence; viii) supply chain management of biologics, and ix) the future up gradation of host cells culture techniques at commercial level. This book series is a latest resource for a wide circle of scientists, students and researchers involve in understanding and implementing the knowledge on applied life sciences to develop biologics for proper health care to continue life in smooth and sustainable pattern without any adverse effect.

More information about this series at http://www.springer.com/series/16344

Basanta Kumara Behera • Ram Prasad • Shyambhavee Behera

Competitive Strategies in Life Sciences

Basanta Kumara Behera Advanced Centre for Biotechnology Maharshi Dayanand University Rohtak, Haryana, India

Ram Prasad Department of Botany School of Life Sciences, Mahatma Gandhi Central University Motihari, Bihar, India

Shyambhavee Behera Department of Community Medicine University College of Medical Sciences New Delhi, Delhi, India

ISSN 2662-348X ISSN 2662-3498 (electronic) New Paradigms of Living Systems ISBN 978-981-15-7589-1 ISBN 978-981-15-7590-7 (eBook) https://doi.org/10.1007/978-981-15-7590-7 © Springer Nature Singapore Pte Ltd. 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Preface

Life sciences is a complex and highly competitive industry resulted from the integrated studies and research of various disciplines like biotechnology, medical sciences, basic biological science, biophysics, biochemistry, molecular biology, and genetic engineering, mainly. With the rapid diversification and development of the industry, strategic blueprint for life sciences needs modern technology, consistent monitoring, and integrated supply chain management systems. Thus, keeping the above facts in mind, the first volume of this series is highlighted in the sequential conceptual development of applied life sciences to make the readers acquainted with the long-term potential of life science industry to meet the health care challenges at the global level. The first chapter narrates the biological significance of bigger size biomolecules and their conceptual development to save life in its most critical stages of development and continuity. Manufacturing of biologics by a typical fermentation process using host cells from plant tissue or animal tissue or mammalian cells is described in detail. In addition, modernization of fermentation technique with the incorporation of a continuous manufacturing process is described with suitable and impressive models for clear understanding. New concept and practice for manufacturing therapeutic proteins through animal bioreactor are discussed in detail. The first chapter also covers the most recent aspects on bio-betters vs biologics and trends in patenting biologics. The second chapter comprises manufacturing process of bigger size biomolecules by the application of tools and techniques of modern biotechnology. The second chapter starts with vision, decision, and planning of manufacturing biologic drugs by explaining how coordinate can be maintain between technical wing and overall administration division for successful lunching of quality biopharmaceutical with safety and security. The most important of this chapter is how to develop and establish upstream process starting from the selection of suitable host cells for a target biologic drug design and production. In this connection, special emphasis is given on how to establish and manage host cells bank in order to get a quality product, consistently. The second chapter also explains the different types of fully v

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Preface

automatic bioreactors for mass scale culture of host cells. In addition, the second chapter explains the continuous and semi-continuous manufacturing process in order to save time, energy, and extra budget. Besides this, the risk of human error involved in product processing from the beginning to end is given. The third chapter is an extended part of the manufacturing process with the detailed coverage on product development by solid–liquid separation, release of intracellular products, concentration, purification by chromatography, formulation, and integration of the different processes. With the aid of modern microchips, technology bioprinting has emerged as a powerful tool for design and building tissue and organ, structurally and functionally compatible with the body. So, Chap. 4 is framed to present precise design and development of tissue and organs of certain parts of body like heart valve, myocardial tissue, trachea, and blood vessels to replace in the body on requirement basis. Application-based research focused on tissue regeneration is presented, as well as the current challenges that hamper the clinical utility of bioprinting technology. The fifth chapter comprises the key phases of life sciences product development on the basis of international regulatory norms for quality assurance. Today, the global market needs sufficient productivity of quality product followed by quality control and quality assurance with safety guarantee. So, various clinical phases of trials are discussed until reaching final approval and licensing. Globally, life sciences industry represents one of the dominant economic sectors. So, the fifth chapter is to bring awareness among the readers the terms and conditions for commercialization of biologic drugs by maintain safety, security of the product for health management and improvement, including medical technology, genomics, diagnostics and digital health. In this connection, the authors have given special emphasis on international regulatory acts for the production of GMP-based quality product with a guarantee on the efficacy of the biologic drugs. In addition, the increased demand for life sciences products at the global level is highlighted with the profile of top players in biotechnology sectors and biopharmaceutical sectors. The authors are highly grateful to Mrs. Asha Sharma, Katyayanee, and Gopal for their help in graphic design, model preparation, and data compiling while preparing the manuscript. The kind cooperation of the publisher is highly acknowledged. Rohtak, India Motihari, India New Delhi, India

Basanta Kumara Behera Ram Prasad Shyambhavee Behera

Contents

1

Large Biomolecules: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Strategies in Life Sciences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.1 Lifesaving Larger Size Biomolecules . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . .

1 2 2 40

2

Management and Manufacturing Process of Biologics . . . . . . . . . . . 43 2.1 What Is Biologic? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 2.2 Vision, Decision, and Planning . . . . . . . . . . . . . . . . . . . . . . . . . . 43 2.2.1 Vision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 2.2.2 Decision and Planning . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 2.2.3 Work Planning Leading to Product Development . . . . . . . . 46 2.3 Upstream Process for Recombinant Protein Production . . . . . . . . . 69 2.3.1 Selection of Host Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 2.3.2 Medium Required for Different Host Cells Culture . . . . . . 70 2.4 Growth Medium for Different Types of Host Cells . . . . . . . . . . . . 78 2.4.1 Medium for Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 2.4.2 Fungal Culture Media . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 2.4.3 Animal and Mammalian Cell Culture Media . . . . . . . . . . . 85 2.5 Preservation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 2.5.1 Cryopreservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 2.5.2 Drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 2.5.3 Preservation by Liquid Drying (L-drying) . . . . . . . . . . . . . 88 2.5.4 Preservation by Vacuum Drying . . . . . . . . . . . . . . . . . . . . 88 2.5.5 Metabolically Active Methods . . . . . . . . . . . . . . . . . . . . . 88 2.6 Bioreactor Used for Recombinant Protein Production . . . . . . . . . . 89 2.6.1 Bioreactor Vessel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 2.6.2 Types of Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

3

Downstream Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 3.2 Conceptual Development of Biopharmaceutical . . . . . . . . . . . . . . 105 vii

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Contents

3.3 3.4

Biologics, Biosimilar, and Biobetter . . . . . . . . . . . . . . . . . . . . . . Product Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1 Initial Stages of Recovery . . . . . . . . . . . . . . . . . . . . . . . 3.4.2 Flotation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.3 Flocculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.4 Filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Release of Intracellular Products . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1 Chemical Methods for Cells Disruption . . . . . . . . . . . . . . 3.5.2 Physical Methods of Cell Disruption . . . . . . . . . . . . . . . . 3.5.3 Biological Methods of Cell Disruption . . . . . . . . . . . . . . 3.6 Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.1 Evaporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.2 Liquid–Liquid Extraction . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Biopharmaceutical Purification by Chromatography . . . . . . . . . . 3.7.1 Column Chromatography . . . . . . . . . . . . . . . . . . . . . . . . 3.7.2 Gel Filtration Chromatography . . . . . . . . . . . . . . . . . . . . 3.7.3 Ion Exchange (IEX) Chromatography . . . . . . . . . . . . . . . 3.7.4 Affinity Chromatography . . . . . . . . . . . . . . . . . . . . . . . . 3.8 Biologic Drug Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8.1 Excipients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8.2 Drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8.3 Spray Drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8.4 Integration of Different Processes . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

. . . . . . . . . . . . . . . . . . . . . . . .

108 109 109 111 111 112 114 115 116 117 118 118 120 121 122 125 125 128 131 131 132 133 133 134

Bioprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Pre-bioprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Bioprinting Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1 Bioink . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Post-Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Types of Bioprinter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.1 Inkjet-Based Bioprinting . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.2 Extrusion-Based Bioprinting . . . . . . . . . . . . . . . . . . . . . . . 4.5.3 Laser Bioprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.4 Laser-Assisted Bioprinting (LAB) . . . . . . . . . . . . . . . . . . . 4.6 4D Bioprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7 Selection of Cell Lines for Bioprinting . . . . . . . . . . . . . . . . . . . . . 4.7.1 Bioprinting of Artificial Blood Vessels . . . . . . . . . . . . . . . 4.7.2 Artificial Liver Through 3D Bioprinting . . . . . . . . . . . . . . 4.7.3 3D Bioprinting of Artificial Bone . . . . . . . . . . . . . . . . . . . 4.7.4 Artificial 3D Dental Tissues . . . . . . . . . . . . . . . . . . . . . . . 4.7.5 Artificial Ovary Through 3D Bioprint . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

137 137 138 140 141 141 143 143 144 144 146 147 148 148 149 151 152 152 153

Contents

5

Biopharmaceuticals: New Frontier . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Biological Product Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1 Reference Product and Biosimilar . . . . . . . . . . . . . . . . . . . 5.2.2 Highly Similar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.3 Interchangeable Product . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.4 Biosimilar vs. Generic Drugs . . . . . . . . . . . . . . . . . . . . . . 5.3 Discovery Process and Development . . . . . . . . . . . . . . . . . . . . . . 5.3.1 Earlier Process (Preclinical Trials) . . . . . . . . . . . . . . . . . . . 5.3.2 Clinical Research Organization . . . . . . . . . . . . . . . . . . . . . 5.3.3 Phase I Clinical Trial . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.4 Phase II Trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.5 Phase III Clinical Trial . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.6 Phase IV Clinical Trial . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Approval of Marketing Application . . . . . . . . . . . . . . . . . . . . . . . 5.4.1 Fast Track . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.2 Priority Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.3 Breakthrough Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.4 Accelerated Approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 Designation of Biologic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6 Good Manufacturing Practice (GMP) . . . . . . . . . . . . . . . . . . . . . . 5.6.1 Other Good Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6.2 CGMP for Biopharmaceuticals . . . . . . . . . . . . . . . . . . . . . 5.6.3 Enforcement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7 Quality Assurance (QA) and Quality Control (QC) . . . . . . . . . . . . 5.7.1 Quality Assurance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7.2 Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.8 International Council for Harmonisation (ICH) . . . . . . . . . . . . . . . 5.9 ICH Biopharmaceutical Documents . . . . . . . . . . . . . . . . . . . . . . . 5.9.1 FDA Guidance for Biologic Drugs . . . . . . . . . . . . . . . . . . 5.9.2 European Medicines Agency (EMA) . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix

157 157 159 160 160 160 160 161 166 167 171 171 171 172 172 172 173 173 173 174 174 175 176 177 177 177 178 178 179 179 180 182

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

About the Authors

Basanta Kumara Behera was a professor of biotechnology at three distinguished Indian universities, where he had been regularly associated with teaching and research at postgraduate-level courses on the topic related to biomass process technology development, biopharmaceuticals, microbial process development drug designing, bio-energy management, and biomass processing technology since 1978. In 2009, he joined an MNC company as an adviser for speciality chemicals production and drug design through microbial process technology. He is associated with national and international reputed companies as technical adviser for the production of biopharmaceuticals under cGMP norms. He has the credit of being an author of several academic books. Ram Prasad is associated with Department of Botany, Mahatma Gandhi Central University, Motihari, Bihar, India. His research interest includes applied and environmental microbiology, plant–microbe interactions, sustainable agriculture, and nanobiotechnology. He has more than 150 publications to his credit, including research papers, review articles, book chapters, five patents issued or pending, and edited or authored several books. He has 12 years of teaching experience and has been awarded the Young Scientist Award and Prof. J.S. Datta Munshi Gold Medal by the International Society for Ecological Communications; FSAB fellowship by the Society for Applied Biotechnology; the American Cancer Society UICC International Fellowship for Beginning Investigators, USA; Outstanding Scientist Award in the field of Microbiology by Venus International Foundation; BRICPL Science Investigator Award and Research Excellence Award, etc. Previously, he served as Assistant Professor, Amity University, Uttar Pradesh, India; Visiting Assistant Professor, Whiting School of Engineering, Department of Mechanical Engineering at Johns Hopkins University, Baltimore, USA, and Research Associate Professor at School of Environmental Science and Engineering, Sun Yat-sen University, Guangzhou, China.

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About the Authors

Shyambhavee Behera is a medical graduate from Lady Hardinge Medical College, New Delhi, and MD degree holder from University College of Medical Science, New Delhi. She has been working in the field of non-communicable disease, immunization, maternal and child health, epidemiology, and health administration to upgrade and bring amendments in the community for sustainable life pattern with good health. She is presently working with Department of Community Medicine, Atal Bihari Vajpayee Institute of Medical Sciences, and Dr. RML as senior resident. Hospital, New Delhi, Govt. of India. She is co-author for a book entitled “Move Toward Zero Hunger”, Springer Nature Singapore Private Limited.

Abbreviations

2D 3D AAV AM APIs ATMPs ATMPs ATPS BAFF BAPs BEA BHI BLA BLyS BMS BST CAD CapEX CBA CBP CDRs CFDA CGMP CHO CLED agar CPAs CQA CROs CT cUTI

Two-dimensional Three-dimensional Adeno-associated virus Additive manufacturing Active pharmaceutical ingredients Advanced therapy medical products Advanced therapy medicinal products Aqueous two-phase systems B-cell activating factor Blood agar plate Bile Esculin Agar Biosynthetic human insulin Biologics license application B-lymphocyte stimulator Bristol Myers and Squib Bovine somatotropin Computer-aided design Capital expenditures Chocolate blood agar Chitin binding protein Complementarities determining regions Chinese Food and Drug Administration Current good manufacturing practices Chinese hamster ovary Cystine–lactose–electrolyte–deficient agar Cryo-protective additives Critical quality attribute Clinical research organizations Computed tomography Complicated urinary infection xiii

xiv

DNA EDTA EMA EMEA ERT FBS FDA FSH GAP GCP G-CSF GDP GFC GH GLP GMP GRP GSK GST GVP HAMA hATTR HFM HGH HIV HPV ICH IEX IF IMAC La BP LAB lGF LIF mAb MBP MHRA MRI NA NDA NHS NRAs PPD PPOs

Abbreviations

Deoxyribonucleic acid Ethylenediaminetetraacetic acid European Medicines Agency European Medicines Evaluation Agency Enzyme replacement therapy Foetal bovine serum Food and Drug Administration Follicle-stimulating hormone Good agricultural practice Good clinical practice Granulocyte-colony stimulating factor Good distribution practice Gel filtration chromatography Growth hormone Good laboratory practice Good manufacturing practice Good regulatory practice GlaxoSmithKline plc Glutathione-S-transferase Good pharmacovigilance practice Human anti-murine antibody Hereditary transthyretin-mediated amyloidosis Hollow-fibre membranes Human growth hormones Human immunodeficiency viruses Human papillomavirus International Council for Harmonisation Ion exchange Interferon Immobilized metal ion affinity chromatography Laser bioprinting Laser-assisted bioprinting Insulin-like growth factor1 Laser-induced forward transfer Monoclonal antibody Maltose binding protein Medicines and Healthcare products Regulatory Agency Magnetic resonance imaging Nutrient agar New drug application National Health Service National Regulatory Authorities Pharmaceutical product development Polyphenol oxidases

Abbreviations

PTMs QA QC QRM rBST rhEPO rHI RP SDRs SDS SFF siRNA SLE SSDNA TCBS agar TLC TNF TPA TPP TPQP TSA VH VL VLPs WHO

xv

Post-translational modifications Quality assurance Quality control Quality risk management Recombinant bovine somatotropin Recombinant human erythropoietin Recombinant human insulin Rapid prototyping Specificity determining residues Sodium dodecyl sulphate Solid free-form technology Small interfering RNA Systemic lupus erythematosus Single-stranded deoxyribonucleic acid Thiosulfate-citrate-bile salt-sucrose agar Thin layer chromatography Tumour necrosis factor Tissue plasminogen activator Target product profile Target product quality profile Tryptic soy agar Variable heavy Variable light Virus-like particles World Health Organization

Chapter 1

Large Biomolecules: An Overview

Abbreviations ATMPs AAV APIs BHI bST BAFF BLyS BMS CHO CDRs DNA ERT FSH GH G-CSF GSK HGH HIV HPV HAMA hATTR IF IGF-1 mAb NHS PPOs PTMs rhEPO rBST

Advanced therapy medicinal products Adeno-associated virus Active pharmaceutical ingredients Biosynthetic human insulin Bovine somatotropin B-cell activating factor B-lymphocyte stimulator Bristol Myers Squibb Chinese hamster ovary Complementarities determining regions Deoxyribonuclic acid Enzyme replacement therapy Follicle-stimulating hormone Growth hormone Granulocyte colony-stimulating Factor GlaxoSmithKline plc Human growth hormones Human immunodeficiency viruses Human papillomavirus Human anti-murine antibody Hereditary trasthyretin-mediated amyloidosis Inerferon Insulin-like growth factor 1 Monoclonal antibody National Health Service Polyphenol oxidases Post-translational modifications Recombinant human erythropoietin Recombinant bovine somatotropin

© Springer Nature Singapore Pte Ltd. 2020 B. K. Behera et al., Competitive Strategies in Life Sciences, New Paradigms of Living Systems 1, https://doi.org/10.1007/978-981-15-7590-7_1

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1 Large Biomolecules: An Overview

SDRs SLE SSDNA TPA VL VH VLPs

1.1

Specificity determining residues Systematic lupus erythematosus Single-stranded deoxyribonucleic acid Tissue plasminogen activator Variable light Variable heavy Virus-like particles

Strategies in Life Sciences

The main strategy for studying life sciences is to understand the major challenges encountered by humans for survival and to resolve various issues on the pattern of life by understanding biological system on the basis of integrated knowledge on genomic, proteomic, and recombinant DNA technology and high-speed computing. By virtue of genetic engineering, crucial protein required for health problems and dietary purposes can be produced safely, affordably, and adequately. This technology can be applicable to resolve a wide range of problems related to health, enhancing food resources that can also solve the problem of hunger and poverty. Today’s biotechnology and biopharmaceutical industry want to bring sustainability in manufacturing process for quality production with utmost safety and security. But, understanding customer landscape, designing operational protocol, and intensifying competition are presenting new organizational and operational hurdles for Life Sciences companies. The complex market dynamics are requiring new level of rigour and insight in business strategy development and problem-solving, both at regional and global level. This could only be possible if the planning strategies on approach to life science are an integrated basis by taking into consideration of a diversified range of subjects like biomedical engineering, systems biology, fermentation technology, genetic engineering, primary healthcare, rural economy development and food security. Proper planning strategies in life sciences would be immensely helpful in the development of therapeutic biomolecule marketing status at regional and global level; rural primary healthcare management; rural economy development by introducing genetically modified crops with higher productivity capacity and development of domestic animal farming with especially breed livestock for quality milk and animal meat.

1.1.1

Lifesaving Larger Size Biomolecules

Generally, chemically synthesized drugs are smaller in size. For example, acetylsalicylic acid (ASA), the active ingredient of aspirin, is having 180 Da or 180 g/mol weight. As an advantage, being small molecule it can be easily processed

1.1 Strategies in Life Sciences

3

for ingestible tablet or capsule. Generally, the small molecules dissolve in the gastrointestinal tract, and the active ingredient is absorbed into the bloodstream via intestinal wall. Due to the tiny size, small molecules can easily travel to the destination. But, biologics or biopharmaceuticals are, mostly, protein-based drugs having more than 1300 amino acids and are more than 150,000 g/mol (or 159 kDa) in molecular weight. Biologics bind specific cell receptors that are associated with the disease process. For example, monoclonal antibodies are specialized in recognizing a very specific structure on the cell surface. In cancer therapy, they bind selectively to the receptors of cancer cells, making it possible to mark and fight specific abnormal cells. Healthy cells are left safely in such attack, so biologics often cause fewer side effects than classic chemotherapy. Since the past two decades, biologic drugs (monoclonal antibody, therapeutics, hormones, and immune system signalling molecules) have been in clinical use for life-threatening diseases like rheumatoid arthritis, diabetes, multiple sclerosis, Crohn’s disease, and whole range of cancers.

1.1.1.1

Recombinant DNA Protein Drugs

Since the past two decades, the demand for recombinant therapeutic proteins is in increasing magnitude. Mostly, recombinant human proteins have replaced the original animal-derived version used in medicine. Presently, more than 300 recombinant DNA proteins are available in the global market (mostly from Europe and the USA). As predicted by Allied Market research (Walsh 2018;Allied Maket Research 2018), it is expected that the recombinant therapeutic market will be valued about $217.591 million billion by 2023. On the basis of geographic location, the main regions having promising sales, revenue, market share (%), and growth rate (%) include the USA, North America (Canada and Mexico), Europe (German, France, UK, Russia, and Italy), Asia-Pacific (China, Japan, Korea, India, and South Asia), South America (Brazil, Argentina, and Columbia), and Middle East and Africa (Saudi Arabia, UAE, Egypt, Nigeria, and South Africa). The variety of therapeutic protein drugs includes monoclonal antibodies, erythropoietin, insulin, interferon, human growth hormones, folliclestimulating hormone, and blood factors. Generally, recombinant proteins are produced in prokaryotic cells (mainly bacterium Escherichia coli) and in some specific mammalian cell lines, such as Chinese hamster ovary (CHO) cells. Insects, yeasts, and algae are also used for therapeutic protein drugs production but are commercially not acceptable, so much. Transgenic Plants system is also a good source of therapeutic protein drugs production. But, they have not yet much commercial use. Followings are important recombinant therapeutic proteins derived from different biological resources. These therapeutic proteins are either used for clinical purpose as drugs or used for diagnosis purpose for human diseases.

4

1 Large Biomolecules: An Overview

Human Recombinant Replaced Animal Growth Hormones for Therapeutic Use Clinical use of growth hormone (GH), such as human growth hormones (rHGH), as drugs is known as growth hormone therapy. Basically, growth hormone is a peptide hormone secreted by the pituitary gland responsible for biological stimulation of growth and development, and cell reproduction. Earlier, growth hormones are directly extracted from pituitary glands. Currently, growth hormones are produced by recombinant DNA technology and expressed through suitable non-human hosts. The best examples of such therapeutic hormones are somatropin (brand Humatrope) (from Lilly) and Serostim (Serono, a biotechnology company). The former hormone is also known as somatreopleopin (INN) and is used as prescribed drug for children’s growth disorders and adult growth hormone deficiency. It is only available in the USA with doctor’s prescription, as many of its side effects are not yet clear (Powers 2005). The latter one with brand name Serostim is also a somatropin marketed for HIV-associated complex syndrome. Human Insulin (BHI) Insulin is an anabolic peptide hormone synthesized by beta cells of the pancreatic islet in the body. Glucose metabolism, especially from blood into liver, is regulated by this hormone. In addition, it also controls glucose regulation in fat and skeletal muscle cells. In liver, the insulin converts glucose into glycogen. In other tissues this hormone converts glucose via glycogenesis or fats (triglycerides) via lipogenesis. Circulating insulin inside body also affects the synthesis of protein in a wide variety of tissues. As it is an anabolic hormone, it promotes the conversion of small molecules in the blood into large molecules inside the cells. Low insulin levels in the blood have reverse effect by promoting catabolism, especially of reserve body fat. Currently, two types of recombinant insulin named Humulin (produced by Lilly) and Novolin (produced by Novo Nordisk) are in the market for human therapy. The earlier one was replaced bovine, and the latter one replaced porcine. Some patients want to prefer animal-derived insulin instead of recombinant insulin. This is mainly due to the induction of hypoglycaemia by using recombinant insulin. Humulin N (recombinant type human insulin isophane) suspension is a human insulin suspension. E. coli is used as host cell for the production of this recombinant. Humulin N is a suspension of crystals from combining human insulin and protamine sulphate under appropriate condition for crystal formation. The amino acid sequence of Humulin is similar to human insulin with molecular weight of 5808. Novolin N is a different brand name of the same insulin NPH. Insulin NPH is an intermediateacting insulin, which lasts longer in the body than natural insulin does. Follicle-Stimulating Hormone Follicle-stimulating hormone (FSH) is a gonadotropin, a glycoprotein polypeptide hormone, secreted from gonadotropic cells of the anterior pituitary gland and

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regulates the development, growth, pubertal maturation, and reproductive processes of the body. This hormone is produced in vitro by recombinant technology. The host cell for the expression is Chinese hamster ovary cells (CHO), due to its complex heterodimeric protein. Brand names of FSH are Goal-f, Follistim, Gonal-F RFF Redi-ject, and Gonal-f RFE. Blood Clotting Factor VIII Recombinant blood clotting factor VIII is a complex protein. Due to its low efficiency of gene transcription, massive intracellular loss of its proprotein during post-translational processing, and the instability of the secretary protein, it has been a tough technology for industrialization. Blood clotting factor VIII is available in the market with different brand names: Gonal-f, Follistim, Gonal-f RFF Redi-ject, and Gonal-f RFF. Kogenate from Bayer replaced blood harvested factor VIII. For Research Applications Ribosomal proteins: For research purpose, currently, ribosomal proteins are produced and purified from recombinant sources (Correddu et al. 2019;Parakhnevitch et al. 2005;Malygin et al. 2003;Tchórzewski et al. 1999) rather than directly extracted (Collatz et al. 1977;Fogel and Sypherd 1968). Lysosomal proteins: Recently, Escherichia coli and Saccharomyces cerevisiae are used as host cells for the production of recombinant lysosomal proteins, both for research and clinical use (Solomon and Muro 2017). Earlier lysosomal proteins were extracted directly due to the problem of number and type of post-translational modifications in glycosylation process. So, it was practiced to produce from mammalian cells (Migani et al. 2017).

Human Gene Recombinants with Mammalian Cells Erythropoietin (rhEPO) Recombinant human erythropoietin (rhEPO) and its analogues are used in the prevention and reversal of anaemia in chronic kidney diseases (CKS), malignancy, and AIDS. Due to the complexity nature of EPO, it is manufactured in mammalian host cells (Fig. 1.1). Transfected bacteria such as E. coli are useful as hosts for the production of non-glycosylated recombinant proteins. Erythropoietin is marketed as epoetin alfa by Amgen Inc. (formerly Applied Molecular Genetics), which is an American biopharmaceutical company. Granulocyte Colony-Stimulating Factor Granulocyte colony-stimulating factor (G-CSF or GC SF) is also known as colonystimulating factor 3 (CSF3). G-CSF also stimulates the survival proliferation, differentiation, and function of neutrophil precursors and mature neutrophils. It is a glycoprotein hormone (functionally it is a cytokine type), stimulates the bone marrow to produce granulocytes and stem cells, and releases them into the bloodstream. It is produced in mammalian cells, and in a non-glycosylated form, in bacterium E. coli through recombinant DNA technology. Filgrastim (recombinant

6

1 Large Biomolecules: An Overview

Fig. 1.1 Host cell suitable for production of non-glycosylated vs. glycosylated biopharmaceutical

human granulocyte colony-stimulating factor, rhG-CSF) and lenograstim are pharmaceutical analogues, produced naturally. Filgrastim is sold as Neupogen from Amgen, and pegfilgrastim sold as Neulasta. Alpha-galactocidase It is a glycoside hydrolase enzyme that hydrolyses the terminal alpha-galactosyl moieties from glycolipids and glycoproteins. The recombinant type of this enzyme is derived from Chinese hamster ovary cell line as alpha-galactosidase A/GLA protein (fabrazyme) by Sanofi Genzyme. Alpha-L-iduronidase It is sold as Aldurazyme that catalyses the hydrolysis of unsaturated alpha-Liduronosidedic linkase in dermantan sulphate by BioMarin Pharmaceutical Inc. and Genzyme. It is produced by recombinant DNA technology in Chinese hamster ovary (CHO) cell line. N-acetylgalactosamine-4-sulfate It is marketed by BioMarin Pharmaceutical as galsulfase. It is a variant form of the polymorphic human enzyme N-acetylgalactosamine-4-sulfatase of recombinant DNA origin. It is a glycoprotein with a molecular weight of about 56 kDa. This recombinant human enzyme is used as replacement enzyme therapy for the treatment

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of adult and children with mucopolysaccharidosis VI, a rare genetic disorder caused by a deficiency of a lysosomal enzyme. Dornase Alfa Dornase alfa has brand name Pulmozyme and is used for cystic fibrosis (CF) patients to improve pulmonary function. Pulmozyme is manufactured by Genentech. Dornase alfa (proprietary name Pulmozyme from Genentech) is a highly purified solution of recombinant human deoxyribonuclease I (rh DNase), an enzyme that selectively cleaves DNA. The protein is produced from genetically engineered Chinese hamster ovary (CHO) cells containing DNA encoding for the native human protein, deoxyribonuclease 1 (DNase). Tissue Plasminogen Activator (TPA) It is available in the brand name Activase (alteplase) and is manufactured and marketed by Genentech, a US-based company that was merged with Roche Pharmaceutical operated in the USA. Activase is prescribed for the treatment of acute ischaemic stroke and acute myocardial infarction (AMI) for the reduction of mortality and reduction of the incidence of heart failure. Alteplase is an enzyme that occurs naturally in human body and responsible for blood clots to dissolves. It is a human-made protein manufactured by recombinant DNA technology. The naturally occurring protein known as tissue plasminogen activator (TPA) is made by ovarian cells from Chinese hamster. Glucocerbrosidase Glucocerbrosidase is marketed with the brand name Ceredase (alglucerase injection). It is a modified form of the enzyme β-glucocerebrosidase (β-D-glucosyl-Nacylsphine glucucohydrolase). The recombinant formulation of human glucocerebrosidase is Imglucerase and is recommended for Gaucher (GD) as enzyme replacement therapy (ERT). Imiglucerase is produced from transduced Chinese hamster ovary cells and modified by sequential deglycosylation of its carbohydrate side chain to expose alphamannosyl residues that mediate the uptake of the intravenous infused enzyme. Interferon (IF) There are three types of Interferons (IFN), alpha (produced in leukocytes infected virus), beta (produced in fibroblast infected with virus), and gamma (induced by the stimulation of sensitized lymphocytes with antigen or non-sensitized lymphocytes with mitogens). Interferon beta-1a is a cytokine in the interferon family used to treat multiple sclerosis (MS). It is produced by mammalian cells, and whole interferon beta-1b is produced in modified E. coli. Interferon-beta-1a is manufactured by Biogen with the brand name Avonex, and Rebi from Seron. Interferon beta-1b is manufactured by Schering with the brand name Betaseron (for the treatment of Guillain–Berre syndrome and multiple sclerosis).

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1 Large Biomolecules: An Overview

Insulin-like Growth Factor 1 (IGF-1) Insulin-like growth factor 1 (somatomedin C) acts as hormone stimulator. It is produced primarily by the liver and is stimulated by growth hormone (GH). It is having similar molecular structure to insulin. It plays an important role in childhood growth and has anabolic effect in adults. IGF-1 with the brand name Mecasermin is a recombinant form of human insulin-like growth factor used for the treatment of growth failure. Rasburicase Elitek is the brand name of Rasburicase (a Urate Oxidase) marketed by Sanofi. It is a recombinant urate oxidase indicated for initial management of plasma uric acid levels in paediatric and adult patients with leukaemia, lymphoma, and solid tumour malignancies.

Animal Recombinant Biologics Bovine Somatotropin (bST) Bovine somatotropin (bST and BST) or bovine growth hormone (BGH) is naturally synthesized in very minor quantity in the pituitary glands of cow. In 1970, Genentech is the first American-based biopharmaceutical company that patented the gene for BST (Allied Maket Research 2018) and developed recombinant DNA technology to produce recombinant bovine somatotropin (rBST), recombinant bovine growth hormone (rBGH), or artificial growth hormone. Currently, Monsanto, American Cyanamide, Eli Lilly, and Upjohn are marketing FDA-approved rBGH (Bijman 1996;Bovine Somatotropin n.d.). Monasanto is the first company to take credit for FDA rBGH. Subsequently, other different countries (Mexico, Brazil, India, Russia, and at least ten others) also approved rBST for commercial use (Dobson 1996). rBST is banned in Canada, Europe, and Japan as it is used to enhance the milk production in cows. Porcine Somatotropin (pST) It is a growth hormone naturally produced in pigs. Porcine–Somatotropin Recombinant is produced in E. coli in non-glycosylated form, but not permitted by the FDA for human, agricultural or oesticidal products, food additive, or household chemicals. Bovine Chymosin It is used to cleave the milk protein kappa casein. Chymosin is commonly known as rennin. Bovine chymosin is widely used to promote milk clotting in cheese manufacturing. Recombinant chymosin is mainly produced by fungi (yeast), but it can also be produced using bacteria.

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Bacterial Recombinant Xylanases Currently, xylanases are used in textile, paper, and pulp industries as well as in clarification of fruit juices, aroma production, animal feed, baking industry, and production of ethanol. Mostly, bacterial strains are used to produce xylanase by recombinant technology. Protease Proteases have a variety of applications in both the industry (food industry) and domestic settings. Protease Recombinant is a fusion protein of glutathione-S-transferase (GST) and human rhinovirus (HRV) type 14 3C protease. Besides plants and animal systems, microbes are preferred in the production of protease by recombinant DNA technology. It is due to their wide substrate specificity and ease of gene manipulation.

Viral Recombinants Hepatitis B Virus Hepatitis B virus is manufactured by Smithkline Beecham and marketed with the brand name Engerix-B. The recombinant Engerix B is a sterile suspension of non-infectious HBaAg for intramuscular administration. It contains purified surface antigen of the virus obtained by culturing recombinant Saccharomyces cerevisiae cells that carry the surface antigen of the hepatitis B virus. It is clinically prescribed for immunization against infection caused by all known subtypes of hepatitis B virus. Human Papillomavirus (HPV) Vaccine HPV vaccine (Gardasii 9) is clinically prescribed for the protection against infection with human papillomavirus (HPV). It is a non-enveloped deoxyribonucleic acid (DNA) virus that infects skin or mucosal cells. It also contains aluminium, sodium chloride (salt), water, L-histidine, polysorbate 80, and borax, to stimulate the immune system and keep the vaccine in stable form. Two prophylactic HPV vaccines have been available since 2006. Both vaccines are prepared from viruslike particles (VLPs) produced by recombinant technology.

Plant Recombinants Laccases Laccases have a wide range of applications in paper and pulp industries, textile industries, xenobiotic degradation, and bioremediation. Currently, laccase has been in use in nano-biotechnology to catalyse electron transfer reactions without additional cofactor.

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1 Large Biomolecules: An Overview

Polyphenol Oxidases (PPOs) It is available in all plant tissues in the form of a mixture of monophenol oxidase and catechol oxidase enzymes. It is also present in bacteria, animals, and fungi and their products are responsible for desiccation tolerance. It also serves a chemical defence for plants from insects’ attack. E. coli is used as host cell for the expression of VvPPO2 gene for the expression of polyphenol oxidase. Cystatins Plant-derived cystatins act as ectopic inhibitors of Cys protease in biological systems. This protein has the potential in crop protection to control herbivorous pests and pathogens. Transgenic plants including soybean have been developed as drought tolerance by inducing expression system for phytocystatins.

Antibody-Based Protein Therapeutics Antibody is a protein complex produced in response to defending the body from the attack of an antigen (a harmful substance). Antigen is a toxin or foreign material (bacteria, fungi, parasites, viruses, chemicals, and other foreign substances), which induces immune response in the body, especially, the production of antibodies. Sometimes the body wrongfully recognizes body’s normal tissue as antigen. This biological process is known as autoimmune conditions such as rheumatoid arthritis and multiple sclerosis. Antibody production is a natural biological process otherwise known as body’s immune system. However, natural antibodies produced in our body can be expressed in mice or another suitable mammalin system by recombinant DNA technology. The mice then are vaccinated with the antigen that scientists want to produce antibodies against. This resulted in production of antibody through immune cells. A monoclonal antibody binds to only one substance. Monoclonal antibodies or MABs are used as biological therapy for curing certain cancer, arthritis, multiple sclerosis, heart disease, lupus, inflammatory bowel disease (Crohn’s disease and ulcerative colitis), psoriasis, and transplant rejection. Currently a diversified range of antibodies is available in the global market (Table 1.1). Polyclonal antibody (pAbs) is defined as a type of antibody that is produced by different clone of plasma B cell lines within the body. It is in contrast to monoclonal antibody that is produced from a single cell line. It is a collection of immunoglobulin molecules identifying different epitopes in the same antigen. The therapeutic antibodies are derived from a variety of sources such as mice, rats, rabbits, non-human primates, or humans and thereafter are engineered to be as human as possible. The final design of human-like antibodies is based on information from natural human antibody, the gene responsible for production of antibody, and even B cell biology. Mostly commercialized antibodies are derived from mouse hybridomas and then humanized. The humanized antibody is made in the laboratory by combining a human antibody with a small part of a mouse or rat monoclonal antibody. The mouse or rat part of the antibody binds to the target antigen, and the

IGF-1R; human IgG1 CD38; humanized IgG1 CCR5; humanized IgG4 CGRP; humanized IgG1 TROP-2; humanized IgG1 ADC HER2; humanized IgG1 ADC Nectin-4; human IgG1 ADC P-selectin; humanized IgG2

(Pending)

(Pending)

(Pending) (Pending)

(Pending)

(Pending) (Pending) (Pending) (Pending) (Pending) Enhertu

Padcev Adakveo

REGNEB3

Narsoplimab

Tafasitamab Satralizumab

Inebilizumab

Teprotumumab Isatuximab Leronlimab Eptinezumab Sacituzumab govitecan [fam]-trastuzumab deruxtecan Enfortumab vedotin Crizanlizumab

CD19; humanized IgG1

CD19; humanized IgG1 IL-6R; humanized IgG2

BCMA; humanized IgG1 ADC EpCAM; humanized scFv immunotoxin Ebola virus; mixture of 3 human IgG1 MASP-2; human IgG4

(Pending) (Pending)

Belantamab mafodotin Oportuzumab monatox

Target; format HER2; chimeric IgG1 GD2; humanized IgG1

Brand name (Pending) (Pending)

INN Margetuximab Naxitamab

Urothelial cancer Sickle cell disease

Hematopoietic stem cell transplantassociated thrombotic microangiopathies Diffuse large B-cell lymphoma Neuromyelitis optica and neuromyelitis optica spectrum disorders Neuromyelitis optica and neuromyelitis optica spectrum disorders Thyroid eye disease Multiple myeloma HIV infection Migraine prevention Triple-neg. breast cancer HER2+ breast cancer

Ebola virus infection

1st indication approved/reviewed HER2+ breast cancer High-risk neuroblastoma and refractory osteomedullary disease Multiple myeloma Bladder cancer

NA Review

NA Review NA NA NA NA

NA

NA Review

NA

NA

NA NA

1st EU approval year NA NA

(continued)

2019 2019

Review Review Review Review Review 2019

Review

Review Review

Review

Review

Review Review

1st US approval year Review Review

Table 1.1 Therapeutic monoclonal antibodies approved/under process in the EU and the USA (With courtesy from: Janice M. Reichert, the Antibody Society)

1.1 Strategies in Life Sciences 11

Brand name BEOVU Polivy Skyrizi Evenity

Cablivi

Ultomiris Gamifant

Libtayo Ajovy Lumoxiti

Emgality Takhzyro Poteligeo Aimovig Ilumya Trogarzo Crysvita IMFINZI Hemlibra

Fasenra

INN Brolucizumab Polatuzumab vedotin Risankizumab Romosozumab

Caplacizumab

Ravulizumab Emapalumab

Cemiplimab Fremanezumab Moxetumomab pasudotox Galcanezumab Lanadelumab Mogamuizumab Erenumab Tildrakizumab Ibalizumab Burosumab Durvalumab Emicizumab

Benralizumab

Table 1.1 (continued)

PD-1; human mAb CGRP; human IgG2 CD22; murine IgG1 dsFv immunotoxin CGRP; human IgG4 Plasma kallikrein; human IgG1 CCR4; humanized IgG1 CGRP receptor; human IgG2 IL-23p19; humanized IgG1 CD4; humanized IgG4 FGF23; human IgG1 PD-L1; human IgG1 Factor IXa, X; Humanized IgG4, bispecific IL-5Rα; humanized IgG1

von Willebrand factor; humanized nanobody C5; humanized IgG2/4 IFNgamma; human IgG1

Target; format VEGF-A; humanized scFv CD79b; humanized IgG1 ADC IL-23p19; humanized IgG1 Sclerostin; humanized IgG2

Asthma

Migraine prevention Hereditary angioedema attacks Cutaneous T-cell lymphoma Migraine prevention Plaque psoriasis HIV infection X-linked hypophosphatemia Bladder cancer Haemophilia A

1st indication approved/reviewed Macular degeneration Diffuse large B-cell lymphoma Plaque psoriasis Osteoporosis in postmenopausal women at risk of fracture Acquired thrombotic thrombocytopenicpurpura Paroxysmal nocturnal haemoglobinuria Primary haemophagocytic lymphohistiocytosis Cutaneous squamous cell carcinoma Migraine prevention Hairy cell leukaemia

2018

2018 2018 2018 2018 2018 2019 2018 2018 2018

2019 2019 NA

2019 Review

2018

1st EU approval year Review Review 2019 2019

2017

2018 2018 2018 2018 2018 2018 2018 2017 2017

2018 2018 2018

2018 2018

2019

1st US approval year 2019 2019 2019 2019

12 1 Large Biomolecules: An Overview

OCREVUS TREMFYA BESPONSA Kevzara Dupixent Bavencio Siliq, LUMICEF Tecentriq Zinplava

Lartruvo Cinqaero, Cinqair Anthim

Taltz Darzalex Empliciti Portrazza Praxbind

Praluent Nucala Repatha Unituxin Cosentyx Opdivo

Ocrelizumab Guselkumab Inotuzumabozogamicin Sarilumab Dupilumab Avelumab Brodalumab Atezolizumab Bezlotoxumab

Olaratumab Reslizumab Obiltoxaximab

Ixekizumab Daratumumab Elotuzumab Necitumumab Idarucizumab

Alirocumab Mepolizumab Evolocumab Dinutuximab Secukinumab Nivolumab

PCSK9; human IgG1 IL-5; humanized IgG1 PCSK9; human IgG2 GD2; chimeric IgG1 IL-17a; human IgG1 PD1; human IgG4

CD20; humanized IgG1 IL-23 P19; human IgG1 CD22; humanized IgG4, ADC IL-6R; human IgG1 IL-4Rα; human IgG4 PD-L1; human IgG1 IL-17R; human IgG2 PD-L1; humanized IgG1 Clostridium difficile enterotoxin B; human IgG1 PDGRFα; human IgG1 IL-5; humanized IgG4 Protective antigen of B. anthracis exotoxin; chimeric IgG1 IL-17a; humanized IgG4 CD38; human IgG1 SLAMF7; humanized IgG1 EGFR; human IgG1 Dabigatran; humanized Fab Psoriasis Multiple myeloma Multiple myeloma Non-small cell lung cancer Reversal of dabigatran-induced anticoagulation High cholesterol Severe eosinophilic asthma High cholesterol Neuroblastoma Psoriasis Melanoma, non-small cell lung cancer

Multiple sclerosis Plaque psoriasis Haematological malignancy Rheumatoid arthritis Atopic dermatitis Merkel cell carcinoma Plaque psoriasis Bladder cancer Prevention of Clostridium difficile infection recurrence Soft tissue sarcoma Asthma Prevention of inhalational anthrax

2015 2015 2015 2015 2015 2015

2016 2016 2016 2015 2015

2016 2016 Review

2018 2017 2017 2017 2017 2017 2017 2017 2017

(continued)

2015 2015 2015 2015 2015 2014

2016 2015 2015 2015 2015

2016 2016 2016

2017 2017 2017 2017 2017 2017 2017 2016 2016

1.1 Strategies in Life Sciences 13

Stelara Cimzia

Ofatumumab Canakinumab Golimumab

Ustekinumab Certolizumab pegol

(Pending) Perjeta Adcetris

IL-12/23; human IgG1 TNF; humanized Fab, pegylated

CD20; human IgG1 IL-1β; human IgG1 TNF; human IgG1

BLyS; human IgG1 CTLA-4; human IgG1 RANK-L; human IgG2 IL-6R; humanized IgG1

Kadcyla

Ado-trastuzumab emtansine Raxibacumab Pertuzumab Brentuximab vedotin

Benlysta Yervoy Prolia RoActemra, Actemra Arzerra Ilaris Simponi

B. anthracis PA; human IgG1 HER2; humanized IgG1 CD30; chimeric IgG1, ADC

Keytruda Cyramza Entyvio Sylvant Gazyva

Pembrolizumab Ramucirumab Vedolizumab Siltuximab Obinutuzumab

Belimumab Ipilimumab Denosumab Tocilizumab

Target; format CD19, CD3; murine bispecific tandem scFv PD1; humanized IgG4 VEGFR2; human IgG1 α4β7 integrin; humanized IgG1 IL-6; chimeric IgG1 CD20; humanized IgG1; Glycoengineered HER2; humanized IgG1, ADC

Brand name Blincyto

INN Blinatumomab

Table 1.1 (continued)

Chronic lymphocytic leukaemia Muckle–Wells syndrome Rheumatoid and psoriatic arthritis, ankylosing spondylitis Psoriasis Crohn disease

Anthrax infection Breast cancer Hodgkin lymphoma, systemic anaplastic large cell lymphoma Systemic lupus erythematosus Metastatic melanoma Bone Loss Rheumatoid arthritis

Breast cancer

Melanoma Gastric cancer Ulcerative colitis, Crohn’s disease Castleman disease Chronic lymphocytic leukaemia

1st indication approved/reviewed Acute lymphoblastic leukaemia

2009 2009

2010 2009 2009

2011 2011 2010 2009

NA 2013 2012

2013

2015 2014 2014 2014 2014

1st EU approval year 2015

2009 2008

2009 2009 2009

2011 2011 2010 2010

2012 2012 2011

2013

2014 2014 2014 2014 2013

1st US approval year 2014

14 1 Large Biomolecules: An Overview

Removab

Soliris Lucentis Vectibix Tysabri Avastin Erbitux Raptiva Xolair Bexxar Zevalin Humira MabCampath, Campath-1H; Lemtrada Mylotarg Herceptin Remicade Synagis

Simulect Zenapax; Zinbryta

MabThera, Rituxan Reopro Panorex Centoxin Orthoclone Okt3

Catumaxomab

Eculizumab Ranibizumab Panitumumab Natalizumab Bevacizumab Cetuximab Efalizumab Omalizumab Tositumomab-I131 Ibritumomab tiuxetan Adalimumab Alemtuzumab

Gemtuzumabozogamicin Trastuzumab Infliximab Palivizumab

Basiliximab Daclizumab

Rituximab Abciximab Edrecolomab Nebacumab Muromonab-CD3

CD20; chimeric IgG1 GPIIb/IIIa; chimeric IgG1 Fab EpCAM; murine IgG2a Endotoxin; human IgM CD3; murine IgG2a

IL-2R; chimeric IgG1 IL-2R; humanized IgG1

CD33; humanized IgG4, ADC HER2; humanized IgG1 TNF; chimeric IgG1 RSV; humanized IgG1

EPCAM/CD3; rat/mouse bispecific mAb C5; humanized IgG2/4 VEGF; humanized IgG1 Fab EGFR; human IgG2 a4 integrin; humanized IgG4 VEGF; humanized IgG1 EGFR; chimeric IgG1 CD11a; humanized IgG1 IgE; humanized IgG1 CD20; murine IgG2a CD20; murine IgG1 TNF; human IgG1 CD52; humanized IgG1

Acute myeloid leukaemia Breast cancer Crohn disease Prevention of respiratory syncytial virus infection Prevention of kidney transplant rejection Prevention of kidney transplant rejection; multiple sclerosis Non-Hodgkin lymphoma Prevention of blood clots in angioplasty Colorectal cancer Gram-negative sepsis Reversal of kidney transplant rejection

Paroxysmal nocturnal haemoglobinuria Macular degeneration Colorectal cancer Multiple sclerosis Colorectal cancer Colorectal cancer Psoriasis Asthma Non-Hodgkin lymphoma Non-Hodgkin lymphoma Rheumatoid arthritis Chronic myeloid leukaemia; multiple sclerosis

Malignant ascites

1998 1995 1995 1991 1986

1998 2016;1999

2018 2000 1999 1999

2007 2007 2007 2006 2005 2004 2004 2005 NA 2004 2003 2013;2001

2009

1997 1994 NA NA 1986

1998 2016;1997

2017;2000 1998 1998 1998

2007 2006 2006 2004 2004 2004 2003 2003 2003 2002 2002 2014;2001

NA

1.1 Strategies in Life Sciences 15

16

1 Large Biomolecules: An Overview

Fig. 1.2 Types of chimeric antibodies

human part makes it less likely to be destroyed by the body’s immune system. Each of the methods has its advantages and disadvantages. Chimeric Antibody Chimeric antibody (cAb) is an antibody made by fusing the antigen-binding region (variable domains of the heavy and light chain, VH and VL) from one species like mouse, with the constant domain (effecter region) from another species such as a rabbit (Fig. 1.2). Chimerization or humanization of antibody is an important biological tool for converting mouse monoclonal antibody into a therapeutic antibody for human use, whereas humanized antibody is made by combining human antibody with a small part of a mouse or rat monoclonal antibody (Fig. 1.3). Humanization of murine monoclonal antibodies has vastly improved their in vivo tolerability. Humanized Antibody Production Without Using Mice Humanization of antibody formed from non-human (mouse, rabbit, goat, horse, and other animals) antibody can be eliminated or can reduce the immunogenicity. In this process, microorganism (as phage display) or even cell-free extracts can be exploited. This technique is convenience for in vitro selection of specific mAbs, greatly facilitating recombinant production of reagents for use in research and clinical diagnostics, as well as for therapeutic use in human. Phage Display Technique Phage display (ADP) is an old technique discovered by Smith in 1985 (Smith 1985). The basis of this technique was to present polypeptides on the surface of lysogenic

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Fig. 1.3 This figure shows the difference between murine, chimeric, and humanized antibodies

filamentous bacteriophages. Currently, this tool has been effectively used for the production of large amount of peptides, proteins, and antibodies. Since then, this method has become one of the most effective ways for producing large amounts of peptides, proteins, and antibodies. Filamentous bacteriophage (genus Inovirus) is defined by its filament-like or rod-like shape. It possesses few genes and is one of the simplest biological systems known so far. It is having genome of circular single-stranded deoxyribonucleic acid (SSDNA) packaged into long filaments. Without killing bacteria (without lysing bacterial cell membrane), these phases secrete into the environment. The most commonly seen filamentous phages are: Pf phages, M13 bacteriophage, f1 phage, fd phage, and Pf phage (infects Pseudomonas aeruginosa). Ff phages: The Ff phages are filamentous type of phages that infect Gramnegative bacteria bearing the F episome. It mainly refers to the closely related group of phages including the f1, fd, and M13 phages. M13 phage: This filamentous bacteriophage is having circular single-stranded DNA (ssDNA) with 6407 nucleotides long encapsulated in about 2700 copies of major coat proteins P8 and capped with five copies of two different minor coat proteins on the ends. The minor coat protein P3 attaches to the receptor at the tip of the F pilus of the E. coli. M13 plasmids are used for recombinant DNA processes, and the virus has also been used for phage display, nanostructures, and nanotechnology applications (Fig. 1.4). f1 phase: This phage is structurally classified as a class I filamentous phage and is closely related to the other Ff phages, such as M13 and phage fd. fd Phage: It is a typical bacteriophage that infects Escherichia coli. It is similar to Enterobacteria phage M13 with respect to many structural and genomic characters. The best example of such case is Escherichia coli K12-infecting Ff phages (f1, fd or M13) that always replicate episomally. Mostly, Gram-negative bacteria are having “lysogenic” or chromosomally integrated filamentous phages. E. coli Ff phage display technology is used for therapeutic recombinant antibodies.

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Fig. 1.4 M13 filamentous bacteriophage (blue: coat protein pill, brown: coat protein pVI, red: coat protein pVII, limegreen: coat protein pVIII, fuchsia: coat protein: piX, purple: single-stranded DNA) (with courtesy from Wikipedia, https://en.wikipedia.org/wiki/M13_bacteriophage)

Adalimumab (Humira) is derived by the application of phage display methodology (Kempeni 1999;Rau 2002;Lee et al. 2007). CDR Grafting Method for Humanized Antibody Without using chimeric antibody as intermediate, humanization (also called reshaping or CDR-grafting) of monoclonal antibody (mAb) derived from non-human sources is made by CDR grafting technology. This is made by inserting the appropriate CDR coding segments (the so-called donor, responsible for the desired binding properties) into a human antibody “scaffold” (the so-called acceptor). The main problem of using murine monoclonal antibodies is their releasing of human anti-murine antibody (HAMA) that has adverse effects on patients. In order to overcome such problem, murine antibodies are genetically tailored with the amino acid residues present in their human counterparts. The complementarity-determining regions (CDRs) are linked with the variable light (VL) and variable heavy (VH) frameworks of human immunoglobulin molecules without any disturbance of the integrity of the antigen-binding site. But, there is a possibility of changing the behaviour pattern as an anti-idiotypic (anti-Id) of the xenogeneic antibodies in patients. In order to reduce the anti-Id response, the grafting of CDR residues is made onto the specificity-determining residues (SDRs) frameworks. The SDRs are identified through the help of the database of the three-dimensional structure of the antigen–antibody complexes of known structure or by mutational analysis of the antibody-combining site. Alemtuzumab is a humanized antibody produced by CDR grafting method. This humanized antibody is manufactured by Genzyme for clinical treatment of B-cell chronic lymphocytic leukaemia and multiple sclerosis (SheikhTaha and Corman 2017;Clerico et al. 2017;Brownlee and Chataway 2017). Global Market The global market for monoclonal antibodies was about USD 115.2 billion in 2018. It is expected to reach US$131.766 billion by 2023 (Fig. 1.5). The continuous increase in market demand of monoclonal antibodies is mainly due to persistent innovation in drug design and discovery, increasing incidence of chronic disease,

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Fig. 1.5 Monoclonal antibodies market (source: grandvieweredearch.com)

and high adoption rate of the therapeutic antibodies in emerging economies. Till November 2016, nineteen monoclonal antibody were marketed, and out of which thirteen monoclonal antibodies were derived from transgenic mice technology. Following are famous companies that involve in using transgenic technology for therapeutic antibody manufacturing process. Medarex It is an American biopharmaceutical company located in Princeton, New Jersey. In 2009, Medarex was purchased by Bristol Myers Squibb. CTLA-4 and PD-1 are two major monoclonal antibodies produced by Medarex. These antibodies act as “brakes” on the T-cells’ anticancer activities. The monoclonal antibodies bind to these proteins and block them by releasing the T-cell to attack cancer cells (CouzinFrankel 2013). In 2009, the FDA approved Simponi, a human monoclonal antibody to tumour necrosis factor alpha, co-developed with Johnson & Johnson Biotech., for the treatment of arthritis. In 2011, FDA approved ipilimumab, a monoclonal antibody to CTLA-4, for treatment of metastatic melanoma. In 2014, FDA approved nivolumab, a monoclonal antibody to PD-1, for treatment of advanced melanoma (BMS Newsroom n.d.). Its use was expanded to the treatment of squamous non-small cell lung carcinoma in 2015 (FDA n.d.). Abgenix Abgenix is a biopharmaceutical company established in 1996 and is headquartered in Fremont, California. It currently operates as a subsidiary of Amgen, Inc. It is engaged in the development and manufacturing of human therapeutic antibodies for

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the treatment of various diseases like cancer, inflammation, and metabolic disease. Its antibody therapeutic product candidates, which are in clinical trials include Panitumumab (ABX-EGF), a human antibody therapeutic product candidate for clinical use of epidermal growth factor receptor and for the treatment of various solid tumours. Abgenix also developed ABX-10241 (ABX-PTH), a fully humanized antibody for the treatment of parathyroid hormone for the treatment of a secondary hyper-para-thyroidism. Regeneron Pharmaceuticals, Inc. Regeneron Pharmaceuticals is an American biotechnology company having headquarters in Eastview, near Tarrytown, New York, and was founded in 1988. Mainly, this company targets at the manufacturing neurotrophic factors and their regenerative potential. The main focus consists of both cytokine and tyrosine kinase receptors. Kymab Kymab is a global biopharmaceutical company mainly involved in development and discovery of fully humanized therapeutic antibody drugs with a proprietary antibody platform having full diversity of human antibody-designing technology. Kymab is a process to develop strong international drug discovery platform mainly related to therapeutic humanized antibody drugs. This company was founded in 2010 having successful track recording in drug discovery and development in the field of immuno-oncology, auto-immunity, haematology, and infectious diseases. The OmniAb The OmniAb therapeutic human antibody platforms with the brand names OmniRat, OmniFlic, OmniMouse, OmniChicken, and OmniClic produce widely diversified, fully humanized antibody drugs, optimized in vivo for immunogenicity, manufacturability, and therapeutic efficacy. Trianni Inc. Trianni mouse involves in transgenic antibody design targeting for entire human antibody repertoire in a single organism. In this mouse, the murine immunoglobulin heavy, kappa, and lambda chain loci are replaced by respective transgenic loci, in which human exons are embedded in murine control regions for therapeutic antibody drug production. Ablexis, LLC Ablexis, LLC was established in 2009. The company offers antibody drug, and other related products, and serves customers in the USA. Ablexis developed fully licensed AlivaMab Mouse, a transgenic mouse that has superior quality antibody production potential as compared to others. Currently, world’s top pharmaceutical companies prefer to use AlivaMab Mouse for antibody drug discovery.

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Biologic Drug Delivery

Mostly, biologics are administered by injection or infusion because if they are taken orally they would be digested in the stomach and intestines and therefore be ineffective. Recently, attempts are in process to develop biologic drug as orally delivering system. Biologics would not survive in acidic pH and digestive enzymes of the gut. Even if survival were possible, the next issue to contend with is absorption; if the protein is not broken down properly, it will not be absorbed into the bloodstream. In order to overcome these problems, it is proposed to develop “robotic pill”, a small device capable of reaching intestinal tract and injecting a biologic drug directly into body. The biologic drug is encapsulated with specific enteric capsule having the quality of reaching lower part of stomach and stays intact at the low pH. When it gets into the small intestine, the outer cover gets dissolved at slightly higher pH of the intestine. This change in pH triggers a chemical reaction that releases carbon dioxide that blows up a tiny “balloon” found within the pill. The forceful emergence of carbon dioxide drives the drug-containing needle into the intestinal wall and delivers the biologic into the bloodstream. The needle gets dissolved, and the balloon is excreted. The development of “robotic pill” drug delivery device is discovered by Rani Therapeutic, based in San Jose, California. The company has already taken trials in animals to deliver drugs like insulin and Humaria. The same type of drug delivery system has been developed, but with modified technique for injecting biologic drug into blood stream. The tip of the needle is made of purely freeze-dried insulin in highly compressed form. The needle shaft is implanted on a tiny compressed spring held in place by a disk of sugar, all within the capsule. When swallowed, water in the stomach dissolves the sugar disk and releases the spring, injecting the needle into the stomach wall. The stomach wall does not have pain receptors, so its unlikely patents would feel the injection. To date, it has been successfully taken trial with animal system (new pill can deliver insulin into the stomach 2019) (https://www.daily-sun.com).

1.1.1.3

Pharmaceutical to Biopharmaceutical

The paradigm shift from pharmaceutical industry to biopharmaceutical industry is a long way journey from nineteenth century to twenty-first century to cover the era of traditional remedies based on centuries of folk knowledge to biological origin therapeutic molecules. Today’s modernized biopharmaceutical industry resulted from the integrated knowledge on genetic engineering, microchips-based process technology, and strict regulatory acts for controlling manufacture technology. This could only be possible due to the industrial revolution from 1760 to 1840 with the spreading of innovative scientific and experimentation, and processing technology. In 1668, Friedrich Jacob Merck purchased the second town pharmacy in Germany (Darmstadt) known as the Engel-Apotheke or Angel Pharmacy, which was in latter

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phase famous as Merk, the world’s oldest operating pharmaceutical and chemical company. In 1827, Heinrich Emanuel Merck had taken the first credit for initiating manufacturing and marketing of alkaloids. Similarly, GlaxoSmithKline came into existence in 1715 and was only in the middle of the nineteenth century that Beecham became involved in the industrial production of medicine and subsequently started marketing patent medicine in 1859. Meanwhile, in 1849, an American-based company named Pfizer started producing fine chemicals, and in later phase manufactured painkillers and antiseptics that had great demand during American civil war. In 1876, Colonel Eli Lilly set up a pharmaceutical business. He was pioneer in linking R&D with manufacturing process. In 1858, another military man Edward Robinson Squibb set up drug manufacturing unit to supply medicine to Union armies in the civil war, laying the basis for today’s Bristol Myers Squibb (BMS). In the second half of the nineteenth century, Switzerland started encouraging the homegrown pharmaceutical industry. Earlier to this Switzerland was centre of the trade in textile and dyes. Swiss manufacturer gradually began to realize that their dyestuffs had antiseptic and other properties and started marketing them as pharmaceuticals, instead of establishing separate pharmaceutical sector. Earlier, Switzerland was lacking any patent granting agency. It was treated as “pirate state”. France is used to call it “the land of counterfeiters”; Sandoz, CiBA-Geigy, Roche, and the Basel hub of the pharmaceutical industry all have their roots in this boom. The country eventually passed patent legislation in 1907, after German’s threat to scrap their bilateral trade treaty. In 1863, Bayer, a dye maker in Wuppertal (home town of Karl Marx’s collaborator Friedrich Engel), expanded its business to medicines by commercializing aspirin around the turn of the twentieth century. During this period, there was no strict demarcation between chemical industry and pharmaceutical manufactures as we have noticed today. Generally, the companies used to focus on cod liver oil, toothpaste, citric acid for soft drinks, and hair gel as on prescription medicines, During World War I, Bayer’s aspirin trademark was seized, and at the same time “American” Merck (now Merck & Co. in the USA or Merck Sharp & Dohme [MSD] elsewhere) was split off from German parent company (Merck KGaA). At the same time, Russian Revolution, Bayer’s subsidiary was seized. This short of disruption in pharmaceutical sector in the early twentieth century takes relative advantage in market, especially in the US market. The period between 1918 and 1939 was remarkable for the discovery of penicillin and insulin from biological systems. The hundred years old pharmaceutical companies like Merck, Eli Lilly, and Roche, and chemical firms like Bayer, ICI, Pfizer, and Sandoz started showing interest in manufacturing penicillin- and insulin-like therapeutic biomolecules during World War II to meet the urgency of healthcare need. As demand for analgesics and antibiotics escalated during World War II, a governmentsupported international collaboration with top pharmaceutical companies like Merck, Pfizer, Squibb, and Lilly was involved in mass production of penicillin. The unprecedented success of this effort developed a new direction for development involving collaboration between companies and the government leading to a modern era of pharmaceutical product development from living system as base.

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Immediately after the World War II, Europe had taken a major step to organize pharmaceutical market sector through social healthcare system such as the OK’s National Health Service (NHS). In 1957, the NHS introduced price fixing scheme as a provision for return on investment for drug manufacturers. This step was taken as initiative to invest in new medicine development and commercialization. In order to meet the demand for therapeutic biomolecule, recombinant DNA technology is proved as blessing for developing new diagnostic kits, monitoring devices, and new therapeutic approach. Synthesis of human insulin, development of erythropoietin by genetically modifying host cells from animals, mammalian, and plant tissues could also possible in terms of quality and quantity (Lomedico 1982; Galambos and Sturchio 1998;Steinberg and Raso 1998). Besides this, productions of new types of experimental mutant mice for research purposes are unique and challenge for safety and secure diagnosis of health and offer new opportunities for innovations to produce a wide range of therapeutic biomolecules. The recombinant DNA technology could be able to develop new era in the history of life science industry development. Series of dramatic events took place in the process of biologic drug development in the field of medical genetic and biomedicine by modifying microorganisms; animal and plant systems; and mammalian cells to produce medically useful substances (Galambos and Sturchio 1998; Steinberg and Raso 1998).

1.1.1.4

Biopharmaceuticals

Biopharmaceuticals (biologics) are derived from transgenic plants, animals, mammalians, and insects. Currently, a wide range of biopharmaceuticals in the form of vaccines, blood components, allergenic, somatic cells, gene therapies, stem cells, and recombinant therapeutic proteins are available as lifesaving drugs. The biologics are complex therapeutic molecules having the composition of sugars, proteins, or nucleic acids. Biological medicinal or therapeutic biological products are often used by regulatory agencies in place of biopharmaceuticals or biologics. The European Medicines Agency uses the term advanced therapy medicinal products (ATMPs) for medicines for human use that are belonging from genes, cells, or tissue engineering, which include gene therapy medicines, somatic stem therapy medicines, tissue engineering medicines, and combination thereof. People claim that biopharmaceutical is the branch of pharmaceutical sciences.

1.1.1.5

Top Biopharmaceutical Companies

The number of biotechnology companies and pharmaceutical industries are in the increase of magnitude since the past decades. In the year 2018, biopharmaceutical sector has witnessed a wide range of prosperities in product revenue, increase in the number of innovative and follow-ons in the global market, diversified stem cell and gene therapy, globalization of biopharmaceutical companies, and development of highly sophisticated and expensive R&D. Besides this, in 2018, there were

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significant developments in the biologics product development. For example, (1) infusion of Onpattro (Alnylam Pharmaceuticals) is the first therapeutic to treat peripheral nerve disease (polyneuropathy) caused by hereditary transthyretinmediated amyloidosis (hATTR) approved by the US FDA. It is supposed to be the first targeted RNA-based gene therapy, (2) the progress of the gene-editing technology called CRISPR, (3) oncolytic viruses that selectively infect and kill cancer cells, and (4) gene therapy. These developments could attract a number of pharmaceutical industries to invest huge amount of money in biopharmaceutical sector. Following are the top ten companies involved in the production of innovative biologics: 1. Pfizer Pfizer Inc. is a multinational pharmaceutical corporation in New York City founded in 1949. In 2018, the Fortune magazine placed Pfizer as No. 57 in the 500 largest US Corporations by total revenue (Collatz et al. 1977). In 2018, Pfizer’s revenues were $53.6 billion over the value of $1.1 billion or 2% in the year 2017. According to Pfizer’s 2019 financial guidance, company anticipates continued strong growth from key product franchises, including Ibrance, Eliquis, Xeljanz, and Xtandi. In 2018, the consumer health division of Pfizer was merged with UK Pharma giant GlaxoSmithKliner, a British company. The company manufactures a wide range of medicines and vaccines as lifesaving drugs for diseases related to immunology, oncology, cardiology, endocrinology, and neurology. The main blockbuster drugs produced by this company are: Lipitor (atorvastatin), used to lower LDL blood cholesterol; Lyrica (pregabalin) for neuropathic pain and fibromyalogia; Diflucan (flucanazole), an oral antifungal medication; Zithoromax (azithromycin), an antibiotic; Viagra (sildenafil) for erectile dysfunction; and Celebrex (Celebra, celecoxib), an anti-inflammatory drug. Pfizer has been manufacturing biosimilars since the last decade. The current commercialized in-market portfolio includes Inflectra (infliximab-dyyb in the USA), the first biosimilar monoclonal antibody approved by Europe and the USA; Nivestim (filgrastim) and Retacrit (epoetin zeta), both approved in Europe, also. On December, 2017, the US FDA approved Ixfliximab-qbtx, also a biosimilar to Remicade (infliximab). Remecade (infliximab, janssen) is a chimeric human-murine anti-tumour necrosis factor monoclonal antibody intended to treat patients with rheumatoid arthritis, Crohn’s disease, ulcerative colitis, ankylosing spondylitis, psoriatic arthritis, and plaque psoriasis. Pfizer is also manufacturing custom-made recombinant adeno-associated virus (AAV) vectors to deliver gene therapy directly to targeted cells. Currently, the company is more concentrating on disease caused by a single-gene alternation. When the vector reaches the targeted cell, the functioning gene is transferred and used as a blueprint to produce the missing or non-functioning protein. Since the last decade, Pfizer has been using animal or adult stem cells for therapeutic purpose in collaboration with leading academic, biotechnology, or pharmaceutical partners around the world who also have experience with currently available, human embryonic stem cell lines that meet the highest ethical standard set by leading scientific authorities.

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2. Novartis In 1996, Ciba-Geigy (Ciba-Geigy originated in the merger of two smaller Swiss firm, Ciba AG and J.R.) merged with Sandoz, the pharmaceutical and agrochemical division of both the companies formed Novartis as an independent entity, and established their headquarters in Basel, Switzerland. Currently, Novartis International AG is one of the largest pharmaceutical companies by both market capitalization and sales. Due to some business problem, Ciba-Geigy and Sandoz business were sold, and Ciba Speciality Chemicals spun off as independent companies. The Sandoz brand disappeared for 3 years but was revived in 2003, and Novartis restricted its generic drugs business into a single subsidiary with the name Sandoz. Following are few major steps taken by Novartis to strengthen its biopharmaceutical production. In 1998, the company expended for biotechnology-based product in collaboration with the University of California at Berkeley, Department of Pant and Medical Biology (Macilwain 1998). The agreement expired in 2003. In 2000, a new company named Syngenta AG was established with the joint collaboration of Novartis and AstraZenica (Sorkin 1999;PRNewsWire 2000). Syngenta is a global company based in Basel, Switzerland, as a biotechnology company involved in genomic research. In 2003, Novartis reorganized its generic business under the brand name of Sandoz (Novartis 2003). In 2006, Novartis acquired the California-based Chiron Corporation. Chiron Corporation was an American multinational biotechnology company based in eighteen countries on five continents and involved in three main areas of biopharmaceutical. Chiron Vaccine, Chiron Blood Testing, and Chiron BioPharmaceuticals are the main units of Chiron Corporation. The biopharmaceutical unit was integrated into Novartis Pharmaceuticals, while the vaccines and blood testing units were developed as Novartis Vaccines and diagnostics division, respectively (Emily Church for Market 2005). In 2006, Sandoz had taken credit of becoming the first company for the production of biosimilar drug with its recombinant human growth hormone drug (Novartis 2006). In 2009, Chinese vaccines company Zhejiang Tianyuan Bio-pharmaceutical Co., Ltd., was acquired by Novartis (Biosimilars News 2011). In 2009, the United States Department of Health and Human Services was pleased to sanction $486 million finds to construct to produce cell-based influenza vaccine, at Holly Spring, North Carolina. In 2013, Novartis has developed new cell culture facilities in Singapore focusing on drug substance manufacturing of clinical and commercial products including monoclonal antibody. In 2015, Novartis in association with BioPharma had completed its acquisition of two Phase III cancer drugs, the MEK inhibitor binimetinib (MEK 162) and BRAF inhibitor encorafenib (LGX 818 (Novartis 2009). In the same year, the company sold its RNAi portfolio to Arrowhead Research (GEN 2015a) and acquired rights to the CD20 monoclonal antibody Ofatumumab from GlaxoSmithKline (Staff 2015;Fierce Pharma n.d.). In 2015, Admune Therapeutics was acquired by Novartis for obtaining licensing PBF-509, an adenosine A2A receptor antagonist that was in Phase I

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clinical trials for non-small cell lung cancer, from Palobiofarma (GEN n.d.;GEN 2015b). In 2018, Novartis has developed expansion for biopharmaceutical manufacturing facilities in French for the development and marketing of monoclonal antibodies, at global level. Besides this, Novartis, also, started the production of several active ingredients including asthma, psoriasis treatment, and anti-injection drug for kidney transplantation and anti-inflammatory, based on recombinant DNA technology. In 2018, Sandoz, a Novartis division, has started joint venture for developing and manufacturing multiple biosimilars in immunology and oncology to capture world market. Biocon Limited is an Indian biopharmaceutical company located in Bangalore. At earlier stage, the company was involved in manufacturing generic active pharmaceutical ingredients (APIs) and marketed at global level. Currently, it has been in manufacturing process of novel biologics, as well as biosimilar insulins and antibodies that are sold in India as branded formulation. Insungen (rh-insulin), Canmab (Trastuzumab), Everrtor (Everolimus), Tacrograf (Tacrolimus), Alzumab (Itolizumab), and Krabeva (Bevacizumab) are some of the recombinant biologics being manufactured by Biocon. In 2019, Novartis is on the way to release five blockbuster candidates in core therapeutic area over the next 2 years. These biologics include Mayzent, Zolgensma, Brolucizaumab (RTH258), Ofatumumab (OMB 157), and Fevipipraant (QAW039). 3. Roche Roche is one of the largest world’s biotechnology company involved in manufacturing biologics for oncology, immunology, infectious diseases, ophthalmology, and diseases of the central nervous system. It was founded in 1896, Basel, Switzerland. Earlier it was involved in pharmaceutical drugs manufacturing process, and at a later stage, in the year 1986 Roche could develop protein-based therapeutics since the introduction of Roferon-A that is an interferon alfa-2a, recombinant, available in the market as a sterile protein for use by injection. Roferon-A is manufactured by recombinant DNA technology that employs a genetically engineered E. coli containing DNA that codes for the human protein. In 1990, Roche acquired a majority stake in Genentech (Collatz et al. 1977). In 2006, Genentech acquired Tanox, which was a biopharmaceutical based in Houston, Texas. Tanox had developed Xolair (a humanized antibody) in collaboration with Novartis and Genentech. In 2009, Roche acquired American biotechnology corporation “Genentech, Inc.”, as sister concern. By this time, Japanese biotechnology company Chugai Pharmaceuticals and the US-based Ventana were owned by Genentech. The Genentech was founded in 1976 by a venture capitalist Robert A. Swanson and biochemist Herbert Boyer. Boyer is considered to be a pioneer in the field of recombinant DNA technology. In 2014, Seragon was acquired by Genentech/Roche for its pipeline of small-molecule cancer drug. In 2019, Lumos Pharma merged to form Biopharmaceutical Company focusing on developing therapeutic to treat rare diseases. Currently, Roche works on next wave of therapeutic proteins giving main stress on rational design, antibody engineering, and new therapeutic modalities.

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4. Johnson & Johnson Johnson & Johnson is an American multinational corporation. It was established in 1886 in Skillman, New Jersey. Immunology, Neurosciences, Infectious Diseases, and Oncology are major franchises of this company. Besides consumer products like Brand-Aid (bandages), Tylenol medications, Johnson’s Baby products, Neutrogena skin and beauty products, Clean & clear facial wash, and Acuvue contact lenses, Johnson & Johnson is involved in the production of biologics, as major lifesaving drugs. It immunology products include the anti-tumour necrosis factor antibodies Remicade (infliximab) and Simponi (golimumab) for autoimmune diseases. Infliximab is a chimeric monoclonal antibody, clinically used for the treatment of number of autoimmune diseases like Crohn’s disease, ulcerative colitis, rheumatoid arthritis, ankylosing spondulites, psoriasis, psoriatic arthritis, and Behcet’s diseases. Golimumab is a human monoclonal antibody, which is used as an immunosuppressive drug. It is mainly used for the treatment of tumour necrosis factor-alpha (TNF-alpha). Stelara (ustekinumab) is the third immunology major product clinically prescribed for interleukin-12 and interleukin-23 and is for the treatment of psoriasis. Johnson & Johnson also manufactures oncology products like Velcade (Bortezomib) and Zytiga (abiraterone). Bortezomib is a recombinant technologybased drug, sold under the brand name of Velcade. It is mainly used for the treatment of multiple myeloma and mantle lymphoma. Bortezommib was approved for medical use in the USA in 2003 and in Europe in 2004. It is on the World Health Organization’s List of Essential Medicines (World Health Organization 2019). 5. Sanofi Sanofi originated in in 1973 as a subsidiary of French petrochemical firm Elf Aquitaine. In 1999, Sanofi merged with Synthelabo, a France-based biopharma and former L’Oréal business company to create Sanofi-Synthelabo, the six largest companies during that time. From 1973 to 1990, Sanofi-Syntheloba became a wellrewound group by acquiring several pharmaceutical groups. In 2004, Sanofi-Aventis was formed by the merger of Sanofi-Synthelabo. In 2007, Sanofi-Aventis revived the relation with Regeneron Pharmaceuticals to develop innovative biopharmaceutical, mainly monoclonal antibody, by using Regeneron R&D as platform, and SanofiAventis as sole marketing right. In 2009, the joint collaboration resulted in developing four antibodies. Among the four antibodies in clinical development, three were antibodies to (a) the interleukin-6, (b) nerve growth factor, (c) delta-like ligand, and (d) (Dll4), being developed for the treatment of advanced malignancies. In 2011, Sanofi acquired Genzyme Corporation, a biotechnology-based company headquartered in Cambridge, Massachusetts, which specializes the treatment of orphan diseases, renal diseases, endocrinology, oncology, and biosurgery. In 2014, Gezyme and Alnylam Pharmaceuticals a US biotechnology company had jointly taken a project on RNAi therapeutics. In 2015, Gezyme acquired right for marketing a rare cancer drug Caprelsa (vandetanib) from AstraZeneca. In the same year, the company had made a global collaboration with Regeneron Pharmaceuticals to discover, develop, and market new immuno-oncology drugs. In 2017, the

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company acquired Protein Sciences, a vaccines biotechnology company. In 2018, Bioverativ Inc., an American multinational biotechnology company, was acquired by Sanofi to develop and therapies for the treatment of haemophilia. In 2019, the company has acquired Synthorx for the production and marketing of THOR-707, a form of interleukin-2 (lL-2) for the treatment of multiple solid tumours. 6. Merck & Co. Inc. Basically, Merck is a German multinational pharmaceutical, chemical, and Life Sciences Company located at Darmstadt and was founded in 1668. Merck & Co. Inc. is an American multinational pharmaceutical company, established in 1891 at New Jersey, USA, as subsidiary of Merck, Germany. At present, Merck & Co. Inc. is the world’s seventh largest pharmaceutical company by market capitalization and revenue. Remicade (infliximab) is one of the major biologic products of Merck & Co. Inc. Originally, this monoclonal antibody was developed by Junming Le at the New York University School of Medicine in collaboration with Centocor (now Janssen Biotech, Inc.). This antibody is a cytokine TNF-alpha and prescribed for the treatment of a wide range of autoimmune disorders, including rheumatoid arthritis, Crohn’s diseases, ankylosing spondylitis, plaque psoriasis, and others. In 2017, Merck developed biosimilar to Remicade, Renflexis. Remicade is also marketed by Jansssen Biotech, Inc. (formerly Centocor Biotech, Inc.) in the USA, Mitsubishi Tanabe Pharma in Japan; Xian Janssen in China; and Schering-Plough (currently, a part of Merck & Co.). Gardasil is marketed by Merck Sharp & Dome. This is also manufactured by GlaxoSmithKline of the United Kingdom and marketed in Japan, as Cervarix. Gardasil is a recombinant human papilloma virus vaccine mainly used for multiple serotypes of human papilloma virus (HPV). Keytruda (pembrolizumab) is also a major product of Merck & Co. It is an immune modulator for the treatment of cancer. In 2014, it was approved by the FDA as Pembrolizumab (MK-3475) as a breakthrough therapy for melanoma treatment. Originally, Pembrolizumab was developed by Organon in 2006. In later phase, this company was acquired by Schering-Plough in 2007, and Merck & Co. acquired Schering-Plough 2 years later. In 2018, Merck applied for a Biologics License Application to the US FDA for investigational vaccine, named V920 for the treatment of Zaire strain of the Ebola virus. V920 is under the category of FDA’s Breakthrough Therapy Designation. V920 is a recombinant, replication-competent Ebola vaccine, consisting of a vesicular stomatitis (virus), which has been genetically engineered to express a glycoprotein from the Zaire ebolavirus, so as to provoke a neutralizing immune response to the Ebola virus. In 2019, the European Commission granted approval to Merck Sharo & Dohme B.V. for its Ebola Zaire vaccine. 7. AbbVie AbbVie is an American biopharmaceutical company established in 2013. Basically, it is spin-off of Abbott Laboratories, which was dealing with medical devices and healthcare located at Abbott Park, Illinois.

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In 2015, AbbVie has acquired Pharmacyclic LLC to deal with ibrutinb, a specialty drug for the treatment of B cell cancer-like mantle cell lymphoma, chronic lymphocytic leukaemia, and waldenstrom’s macroglobulinemia. Pharmacyclic LLC was an America biopharmaceutical company based in Sunnyvale, California, dealing with the development of cancer therapy. In 2017 the FDA approved ibrutinib as a second line treatment for graft vs. host disease. In 2019, AbbVie acquired Mavupharma, mainly dealing with oncology drugs. Mavupharma have been involved in development of novel approaches to target the STING (Stimulator of Interferon Genes) pathway. Signalling linked to this pathway is known to play an important role in generating an immune response against tumours. 8. Amgen Amgen Inc. is a biopharmaceutical company located in Thousand Oaks, California. It was earlier known as Applied Molecular Genetics Inc. Amgen is one of the world’s largest independent biotechnology company established in the year 1980. Amgen mainly focuses on molecular biology and biochemistry having the goal to develop recombinant DNA technology-based biologics. In 2018, the company had record sell of Neulasta, an immunostimulator used to prevent infection in patients undergoing cancer chemotherapy, and Enbrel, a tumour necrosis factor blocker used in the treatment of rheumatoid arthritis and other autoimmune diseases. Amgen has also procured global market of the following biologics: Epogen: Epoetin alfa is a human erythropoietin produced in cell culture using recombinant DNA technology. It is prescribed for the treatment of anaemia, commonly associated with chronic renal failure and cancer chemotherapy. Epoetin is manufactured by Amgen under the trade name Epogen. Janssen Biotech (formerly Ortho Biotech Products, LP), a subsidiary of Johnson & Johnson that, also, markets the same products, but under the brand name of “Procrit”. Aranesp: It is like Epogen and Procrit, an erythropoiesis-stimulating agent used to treat anaemia suffering from long-term serious kidney disease (chronic renal failure) and people receiving chemotherapy for some types of cancer. Sensipar/Mimpara: Cinacalcet is marketed in the brand name Sensipar and Mimpara used to treat hyperparathyroidism due to kidney failure and high blood calcium due to parathyroid carcinoma. In 2014, Cinacalcet was record selling in the market occupying 76th position out of the top 100 drugs in the list of largest selling pharmaceutical products. Nplate: In 2019, The US Food and Drug Administration (FDA) approved Amgen’s Supplemental Biologics License Applications (sBLA) for Nplate (romiplostim) as prescribed drug for serious autoimmune diseases characterized by low platelet counts. Vectibix (Panitumumab): It is a fully human monoclonal anti-epidermal growth factor receptor (EGFR) antibody approved by the FDA as first-line therapy for colorectal cancer (mCRC), in 2017. Prolia (Denosumab): It is a new class of drugs called anti-RANKL agents for the treatment of osteoporosis. Prolia is the brand name of Denosumab (injection).

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1 Large Biomolecules: An Overview

Xgeva (Denosumab): It is a monoclonal antibody made to target and destroy only certain cells in the body to protect healthy cells. Xgeva is the brand name of denosumab used for the treatment to prevent bone fractures and other skeletal conditions in the patient with multiple myeloma. It is also used to treat high blood levels of calcium caused by cancer. In 2018, Xgeva got approval from the FDA. 9. GlaxoSmithKline plc (GSK) It is a British multinational pharmaceutical company located in Brentford, London. It was established in 2000 by the merger of Glaxo Wellcome and Smithkine Beecham. Currently, the main strategy of GSK is to develop innovative, high-quality medicines focusing on life-threatening diseases including HIV, respiratory problems, oncology, and immuno-inflammation. In 2009 GSK with Pfizer jointly crated therapies for HIV infection with ViiV Healthcare. In 2012 Shionogi joined the company. Currently, GlaxoSmithKline is a major shareholder of this company followed by Pfizer and Shionogi. In 2013 GSK acquired Human Genome Science (HGS) for developing lupus drug Belimumab (Benlysta), Albiglutide for type 2 diabetes, and Darapladib for atherosis. Lupus (systemic lupus erythematosus, SLE) is an autoimmune disease in which the body’s immune system mistakenly attacks healthy tissue in many parts of the body. Belimumab is a human monoclonal antibody that inhibits B-cell activating factor (BAFF), also known as B-lymphocyte stimulator (BLyS). It is approved in the USA, Canada, and Europe to treat systematic lupus erythematosus (SLE). Albiglutide is glucagon-like peptide-1 agonist drug marketed by GSK for the treatment of type 2 diabetes. As of 2017, it is unclear regarding the life safety issue of this drug. But it was withdrawn from the worldwide market by July 2018 for economic reasons. Darapladip is an inhibitor lipoprotein-associated phospholipase A2 (Lp-PLA) that is in development as a drug for treatment of arthrosclerosis. But in 2013, GSK had press released regarding failure of this drug in Phase III trials. In 2014, Noratis and Glaxo with a mutual understanding deal in which Novartis sold the vaccine business to GSK and purchased its cancer division. In 2015, GSK acquired GlycoVaxy, a Swiss pharmaceutical company. In the same year, GSK had deal with Pfizer by selling two meningitis drugs (Nimenrix and Mencevax). In 2018, the company divested its portfolio of gene therapy drugs to Orchard Therapeutics. In the same year, GSK acquired Tessaro, a public pharmaceutical company mainly focused on drug development for cancer. By this deal, SKG got advantage in dealing ovarian cancer drug (Zejula) marketing. Zejula is the trade name of Niraparib, which was granted fast track designation by the US FDA. 10. Bristol Myers Squibb In 1989, Bristol Myers and Squibb merged together and formed Bristol Myers Squibb. It is an American pharmaceutical company located at New York City. The BMS mainly deals with prescription of pharmaceuticals and biologics, covering a wide range of therapeutic areas, including cancer, HIV/AIDS, cardiovascular diseases, diabetes, hepatitis, rheumatoid arthritis, and psychiatric disorders.

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In 2009, BMS diversified its activities mainly focusing on pharmaceutical business and biologic drugs. In 2011, the company accelerated its activity to produce biopharmaceuticals by opening dozens of manufacturing facilities and six product development sites. In 2014, the company procured FDA approval for the use of the PD-1 inhibitor nivolumab (Opdivo) in treating patients whose skin cancer cannot be removed or have not responded to previous drug therapies. PD-1 is known as programmed cell death protein 1 present on the surface of the cells that has a role in regulating the immune system’s and promoting self-tolerance by suppressing T-cell inflammatory activity. This prevents autoimmune diseases. In 2015, the company joined with Rigel Pharmaceuticals and got licence and commercialized PROSTVAC, Bacarian Nordic’s phase III prostate-specific antigen-targeting cancer immunotherapy.

1.1.1.6

Living System as Source of Biologics

Living system is used as host for the production of biologics. Microorganisms or plant and animal cells or mammalian cells are the most suitable for the expression of biologics by using recombinant DNA technology (Fig. 1.6). Most biologics are very large and complex molecules or mixtures of molecules.

Fig. 1.6 Expression of biologics by using recombinant DNA technology

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1 Large Biomolecules: An Overview

Fig. 1.7 Bacterial protein expression system, Escherichia coli

The biological host system used to produce biologics can be sensitive to very minor changes in the manufacturing process. In order to ensure consistency in manufacturing process over time, the upstream process for host cells culture and maintenance is strictly followed under the cGMP manufacturing process. The manufacturing process, especially handling of host cells in bioreactor, varies from the nature of recombinant host cells. 1. Microbes for biologics Bacteria as host cells: A variety of microbes such as bacteria, fungi, and even viruses are used for the production of biologic drugs like anticancer, antiinflammatory activities, and antibodies, through recombinant DNA technology. The first biologic drug, insulin, was produced using E. coli cells. The enterobacterium E. coli is commonly used for the production of recombinant proteins and widely used for primary cloning, genetic modification, and laboratory R&D purpose (Fig. 1.7). But, soon it was realized that every therapeutic cannot be produced in every bacterial cells. Especially, production of monoclonal antibody and certain enzyme faces two main obstacles. Bacterial cells are unable to correctly fold these complex proteins, nor able to confer required post-translational modifications (PTMs) that are present in most of the eukaryotic proteins (Jenkins 2007). Although glycosylation is

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the most common PTM (Walsh and Jefferis 2006), but other modification such as disulfide bond formation, phosphorylation, and proteolytic processing might be essential for biological activities. It is now possible to attach or bind synthetic PTMs in the case of pegylated products such as human growth hormone, granulocyte colony-stimulating factor, and interferon alfa-2a and alfa-2b (Bailon and Won 2009). Yeasts as Host Cells Yeasts are unicellular eukaryotic host cells having unique characters to be used for the expression of biologic drug in commercial level. It is mainly due to robust growth with high compatibility in accommodation of foreign gene for therapeutic protein expression. Mostly, Saccharomyces cerevisiae, a traditional baker’s yeast, has been used as host cells for the production of biologic drugs. Yeast system is very handy to use and get desired post-translational modifications. Currently, lot of biopharmaceuticals including vaccines and blood factors have been gradually dominating the world market. Several nonconventional yeasts species like Hansennula polymorpha, Pichia pastoro, and Yarrowia lipolyticca are also in use as an alternate option for the commercialization of biopharmaceuticals (Krainer et al. 2012; Martinez et al. 2012;Saraya et al. 2014). This is mainly due to their faster growth on cheap carbon sources and higher secretion capacity than S. cerevisiae. In 2008, H. polymorpha, a non-conventional yeast, was used for the production of hepatitis B vaccine (Seo et al. 2008). Option for using yeast as host cells is taken when some biologic is not produced in soluble form in the prokaryotic system or a specific PTM. Another methylotrophic yeast P. Pastoris was for Kallikrein inhibitor (Kalbitor) and was approved by FDA in 2009. The main reason of using yeast as host cells for heterogenous protein expression is its similarity with animal secretion pathway of recombinant protein. In yeast system only limited endogenous proteins are secreted. So, it is easy to purify the secreted recombinant protein. Generally, secretary protein production is a complex process with the sequential steps including transcription, translation, translocation, post-translational modifications and protein folding, peptide cleavage and additional glycosylation, sorting, and secretion (Hou et al. 2012a, b). But, in yeast system several limiting factors like different glycosylation process and proteolytic degradation are often encountered during secretory production of heterogenous proteins. 2. Insect as cell lines as host Currently, insect cell lines are used as host for recombinant baculovirus infection. This recombinant technology was developed in the 1990s as an easy and conventional system as compared to mammalian expression system. Insect cell lines are presently being used for the expression of a wide variety of proteins, including cytosolic enzymes and membrane-bound proteins. The main advantages of insect cells are high level of protein expression with post-translational modification as in mammalian cells, ease of scale-up, and simplified cell growth with high density. The

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1 Large Biomolecules: An Overview

recombinant protein expressed in insect cell lines is more antigenically, immunogenically, and functionally similar to the native mammalian protein. Baculovirus expression systems are powerful and versatile delivery and expression vehicles for producing high levels of recombinant protein expression (about 500 mg/ L) in insect cells. At present, more than 100 insect cell lines are available for recombinant protein production with lines derived from Bombyx mori, Mamestra brassicae, Spodoptera frugiperda, and Drosophila melanogaster. The most common insect cell expression consists of two parts: an insect host cell and a baculovirus Autographa californica. It is a multiple nuclear polyhedrosis virus (AcMNPV) vector only targeting to insects, which is safe for humans. In the baculovirus backbone, the gene of interest is attached with a suitable and strong promoter (polyhedron or p10). Transduction of the baculovirus genomes into the insect cells results in expression of both viral genes and product genes. The infected cells having new viral particles infect new cells. The life of infected cell is for a short period and will eventually lyse. After a post-infection period of about 48 h, the protein production starts and further continues. Fall army worm Spodoptera frugiperda (Sf9, Sf21, express+) and cabbage looper Trichoplusia ni (BTI-TN5B1-4 High five). A stable, inducible insect system based on the fruit fly Drosophila melanogaster (D. Mel-2) is also available. 3. Hybridoma cell lines Currently, hybridoma cell lines are widely used to produce monoclonal antibody. This is developed by immunizing a mouse with a target antigen, which induces immune response. The B lymphocytes are isolated from the spleen of immunized mouse to produce desired antibodies to the antigen. The B lymphocytes are fused with an immortal myeloma cell line, resulting in the production of desired quantity of antibody. This hybridoma of murine B lymphocytes and myeloma cells is then screened to find out its efficacy in expression of potential therapeutic antibody production. The main disadvantages with this biological process technique is the prolong time taken for cultivation and harvest the B lymphocytes from the immunized mice, and also assure on the mouse generating specific antibodies of interest. 4. Hamster cell lines Epithelial cell line derived from the Chinese hamster ovary (CHO) is a unique host cell used for the production of biologic drug both in laboratory purpose and industrial level. CHO cells are the most common mammalian cell line used for mass production of therapeutic protein on the scale of 3–10 g/l of culture. The main advantage of this expression system is that cells can be cultured in serum-free media (SFM). Mainly, the presence of bovine spongiform encephalopathy (BSE) in bovine serum albumin caused infection to CHO while growing in culture medium containing serum. Post-translational modification (PTM) in this expression system is almost similar as in human cell lines.

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5. Human cell lines Human cell lines are the most reliable host cells for the recombinant production of therapeutic protein production as the post-translational modifications (PTMs) that are consistent with those seen on endogenous human proteins. Other than human cell lines, the PTMs process in other non-human mammalian cell lines also produce non-human PTMs such as galactose-α1, 3-galactose, and N-glycolylneuraminic acid, which are potentially immunogenic. Besides this, human cell lines are grown easily in a serum-free suspension culture. The disadvantage of using human cell lines is that it is prone to viral contamination. But this problem can be mitigated with multiple viral inactivation or clearance steps. 6. Transgenic animals Transgenic animals such: goats, cattle, pigs, rabbits, and chickens can be used as sterile bioreactor to produce large complex proteins. The biological process of target gene transfer and expression of therapeutic protein can be directed to occur in the mammary gland of large and small farm animals. The mammary gland is considered as the best sterile bioreactor. The alveoli are the basic secretary gland of milk. It is composed of epithelial cells capable of synthesizing the fats, carbohydrates, and proteins, expelling the product to the inside the lumen of the alveoli. Mainly, two types of proteins secreted by the mammary gland are: caseins and serum proteins. The genes that encode these proteins are transcribed at high levels, specifically in the mammary gland during the pregnancy and lactation (Clark 1998). By means of genetic engineering techniques, these genes are manipulated to produce recombinant protein in the milk of transgenic animals (Gordon et al. 1987;Simmons et al. 1987). The promoters and regulatory regions of genes of specific milk proteins are utilized to direct the gene expression. The main advantages of using transgenic animal to produce recombinant therapeutic drugs include: (1) low capital investment as compared with other methods of therapeutic protein production, (2) high production yield, and (3) the PTMs are similar to the biological processes involved in human cell lines. Compared to large domestic animals like cow, goat system is better option for the purpose of recombinant therapeutic protein drugs. It is mainly due to that the milk contains high yield of purified therapeutic protein with relatively short generation interval and production of multiple offspring, as compared to large ruminants like cattle. A large number of transgenic animals (TAs) have been reported for therapeutic protein production (Dyck et al. 2003;Echelard 1996;Houdebine 2009;Redwan 2009; Rudolph 1999;Soler et al. 2006). The first two therapeutic agents isolated from the milk of transgenic animals are: C1 inhibitor (ruconest) and antithrobin (ATryn). Ruconest is analogous of the human C1-INH protein (conest alfa) and is purified from the milk of rabbits, manufactured

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and distributed by Swedish Orphan Biovitrum AB (SOBI, Stockholm, Sweden) in Europe. Ruconest was approved by the US FDA in 2014 for the treatment of acute angioedema attacks in adult and adolescence patients with hereditary angioedema (HAE). ATryn is the brand name of the anticoagulant antithrombin manufactured by Massachusetts-based US company eEVO Biologics (formerly known as GTC Biotherapeutics). It is derived from the milk of goats that have been genetically modified to produce human antithrombin, a plasma protein with anticoagulant properties. In 2009, ATryn was approved by the FDA for treatment of patients with hereditary antithrombin deficiency who are undergoing surgical or childbirth procedure. Some life sciences-based industries: PPL Therapeutics (England), GTC Biotherapeutics (USA), ZymoGenetics (USA), Nexia Biotechnologies (Canada), Pharmining (Netherlands), BioProtein Technologies (France) Avigenics (USA), Viragen (USA), and TranXenoGen (USA) are actively engaged in developing transgenic animal for therapeutic protein drug through mammary gland. 7. Transgenic plants for therapeutic protein Transgenic plants are also an attractive and conventional system for the production of biopharmaceuticals such as vaccines, antibodies, antibody derivatives, and some serum-derived protein. Besides this, transgenic plants have been used to produce bioactive and immunogenic peptides. Foreign genes can be introduced in plant system through Agrobacterium tumefaciens and A. rhizogenes based vector-mediated gene transfer technique. When these bacteria are infected, a proportion of Agrobacterium Ti plasmid is trans-located to the plant cell and integrated into the plant cell genome. The following are the advantages and disadvantages of these systems making them as potentially attractive recombinant protein producer : The advantages for using transgenic plants as host system is cost-effective, scalable, and safe means of producing biologics as compared to the fermentationbased method, as presently widely used in commercial purpose. The fermentation process of production needs massive investment and high operational cost. The main disadvantages associated with transgenic plants are: (1) low protein expression, (2) the post-translation gene often remains in silence mode (a sequencespecific mRNA degradation mechanism), (3) glycosylation process is different from that in human, and (4) most of the plant’s growth is seasonal and based on geographical location basis. In 2012, the FDA approved Taliglucerase alfa (Elelyso), the first recombinant plant-derived therapeutic for Gaucher disease. It was manufactured by Protalix Biothrapeutics, Israel. This company mainly manufactures plant-based therapeutic protein drugs. The enzyme Taliglucerase alfa is a carrot-expressed human recombinant β-glucocerebrosidase. Tobacco has been the most widely used crop

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for molecular farming. The main advantages with tobacco crop is its high biomass yield, well-studied genetic engineering related to expression process, year-round growth and harvesting, and availability of detailed protocol on crop growth and development. Mapp Pharmaceutical has developed ZMapp for Ebola treatment through genetically engineered tobacco plants. The entire development process of this biologic was carried out at Kentucky Bioprocessing, a unit of tobacco giant Reynolds American. This biologic drug was designed with the genes coding for chimeric MAbs that were inserted into viral vectors, and tobacco plants are infected with the viral vector encoding for the antibodies using Agrobacterium culture. Subsequently, antibodies are extracted and purified from the plant.

1.1.1.7

Biobetter vs. Biosimilar

Both the biosimilars and biobetters are ultimate version of original biologic but have differences in designing and similar in function like a biologic as reference molecule. A biosimilar is a near-perfect version of an approved biologic as reference molecule. In reality, biosimilars are officially approved version of original biologics having expiry patent right. So, we can say biosimilars are generic version of biologics whose patent right tenure has been completed. An example of an approved biosimilar is Amjevita (adalimumab-atto) used to treat rheumatoid arthritis and psoriasis. It is an anti-TNF-α monoclonal antibody biosimilar to Humaria (adalimumab). Generics are copies of synthetic drugs, while biosimilar are modelled after drugs that use living organism as important ingredients. Both the generics and biosimilars lower the cost of biologic drugs. Biobetter refers to a recombinant therapeutic protein drug belonging to the same class as an existing biopharmaceutical but is not identical. It is an improved version over the original biologic, as reference. Biobetter is designed on the success of an existing, approved biologic. Biobetters are not entirely new drugs and they are not generic versions of biological drugs. The basic interest for developing biobetter is to reduce the risk of immunogenicity and to make the drug safe and more body friendly. Currently, lot of reputed companies like Novo Nordisk, Biogen Idec, Roche Group, Sanofi-Aventis, GlaxoSmithKline, and Eli Lilly are busy in developing biobetters. Few companies even have acquired smaller, innovative biopharmaceutical companies that have promising pipelines. For example, British pharma AstraZenica purchased the biotech company Medimmune that intends to focus on biobetter R&D. The Food and Drug Administration (FDA) has developed clear regulatory act for the safe and effective use of biosimilar products for healthcare of patients (Table 1.2).

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Table 1.2 List of biosimilar approved by FDA (with courtesy from: https://www.fda.gov) Biosimilar name Avsola (infliximabaxxq) Abrilada (adalimumabafzb) Ziextenzo (pegfilgrastimbmez) Hadlima (adalimumabbwwd) Ruxience (rituximabpvvr) Zirabev (bevacizumabbvzr) Kanjinti (trastuzumabanns) Eticovo (etanerceptykro) Trazimera (trastuzumabqyyp) Ontruzant (trastuzumabdttb) Abrilada (adalimumabafzb) Amjevita (Adalimumab -atto) Avsola (infliximabaxxq) Cyltezo (Adalimumabadbm) Cyltezo (Adalimumabadbm)

Approval date December 2019

Reference product Remicade (infliximab)

November 2019

Humira (adalimumab)

Abrilada information

November 2019

Neluasta (pegfilgrastim)

Ziextenzo information

July 2019

Humira (adalimumab)

Hadlima information

July 2019

Rituxan (rituximab)

Ruxience information

June 2019

Avastin (bevacizumab)

Zirabev information

June 2019

Herceptin (trastuzumab)

Kanjinti information

April 2019

Enbrel (etanercept)

Eticovo information

March 2019

Herceptin (trastuzumab)

Trazimera information

January 2019

Herceptin (trastuzumab)

Ontruzant information

November 2019

Humira (adalimumab)

Abrilada information

September 2016

Humira (adalimumab)

Amjevita information Press release: FDA approves Amjevita

December 2019

Remicade (infliximab)

Avsola information

August 2017

Humira (adalimumab)

Cyltezo information

August 2017

Humira (adalimumab)

Cyltezo information

More information Avsola information

(continued)

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Table 1.2 (continued) Biosimilar name Erelzi (Etanerceptszzs) Eticovo (etanerceptykro) Fulphila (pegfilgrastimjmdb)

Approval date August 2016

Reference product Enbrel (etanercept)

April 2019

Enbrel (etanercept)

Eticovo information

June 2018

Neluasta (pegfilgrastim)

Hadlima (adalimumabbwwd) Herzuma (trastuzumabpkrb) Hyrimoz (adalimumabadaz) Inflectra (Infliximabdyyb) Ixifi (infliximabqbtx) Kanjinti (trastuzumabanns) Mvasi (Bevacizumabawwb) Nivestym (filgrastimaafi) Ogivri (trastuzumabdkst)

July 2019

Humira (adalimumab)

Fulphila information Press Release: FDA approves first biosimilar to Neulasta to help reduce the risk of infection during cancer treatment Hadlima information

December 2018

Herceptin (trastuzumab)

Herzuma information

October 2018

Humira (adalimumab)

Hyrimoz information

April 2016

Remicade (infliximab)

Inflectra information Press release: FDA approves Inflectra

December 2017

Remicade (infliximab)

Ixifi information

June 2019

Herceptin (trastuzumab)

Kanjinti information

September 2017

Avastin (bevacizumab)

July 2018

Neupogen (filgrastim)

Mvasi information Press release: FDA approves first biosimilar for the treatment of cancer Nivestym information

December 2017

Herceptin (trastuzumab)

Ontruzant (trastuzumabdttb) Renflexis (Infliximababda) Retacrit (epoetin alfaepbx)

January 2019

Herceptin (trastuzumab)

May 2017

Remicade (infliximab)

Renflexis information

May 2018

Epogen (epoetin alfa)

Retacrit information press release: FDA approves first epoetin alfa biosimilar for the treatment of anemia

More information Erelzi information Press release: FDA approves Erelzi

Ogivri information Press release: FDA approves first biosimilar for the treatment of certain breast and stomach cancers Ontruzant information

(continued)

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Table 1.2 (continued) Biosimilar name Ruxience (rituximabpvvr)

Approval date July 2019

Reference product Rituxan (rituximab)

More information Ruxience information

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Seo HS et al (2008) Analysis and characterization of hepatitis B vaccine particles synthesized from Hansenula polymorpha. Vaccine 26:4138–4144 Sheikh-Taha M, Corman LC (2017) Pulmonary Nocardia beijingensis infection associated with the use of alemtuzumab in a patient with multiple sclerosis. Mult Scler J 23(6):872–874 Simmons JP, McClenaghan M, Clark AJ (1987) Alteration of the quality of milk by expression of sheep beta-lactoglobulin in transgenic mice. Nature 328:530–532 Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228:1315–1317 Soler E, Thepot D, Rival-Gervier S, Jolivet G, Houdebine L-M (2006) Preparation of recombinant proteins in milk to improve human and animal health. Reprod Nutr Dev 46:579–588 Solomon M, Muro S (2017) Lysosomal enzyme replacement therapies: historical development, clinical outcomes, and future perspectives. Adv Drug Deliv Rev 118:109–134 SorkinAR (1999) The New York Times. AstraZeneca and novartis to shed agricultural units. Accessed 27 May 2013 Staff (2015) Novartis Sells RNAi R&D Portfolio to Arrowhead in $35M Agreement. Genetic Engineering & Biotechnology News. Retrieved 8 June 2016 Steinberg FM, Raso J (1998) Biotech pharmaceuticals and biotherapy: an overview. J Pharm Pharm Sci 1(2):48–59 Tchórzewski M, Boguszewska A, Abramczyk D, Grankowski N (1999) Overexpression in Escherichia coli, purification, and characterization of recombinant 60S ribosomal acidic proteins from Saccharomyces cerevisiae. Protein Expr Purif 15:40–47 Walsh G (2018) Biopharmaceutical benchmarks 2018. Nat Biotechnol 36:1136–1145 Walsh G, Jefferis R (2006) Post-translational modifications in the context of therapeutic proteins. Nat Biotechnol 24:1241–1252 World Health Organization (2019) World Health Organization model list of essential medicines: 21st list 2019. World Health Organization (WHO). hdl:10665/325771

Chapter 2

Management and Manufacturing Process of Biologics

2.1

What Is Biologic?

The definition of biologic has persistently being updated with the marketing of new product derived from engineered living resources. However, in broader sense biologic can be defined as any therapeutic complex biomolecule manufactured in, extracted from, or semi-synthesized from genetically engineered biological sources. Types of biologic drugs include vaccines, blood, blood components, cells, allergens, somatic cells, tissues, and recombinant proteins. The ward biologic is often referred as biopharmaceutical. Biologics can be composed of sugars, proteins, nucleic acids, or complex combinations of these substances. The host cells for biologics include microbes, insects, animal cells, mammalian cells, and plant cells. Biologics are distinguished as larger size molecules with high cost as compared to chemically synthesized therapeutic drugs. Various terminologies have been in use for the expression of biologics. Some regulatory agencies use the terms biological medicinal products or therapeutic biological products to express larger size biomolecules such as protein- or nucleic acid-based drugs differentiating them from products like blood, blood components, or vaccines, which are commonly isolated directly from a biological source. The Advanced therapy medical products (ATMPs) are being used by the European Medicines Agency to express biopharmaceutical products.

2.2 2.2.1

Vision, Decision, and Planning Vision

The main vision lying behind for developing biopharmaceutical company is to manufacture well-designed biological origin therapeutic molecules which, can be © Springer Nature Singapore Pte Ltd. 2020 B. K. Behera et al., Competitive Strategies in Life Sciences, New Paradigms of Living Systems 1, https://doi.org/10.1007/978-981-15-7590-7_2

43

44

2 Management and Manufacturing Process of Biologics

body friendly to patients for restoring health and transforming the lives to a normal stage. In order to develop quality biologic drugs, a biopharmaceutical company has to follow guidelines (Good Manufacturing Practice, GMP) as laid down by the World Health Organization (WHO) (World Health Organization 1992). GMP for biologic drugs was first published by the WHO in 1992. This provides risk-based approaches for manufacturing biologic drugs. Brief information on WHO guidelines for biologic drugs is explained in Table 2.1. The WHO guidelines are advisory in nature having scientific and technical justification (World Health Organization 1992). National regulatory authorities (NRAs) of any country can follow WHO guidelines in order to produce and maintain harmony in quality products for human use with safety and security. As the biologic drugs are originated from living system, maximum care is required during manufacturing process and product marketing for end users. So, WHO guidelines cover the following facts in order to maintain risk-free manufacturing process: • Host cell management and preservation (including seed lots, cell bank, and intermediate managements) • Microbes’ propagation through different living systems • Extraction, purification, crystallization of biologics of interest (including human, animal, insect, plant tissues, bacteria, and fungi) • Control and testing of biological products • Recombinant DNA/protein engineering development for target biologics • Hybridoma technique development • Packing, logistic system management, and supply chain management • Disposal of rejected biologic drugs

2.2.2

Decision and Planning

The basic approach for planning a biotechnology company is to have deep survey on the necessity of specific therapeutic molecule, and its assessment for public acceptability, and suitable resource needed to achieve the targeted objectives. For biopharmaceutical product development, the blue print is designed in phase wise by ensuring success in the completion of project to its final form. The overall planning process for developing blue print of a biopharmaceutical company includes the followings: • Strategic plans for the market survey on a target biologic drug in contest with public demand. • Pilot level R&D work with research institute or in-house laboratory on drug design and development. • Planning for developing infrastructure for manufacturing process, including all types of civil constructions. • Contract basis manufacturing of turnkey project, for long-term basis.

2.2 Vision, Decision, and Planning

45

Table 2.1 WHO guidelines for manufacturing biologic drugs No 1

Nature of assignment development/manufacturing Biological product development and manufacturing

2

Pharmaceutical products

3

Manufacturing practices for sterile pharmaceutical products

4

Active pharmaceutical ingredients

5

Good manufacturing practice for medicinal products

6

Good manufacturing practice for medicinal products

7

EU guidelines for good manufacturing practice for medicinal products for human and veterinary use EU guidelines for good manufacturing practice for medicinal products for human and veterinary use Current good manufacturing practice for finished pharmaceuticals

8

9

10

WHO good practices for pharmaceutical quality control laboratories

Purpose World Health Organization (1992) Annex 1 (WHO Technical Report Series, No. 822). http://www.who.int/ biologicals/publications/trs/areas/vaccines/gmp/WHO_ TRS_822_A1.pdf?ua¼1. Accessed 8 November 2015 2013: Annex 2 (WHO Technical Report Series, No. 986; http://www.who.int/medicines/areas/quality_ safety/quality_assurance/TRS986annex2.pdf?ua¼1. Accessed 8 November 2015 2011: Annex 6 (WHO Technical Report Series, No. 961. http://www.who.int/medicines/areas/quality_ safety/quality_assurance/ GMPSterilePharmaceuticalProductsTRS961Annex6. pdf?ua¼1. Accessed 8 November 2015 2010: Annex 2 (WHO Technical Report Series, No. 957. http://www.who.int/medicines/areas/quality_ safety/quality_assurance/ GMPActivePharmaceuticalIngredientsTRS957Annex2. pdf?ua¼1. Accessed 8 November 2015 Part I, Annex 2. Manufacture of biological medicinal substances and products for human use. Pharmaceutical Inspection Convention and Pharmaceutical Inspection Co-operation Scheme (PIC/S); 1 March 2014 (http:// www.fda.gov.ph/attachments/article/224762/pe-00911-gmp-guide-xannexes.pdf. Accessed 8 November 2015 Part II. Pharmaceutical Inspection Convention and Pharmaceutical Inspection Co-operation Scheme (PIC/S). 1 March 2014 (http://www.medsafe.govt.nz/ regulatory/Guideline/PE_009-8_GMP_Guide%20_ Part_II_Basic_Requirements_for_API.pdf. Accessed 8 November 2015) Annex 2: Manufacture of biological active substances and medicinal products for human use. Brussels: European Commission (2013) Part I. Chapter 6: Quality control. Brussels: European Commission; 2005

Code of Federal Regulations Title 21, Vol. 4, revised 1 April 2014. Silver Spring, MD: United States Food and Drug Administration; 2014 (http://www.accessdata. fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm? CFRPart¼211&showFR¼1. Accessed 4 July 2015) In: WHO Expert Committee on Specifications for Pharmaceutical Preparations: forty-fourth report. Geneva: World Health Organization; 2010: Annex 1 (WHO Technical Report Series, No. 957 (continued)

46

2 Management and Manufacturing Process of Biologics

Table 2.1 (continued) No 11

Nature of assignment development/manufacturing Good manufacturing practice for drugs (2010 revision)

12

WHO guidelines on good manufacturing practices for blood establishments

13

WHO good distribution practices for pharmaceutical products

14

WHO guidelines on quality risk management

Purpose Beijing: China Food and Drug Administration; 2011 (http://eng.sfda.gov.cn/WS03/CL0768/65113.html. Accessed 8 November 2015) In: WHO Expert Committee on Specifications for Pharmaceutical Preparations: forty-fifth report. Geneva: World Health Organization; 2011: Annex 4 (WHO Technical Report Series, No. 961; http://www.who.int/ bloodproducts/publications/GMP_ Bloodestablishments.pdf?ua¼1. Accessed 2 February 2016) In: WHO Expert Committee on Specifications for Pharmaceutical Preparations: forty-fourth report. Geneva: World Health Organization; 2010: Annex 5 (WHO Technical Report Series, No. 957; http://www. who.int/medicines/areas/quality_safety/quality_assur ance/GoodDistributionPracticesTRS957Annex5.pdf? ua¼1. Accessed 2 February 2016) In: WHO Expert Committee on Specifications for Pharmaceutical Preparations: forty-seventh report. Geneva: World Health Organization; 2013: Annex 2 (WHO Technical Report Series, No. 981; http://www. who.int/medicines/areas/quality_safety/quality_assur ance/Annex2TRS-981.pdf?ua¼1. Accessed 2 February 2016)

Program planning involves the meticulous analysis of the current status of the subjects carrying the main theme of the project to be undertaken. Besides this the process of planning also includes the clear objective, timely action plan, and strategies on timely outcome of successful results. Mostly, work planning is immediately followed by program planning. Work planning is the short-term planning involved in arrangement of adequate budget, preparation of CapEX (Capital expenditures), fixation of responsibility, and indicators for monitoring and measuring progress and results. The success of a company mainly depends on how meticulously the CapEX is designed to acquire, upgrade, and maintain physical assets such as properties, buildings, an industrial plant, technology, and equipment. An example CapEX preparation is given in Table 2.2.

2.2.3

Work Planning Leading to Product Development

Work planning is mainly to augment and modernize the target biopharmaceutical development on quality base to reach the user with minimum possible cost. The

2.2 Vision, Decision, and Planning

47

Table 2.2 An example of CapEX (capital expenditure) preparation of a typical biological product (Thiocolchicoside, TCS) is given before planning for manufacturing Capex workings for Bio CCS Crude Prepared by : Reviewed by : Sr. No. 1 Annexture-

Dr.Kumaran Dr B.K.Behera

Dated

03 th Rev -01 July2013 Released for review with Mr Anil Bansali Basis: TCS 1000 Kg, CCE-GMP 0 Kg, Bio CCS crude 800 Kg /Annum

Descripon

Basis

Remarks

Equipment cost Process Equipments

Up stream

1123.30

Down stream

245.60

1

Annexture2

2

Ulity Equipments

413.00

QC Equipments Total cost

115.50 1897.40

Piping & Valve cost Process

10%

112.33

Ulity

20%

131.72

A

Total - Eqpt. & piping

2141.45

Annexture9

Electrical cost

125.00

Annexture3

Instrumentaon cost

50.00

B

Total Material Supply cost

2316.45

5

Packing & Forwarding charges

1% of B

23.16

6

Excise Duty

-

-

7

VAT

-

-

D

Total Supply cost including P&F

8

Transportaon cost

9

Civil/ Structural cost

Most of the upstream process equipments piping are part of equipment and factored in equipment cosng

Lumpsum amount

2339.61 2% 46.79 861.25

Needs further finetuninig with Dr.Bekhara

(continued)

48

2 Management and Manufacturing Process of Biologics

Table 2.2 (continued) 10

Effluent treatment Plant

50.00

Annexture5

Fire Protecon system

50.00

12

Electrical Installaon cost

20% of 3

25.00

13

Insulaon & Painng cost

3% of A

64.24

Annexture4

HVAC systems

239.50

E

Total cost of supply & Installaon

3676.40

14

Engineering Fees

5% OF E

183.82

15

Conngency

15% of E

551.46

F

Total cost with conngency

17 18

Insurance Furnitures for Bio Plant Total Project Cost With Insurance

G

4411.68 1% of D

23.40 75.00

On Supply & Installaon

4515.08

(+/- 15%)

Assumpons: 1. The above cosng is only an esmate without any detailed engineering 2. Equipment cost is based on the budgetry quote obtained end of 2012 3. Equipments sizing are done considering 1000 Kg TCS ,0 Kg CCE-GMP and 800 Kg Bio CCS crude /Annum 4. Equipments idenficaon and Cosng is done based on understanding of the process in Shake flask level and 30 l fermenter level. 5. No cost considered for ETP system.

(continued)

2.2 Vision, Decision, and Planning

49

Table 2.2 (continued) PROCESS EQUIPMENTS SPECIFICATION AND COST ANNEXTURE: I

Capacity

Specificaon

Unit Cos Rs. Lacs

20

Bio safety cabinet room

Refregirator (-40 deg C),(-10 deg C)&(-80 degC) Incubator (360 lit) -3 Nos (Temp 28,33,37 degC res.) Double deck Shaker (150 flasks holding capacity,pilot shaker(300 flask holding capacity),Incubator shaker(300 flask holding capacity) Bio saey cabinet -2 Nos (Harizontal )

Autoclave room

Autoclave-2Nos (Capacity-150L)

3.5

Media Preparaon Vessel jacketed with agitator

Semi -Automated system, Top driven agitator with marine impeller Single mechanical seal Suitable for CIP & SIP Handhole for powder addion Jacket: Plant steam supply, Chilled water supply & return with automated valves,Sensors: Temperature, Level

Sr. No

Equipment Descripon PROCESS

1.0

Media Preparaon & Fermentaon Germ plasm

W.V (L)

G.V(L )

Incubator room Shaker and laminar flow area

1.1

200 0

2250

22.5

7

30

Semi -Automated system,

Media Preparaon Vessel jacketed with agitator 1.2

10

500

625

Top driven agitator with marine impeller Single mechanical seal Suitable for CIP & SIP Handhole for powder addion

19

(continued)

50

2 Management and Manufacturing Process of Biologics

Table 2.2 (continued) Jacket: Plant steam supply, Chilled water supply & return with automated valves Sensors: Temperature, Level Semi -Automated system,

Seed Vessel -I , Skid mounted , Boom mounted Agitator

Top driven agitator with two set rushton turbine impellers Single mechanical seal

1.3

5

10

Suitable for SIP

28

Jacket: Plant steam supply, Chilled water supply & return with automated valves. Automated temp control loop with heat exchanger & Pump in the jacket Automated pH control Seed Vessel -I , Skid mounted , Boom mounted Agitator

Semi -Automated system, Top driven agitator with two set rushton turbine impellers Single mechanical seal

1.4

5

10

Suitable for SIP

14

Jacket: Plant steam supply, Chilled water supply & return with automated valves. Automated temp control loop with heat exchanger & Pump in the jacket Automated pH control Seed Vessel -II , Skid mounted , Boom mounted Agitator

Semi -Automated system, Boom driven agitator with three set rushton turbine impellers Double mechanical seal with Thermo siphon Suitable for CIP/SIP

1.5

50

75

Jacket: Plant steam supply, Chilled water supply & return with automated valves.

72

Automated temp control loop with heat exchanger & Pump in the jacket Automated pH control Air flow control Flow meter & Control Valve

(continued)

2.2 Vision, Decision, and Planning

51

Table 2.2 (continued) Sensors: Temperature, pH, DO Seed Vessel -III, Skid mounted, Boom mounted Agitator

1.6

Semi -Automated system, Boom driven agitator with three set rushton turbine impellers Double mechanical seal with Thermosiphon Suitable for CIP/SIP 500

750

Jacket: Plant steam supply, Chilled water supply & return with automated valves. Automated temp control loop with heat exchanger & Pump in the jacket Automated pH control

102

Air flow control Flow meter & Control Valve, Automated pressure control Sensors: Temperature, pH, DO Producon Fermenter, Skid Mounted, (3-4 VVM) - 2 Nos

1.7

Semi -Automated system, Boom driven agitator with three set rushton turbine impellers Double mechanical seal with Thermosiphon Suitable for CIP/SIP 500 0

8500

Jacket: Plant steam supply, Chilled water supply & return with automated valves. Automated temp control loop with heat exchanger & Pump in the jacket Automated pH control

250

Air flow control Flow meter & Control Valve, Automated pressure control Sensors: Temperature, pH, DO Producon Fermenter, Skid Mounted, (3-4 VVM) -1 No

Semi -Automated system, Boom driven agitator with three set rushton turbine impellers Double mechanical seal with Thermosiphon Suitable for CIP/SIP

(continued)

52

2 Management and Manufacturing Process of Biologics

Table 2.2 (continued) 1.8

500 0

8500

Jacket: Plant steam supply, Chilled water supply & return with automated valves. Automated temp control loop with heat exchanger & Pump in the jacket Automated pH control

125

Air flow control Flow meter & Control Valve, Automated pressure control Sensors: Temperature, pH, DO 1.9

1.10

Sterile Filtraon unit (Pre & Final 0.2 M) for Glucose Media & Nutrient soluon Metering Pump for Glucose Feed, Plunger/ Diaphragm type, 0 to 100LPH or by Air pressure Alkali vessel, Suitable for SIP, with Sterile Air Filter

1.11

4 nos 100 LPH

12 4 nos

100 LPH

8

Semi -Automated system,

500

625

Acid dosing Vessel, Nalgen Bole with Peristalc Pump 1.12

500

625

Anfoam Vessel, Suitable for SIP, with Sterile Air Filter 1.13

0.45micron and 0.2 micron

Top driven agitator with marine impeller Single mechanical seal Suitable for CIP & SIP Handhole for powder addion Jacket: Plant steam supply, Chilled water supply & return with automated valves Sensors: Temperature, Level , pH Semi -Automated system, Top driven agitator with marine impeller Single mechanical seal Suitable for CIP & SIP Handhole for powder addion Jacket: Plant steam supply, Chilled water supply & return with automated valves Sensors: Temperature, Level , pH

15

15

Semi -Automated system,

500

625

Top driven agitator with marine impeller Single mechanical seal Suitable for CIP & SIP Handhole for powder addion Jacket: Plant steam supply,

15

(continued)

2.2 Vision, Decision, and Planning

53

Table 2.2 (continued) Chilled water supply & return with automated valves Sensors: Temperature, Level Harvested broth vessel with agitator 1.14

Semi -Automated system,

600 0

7500

CIP Staon

200 0

2500

1.16

Mobile CIP system, Pumps

3 nos

1.17

Water for injecon holding tank Glasswares for lab process Sub Total Upstream

1000 L

2

2.2

Single tank with provision for dosing acid & alkali Heat exchanger & Circulaon pump for supply & recirculaon Sensors: Conducvity, Temperature Mobile trolley with Centrifugal Pump, PUMP with VFD Sensors: Conducvity Sensor

50

15

5 3

Down Stream Process TFF filter system

2.1

47

Fully Auotmated system

1.15

1.18

Top driven agitator with marine impeller Single mechanical seal Suitable for CIP & SIP Handhole for powder addion Jacket: Plant steam supply, Chilled water supply & return with automated valves Sensors: Temperature, Level , pH

100 100 0 LPH

Buffer vessel for TFF system -2 Nos Solid Mass collecon tank/Biomass Kill tank

2.3

500

150 0 Permeate collecon tank

650

1750

10 Semi automated, SS316, Ra 0.8, Jacketed, Anchor type agitator, CIP able Sensors: Level & Temperature

25

Semiautomated, SS316, Ra 0.8, Jacketed,CIP able

(continued)

54

2 Management and Manufacturing Process of Biologics

Table 2.2 (continued) 2.4 2.5

600 0 LPH

7500

1200

10

1200

2

7500

2.6

Permeate Feed Pump to Resin Column Water feed tank

2.7

Methanol feed tank

2.8

XAD Resin

2.9

XAD column

2.10

Holding tank

100 0 100 0 200 kg 150 X 22c m 500

2.11

Aqueous fracon collecon tank

600 0

2.12

Finished Material Processing system( Milling, )

2.13

Finished Material Processing system ( Shiter)

2.14

Finished Material Processing system ( Packing)

2.15

Process pumps

Sensors: Level & Temperature

6 2.5

LS

LS

LS

Amberlite Resin XAD 1180,

36

4 glass columns each having 50 kg capacity

15

Intermienet holding tank for Aq layer Semi automated, 304 Nonjacketed, CIP able Sensors: Level & Temperature

1.8

Equipment required for Finished dried product processing units: IC Bins, Split valves, Blender, Milling, Fillling and Packing ( cGMP quality) Equipment required for Finished dried product processing units: IC Bins, Split valves, Blender, Milling, Fillling and Packing ( cGMP quality) Equipment required for Finished dried product processing units: IC Bins, Split valves, Blender, Milling, Fillling and Packing ( cGMP quality) As per requirements (around 15 pumps)

5

Intermient holding tank ofmethanol Ext mixture Reactor for methanol concentraon Condenser for the above

2

Reciever for the Methanol cponcentraon reactor For Seperaon of CCS and CCE

2

5

2

5

10

Sub Total - Down Stream Down stream process eqpts 1.0

SS holding tank

500

2.0

SS reactor

1000

3.0

SS condenser

4.0

Reciever

10 m2 500

5.0

Glass column

9"*3.

12 2.5

6

(continued)

2.2 Vision, Decision, and Planning

55

Table 2.2 (continued) 5m 6.0

Mixing vessel

500

For silica and solvent slurry preperaon For dosing tank of solvent (CCS & CCE) Intermient holding tank of Ext mixture For CCS Ext concentraon

7.0

Holding tank

500

8.0

Holding tank

200

9.0

SS reactor

1000

10.0

Condenser

11.0

Reciever

10 m2 500

12.0

Holding tank

100

13.0

SS crystalliser

14.0 15.0

6

12

For the above

2.5

For the above

2 1

500

For the Ext concentrate collecon (CCS) For CCS crystallisaon

6

Condenser

5 m2

For the above

2

Reciever

200

For the above

1.5

16.0

Holding tank

50

1

17.0 18.0

Agitated Nutrsche filter drier Holding tank

For charging DNS for crysatllisaon For filtering CCS

100

For collecng CCS ML

1

19.0

Holding tank

2000

4

20.0

RVD

12 trays

For collecng and preperaon of Ext (CCS) For drying the CCS

21

2 1.5

10

CCE 21.0

SS reactor

1000

For CCE Ext concentraon

12

22.0

Condenser

For the above

2.5

23.0

Reciever

10 m2 500

For the above

2

24.0

Holding tank

100

1

25.0

SS crystalliser

500

For the Ext concentrate collecon (CCE) For CCE crystallisaon

6

26.0

Condenser

5 m2

For the above

2

27.0

Reciever

200

For the above

1.5

28.0

Holding tank

50

1

29.0

10

30.0

Agitated Nutrsche filter drier Holding tank

Charging Ethyl acetate for crysatllisaon For filtering CCE

100

For collecng CCE ML

1

31.0

Holding tank

2000

For collecng and preperaon of Ext (CCE)

6

(continued)

56

2 Management and Manufacturing Process of Biologics

Table 2.2 (continued) 32.0

Holding tank

2000

Methanol day tank

6

33.0

Holding tank

500

DNS ML collecon tank (CCS)

2

34.0

Process pumps

8

For transfering the contents between the process vessels

9.6

35.0

Scrubber

10

37.0

Disllaon column

75 1368. 9

(continued)

2.2 Vision, Decision, and Planning

57

Table 2.2 (continued) UTILITY EQUIPMETS SPECIFICATION ANNEXTURE- 2 1

BLACK UTILITY

Capacity

Process load

2

Plant Steam generaon ( Boiler, Water tank, Fuel tank) Feed water pump

4

Chilled water generaon system, 3x100TR & Pumps Chilled water circulaon pumps Chilled water circulaon pumps Holding tanks-Primary and secondary(Insulated) Chiller compressor

300TR

Chilled Brine & Pumps

20TR

Cooling water & Pumps

78.15

75

Power Power consumpon consumpon (KWH)-Single (KWH) frementer 43.96

Cost in lacs

100

5

75

2.81

5

75

2.81

120

100

20

15

2

50

100

37.5

5 5

Chilled brine compressor Holding tanks-Primary and secondary(Insulated) Chilled brine circulaon pumps 5

Usage %

3 Ton

Furnace oil pump

3

HP

300

30

67.5

30

50

11.25

20

100

15

300 TR * 2 nos

45

16

Fan for cooling tower

50

100

37.5

Circulaon pumps for cooling tower

100

100

75

(continued)

58

2 Management and Manufacturing Process of Biologics

Table 2.2 (continued) 6

Compressed Instrument Air (Generaon, Filter, Dryer, Receiver),7.5 Kg/cm2 Air compresser reciever Compressor for Instrument air

500NCFM 50

70

26.25

21

7

Compressed Process 500NCFM 50 Air (Generaon, Filter, Compressor for process air

70

26.25

26

8 9

Air dryer for compressed air Purified water system

500 NCFM 6 KL per day

10

Vacuum pumps

3

20

6

11

Hot water system

30 m3

10

22

45

Total Electrical load in ulity HVAC systems

868

Ttal cost

948

80

100

60.0

413

(continued)

2.2 Vision, Decision, and Planning

59

Table 2.2 (continued) INSTRUMENTS FOR QUALITY CONTROL – ANNEXTURE - 3 Instrumentaon

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10. 11

Instruments related to strain improvement Gel documentaon Electroporer Electrophrosis vercal Electrophrosis Horizandal MicroCentrifuge

Specificaon

16000 rpm

PCR Microscope (stereo) with camera Double beam double wave length spectro photometer pH meter Metler balance(3) HPLC Total

Amount in Rs 5 7 2.5 2.5 2 7 7 7.5 2 3 5 50

HVAC Facility ANNEXTURE- 4 S. No 8 8.1 8.2 8.3

Acivity/Parameter

Specificaon

Clean Room Facility HVAC & Ducng, Piping, BMS, Clean Room Panels,Doors, Furnitures, Inst etc

Amount in Rs

158

Other Misc Total

81.5 239.5

Fire protecon systems ANNEXTURE- 5 S. No 5

Item EHS ( Environment, Health & Safety)

Qty

Remarks/Sepcificaon

Amount in Rs 25

(continued)

60

2 Management and Manufacturing Process of Biologics

Table 2.2 (continued) 5.1

Fire Alarm system & Public Address system

5.2 5.3 5.4

Venng & scrubber Sytem Drainage system Fire Hydrant System

5

C Total

ANNEXTURE-6 Effluent treatment system

10 5 5 50

50

ANNEXTURE-7 Furnitures for Bio Plant Furniture for:

Specificaon

Amount in Rs 25

1.0

Molecular biology and strain improvement

4 working table,1 wall rack laboratory stool-8

2.0

Analycal laboratory

2-fume hood,2-working table,8-stool,1-wall rack

15

3.0

Immobelised reactor room

5

4.0

Gel Bead preperaon

5.0

Instruments room

6.0

Wash area

7.0 8.0

Office room Central control room (DCS panel room) & documentaon room

working table-1,1-Wall rack working table-1,1-Wall rack Working table-04,1-Wall rack Working table(SS) -01,1Wall rack Computer,table,chairs etc Chairs ,tables etc Total

75

5 5 5 10 5

(continued)

2.2 Vision, Decision, and Planning

61

Table 2.2 (continued) BIO Plant Civil Construction Cost ANNEXTURE- 8

S.NO 1 2 3

Room

Class

Length in M

Utility Block (MCC room,DG, Process block

Breadth in M

Floors

Area m2

20

20

1

400

37.5

30.5

3

3431.25

10

10

1

100.00

Warehouse (Extension of warehouse)

Total Area

3931.3

Cost per Sq.M

20000

Total cost in Rs

78625000

Structural cost (Utility pipe racks)

7500000 Total cost in Lacs

861

Cost Estimation –Electrical ANNEXTURE- 9 S.No

Description

Qty

Unit cost in laks

Aprox.cost in laks

1 2 3 4 5 6 7 8 9 10 11 12 13 13a

Main changeover panel Sub Switch board MCC Panel(Process,Utility&HVAC) MPDB MLDB PDB,s LDB,s Power Cables Cable Trays and MS support Termination Materials Lighting material FLP items Earthing Materials Capacitor panel DG set (600kva)and Control panel with AMF Statutory Requirements Miscellaneous Tansformer yard extension

1 1 3 1 1 3 3 lot lot lot lot lot lot 1

10 7.5 4 0.5 0.5 0.25 0.25 15 4 3 5 5 5 3

10 7.5 12 0.5 0.5 0.75 0.75 15 4 3 5 5 5 3

1

45

45

lot lot

5 3

5 3

14 15 16 17

Total Electrical Cost

125

(continued)

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2 Management and Manufacturing Process of Biologics

Table 2.2 (continued) First floor Condenser Reciever Condenser

Condenser Reciever

Reciever

Condenser

Reciever

2 Down stream process area (Reactor,RVD,Glass column &Crystallisers) 50 L HT(DNS)1 KL reactor 500 L crystalliser Holding tank 500LCrystalliser 1 KL Reactor

10 Mixing vessel

Bio process -Technology-02 1 KL Reactor 15.5 1.5

Corridor

10

5000 L Fermenter section

Corridor

Corridor

Corridor

5 L &50 LFermanter

500 L Fermanter

Pilot shaker

Incubator

Germ plasm

Service corridor

2

7 10

6

4

1.5

4

3 2

24

37.5

(continued)

2.2 Vision, Decision, and Planning

63

Table 2.2 (continued) Ground floor 2 Service area Water holding tank RVD CCE Buffer Hol tank

CCS

Intermittent colln tank

10

solvent hol tank TFF filter ANFD Glass columns For Cocn tank

25.5

For soll elution 1.5

XAD Resin column

DNS ML coll tank

Broth coll Filtrate tank Hol tank

service area

Intermittent colln tank

Spent Aq. Layer -5KL 10 Solvent mixture preperation tanks

Second change room

First change room

5 KL Fermenters DNS ML coll tank(Cumulative) 2

Service area

7 10

6

4

1.5

4

3 2

24

37.5

(continued)

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2 Management and Manufacturing Process of Biologics

Table 2.2 (continued) Second floor 2 Service area

10

HVAC systems and accessories Purified water holding tank

25.5

service area

1.5

Nutirent preperation vessel

Purified water system

10

Warehouse storage space

HVAC systems and accessories

Media preperation 2

Service area

10

1.5

2 24

37.5

(continued)

2.2 Vision, Decision, and Planning

65

Table 2.2 (continued) Utility Block

Hot water system

Air compressor -01

Air compressor-02 Vacuum pumps Instrumentation room

3 T Boiler

Passage

Passage

20

Chilled water system

Chilled Brine system

MCC panels

20

specific steps to be followed by a promoter for establishing a biopharmaceutical manufacturing industry depend on the type of biologic and its immediate availability in the global market to meet the emergency of clinical requirement. The steps in work planning process are: • • • • • •

Design and development of biologic of interest Organization of tasks force Availability of livestock Management of technical wings and administrative cells Pathway for manufacturing product and supply chain management Development of effluent treatment plant and waste recycling

2.2.3.1

Design and Development of Biologic of Interest

The biologic drugs are highly sensitive to the nature of manufacturing process and temperature, due to their biological origin. So, quality by design (QbD) is the most essential aspect while launching a biologic in the global market (Fig. 2.1). QbD is not merely a regulatory aspect of manufacturing biopharmaceutical product development and testing but to assure the overall performance of the product in depth on

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2 Management and Manufacturing Process of Biologics

Quality by design

Survey, idenficaon selecon of product of interest

Design/formulaon to meet the necessity of end user

Development of protocol as prescribed in regulatory acts

On CQA basis

On CQA basis Q8

Idenfying quality variability during manufacturing process

Manufacturing parameters on product quality

On CQA basis

Upgrading process for consistency in quality

ICH Q6A

Risk assessment and risk control Q9 Fig. 2.1 Quality by design

quality assurance point of view and its long-term side effect to the user from safety and security aspects. Besides this, maximum attention and precaution must be taken for product performance profile (target product profile [TPP], target product quality profile [TPQP], and critical quality attribute [CQA]).

2.2.3.2

Organization of Task Force

Development of efficient, sincere, and dedicated task force is the primary requirement to bring sustainability in quality production. The task force requires a biopharmaceutical company composed of skilled and non-skilled groups belonging to scientists, engineers, technicians, program experts, logisticians, sales persons, and other global health professions to meet the objectives defined.

2.2 Vision, Decision, and Planning

2.2.3.3

67

Availability of Livestock

Timely availability of quality and cheap feedstock is primarily important for a biotechnology or biopharmaceutical industry. The biopharmaceutical products are of microbes, plants, animals, insects, and mammalian cells origin and need different types of feedstocks. Generally, glucose is the basic energy source for the growth and development of variety of host cells, especially bacteria and fungi, responsible for the production of biologics. Molasses is one of the cheap feedstock used as the source of carbohydrate and other minerals. Besides this, starch is also used as a source of carbohydrate, by enzymatic hydrolysis. Million ton of molasses is used for bio-fuel (ethanol) and organic acids (lactic acid, acetic acid, formic acid, oxalic acid, uric acid, malic acid, and amino acid) production through fermentation process. Mostly, E. coli, Corynebacterium glutamicum, Pseudomonads, Bacilli, and Baker’s yeast are used as host cells for the above purposes. But, the first-generation feedstock is derived from crop plants as energycontaining molecules like sugars, oils, and cellulose with limited availability. So, scientists are in process to utilize non-food feedstock like straw, bagasse, and forest residues as second-generation feedstock. Still, it has been in process to use algal biomass as third-generation feedstock as carbon source. With the improvement in tools and technology for harvesting sunlight, development of photobiology solar cell and electrofuels is supposed to bring new revolution in carbohydrate feedstock development from solar light, water, and CO2 available as exhaust gas from industries (Cameron et al. 2014). Currently, instead of using animal-derived feedstock; people have started using non-animal origin hydrolysates such as plant hydrolysates or yeast hydrolysates for recombinant protein and antibiotic production. These sources of hydrolysates are free from any pathogenic contamination (bacteria or viruses) and support cell growth, in better as compared to animal origin. Natural medium like animal body fluids or medium of tissue extraction like plasma, serum, lymph, and chicken embryos leaching solution is used as feedstock for mammalian cell culture. This natural culture medium contains nutrients, various somatomedin, and hormones for supporting growth and development of mammalian cells. This medium is complex in nature, and the overall composition is vary from batch to batch during extraction process. Currently, serum from cattle is widely used for animal cell culture. The main advantages of cattle (bovin calf serum, newborn calf serum, and foetal serum) serum are availability of adequate resource, easy way of preparation, and long application time.

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2 Management and Manufacturing Process of Biologics

2.2.3.4

Management of Technical Wings and Administrative Cells

Biopharmaceutical company needs well-defined manpower for administration control, manufacturing process, product quality maintenance, etc. In traditional pharmaceutical industries, it has been easy to maintain consistency in product quality due to use of well-defined chemical and physical techniques. But in biologic drugs manufacturing process, the involvement of biological processes becomes question of challenge to maintain consistency in product manufacturing process and development. The biological properties of host cells are inherently variable in features. So, it is obvious to strictly maintain quality risk management (QRM) in biologic drugs production process. So, a consortium of scientists and technicians is needed for bringing sustainability in biologic drugs production on quality base. Before engaging manpower, it is necessary to ensure that the employees are free from any conterminous disease. The manpower working in the core units should have regular health check-up practice. Workers having contaminated diseases are threat to biologic drugs quality control. Employees associated with production division should be updated with training in disinfection, personal hygiene, and microbial risk management. Non-skilled labours, administrative staff, technical experts in machineries management, and employees indirectly associated with manufacturing process should be kept away from biologic drugs production division. Personnel working in animal maintenance division should have restricted movement in production division in order to have control over cross-contamination. Staff assigned duty in BCG (Bacillus Calmette-Guerin) or any such products should be restricted for working with any other infections’ agents.

2.2.3.5

Pathway for Manufacturing Product and Supply Chain Management

The detailed blueprint of manufacturing of biologic drugs includes the starting point for suitable host cell selection, isolation, cultivation, and preservation in cell bank followed by pilot-level production of targeted biologic and development of turnkeyscale production for commercialization. The completion of entire manufacturing process is organized under the strict guidelines of good manufacturing process (GMP). The overall process for biologic drugs manufacturing is strictly governed by good manufacturing process (GMP). On the basis of product types, end-user and purpose of use, the biologic drugs are categorized into following: • • • •

Monoclonal antibodies, Recombinant therapeutic proteins, Growth hormones, and Therapeutic enzymes.

2.3 Upstream Process for Recombinant Protein Production

69

The market segments that cover biologics are hospitals, specialized clinics, and research and development institutes.

2.2.3.6

Development of Effluent Treatment Plant and Waste Recycling

Handling of wastes from biopharmaceutical industries has been a big challenging task because they contain biological wastes, other than the toxic effluent containing variety of chemicals. The biological wastes from biopharmaceutical industry include dead host cells, debris from genetically engineered host cells, unwanted organic molecules (proteins, amino acids, lipids, and nucleic acids), and wastes biological liquid (serum-like fluid). Hazardous biological wastes are often more harmful as compared to other chemical pollutants. Before disposing the wastes from biological origin, they are to be completely sterilized and processed for further disposal.

2.3 2.3.1

Upstream Process for Recombinant Protein Production Selection of Host Cells

The upstream process is initiated with the selection of suitable host cells for the heterologous expression of therapeutic biologic drugs. The host cells presently used for biologic drugs production are belonging to bacteria, fungi, plant tissues, animal cells, insect cells, and mammalian cells. Before using such host cells, recombinant DNA technology is applied to develop engineered host cells having foreign gene responsible for target biologic expression. The genetically modified host cells are left for a few generations, creating numerous identical copies. The cell suspension is subjected to centrifuge under low temperature to obtain a residue mass of host cells, which is transferred to small vials for cryopreservation under 196  C in liquid nitrogen. The cell banks are located in three different secure places from any unwanted circumstances. A manufacturer creates a working cell bank by thawing selective vial from master cell bank. Specific host cells in which target biologic drug is to be expressed must be an easy and conventional system for rapid growth and adequate quantity of protein expression (Fig. 2.2). Following are some common expression systems that mainly depend on: • • • • •

time taken for expression the target protein, easy and conventional for handling, availability of adequate amount after purification, nature of post-translational modification, and shortest possible pathway of recombinant protein expression. Before the selection of suitable host, one has to ensure the following:

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2 Management and Manufacturing Process of Biologics

cDNA Selection of suitable host cells for heterologous protein expression

Stable transfectants

Expression Vector

Cloning

Mammalian cells

Selection of positive Clones

SCALE UP PROCESS

Bacteria/Yeast/ insect/CHO Fig. 2.2 Steps involved in optimizing protein expression system to produce desired recombinant protein

• • • •

identification of foreign gene encoded with the target protein, production of cDNA from the related mRNA, selection of appropriate vector belonging to suitable expression system, and scale-up of process.

2.3.2

Medium Required for Different Host Cells Culture

2.3.2.1

Bacteria as Host Cells

Both the Gram-positive and Gram-negative bacteria are used for broad range of heterologous protein (biologic drugs) expression (Rosano and Ceccarelli 2014). The main advantages with bacteria as host cells are their fast growth, high level of protein expression, relative genetic simplicity, and genetic stability (Baneyx 2004). In Gram-positive bacteria such as Bacillus subtilis, Escherichia coli, and Streptomyces lividans, the synthesized protein directly releases into the culture broth through the general secretary pathway, due to lack of outer membrane (OM) (Baneyx 2004). Bacteria are better option for non-glycosylated biologic drugs production with high titer values (Hatada et al. 2004; Kang et al. 2014; Öztürk et al. 2016a). A wide range of biologic drugs like insulin, interleukin-2 (IL-2), and human growth hormone (hGH) have been in commercial production line with great challenges (Reed and Chen 2013a; Wang et al. 1984; Azam et al. 2015; Baneyx 1999). Production of recombinant insulin from genetically engineered

2.3 Upstream Process for Recombinant Protein Production

71

E. coli, in 1980, was the most remarkable milestone in the history of heterologous protein expression (World Health Organization 1992). Plenty of information on production of biologic drugs is available in several reviews (Öztürk et al. 2016b; Park and Schumann 2015; Westers et al. 2004; Anné et al. 2012). In Gram-negative bacteria most of the secretary proteins accumulate in the periplasm location, after synthesized in cytoplasm matrix. Periplasm is concentrated gel-like matrix located between the inner cytoplasmic membrane and the bacterial outer membrane (Fig. 2.3). The proteins are also associated with denaturized recombinant protein. So, in protein isolation process, the Gram-negative bacteria are processed for enzymatic lysis before subjecting for downstream process. Gram-negative bacteria are having seven secretary systems (type I–VI, and type VIII) for secreting protein to the extracellular space (Costa et al. 2015; Griffin et al. 2007). Out of these seven pathways for secretion of protein, the recombinant protein secretes through five systems that include type I (T1SS), type II (T2SS), type III (T3SS), type V (T5SS), and type VIII (T8SS) (Reed and Chen 2013b; Natale et al. 2008). Mostly, these secreted proteins (unfold) first detain inside the cell. In Grampositive bacteria the protein migrates to culture medium. But, in Gram-negative bacteria the secreted proteins migrate to cytoplasm or periplasm zone of the host cells. The overall process for heterologous protein expression (human protein expression) in E. coli is shown in Fig. 2.4. The cost of therapeutic protein mainly depends on the new tools and techniques involved in downstream processing. Secretion pathway of recombinant protein in Gram-negative bacteria is a complex process, and the resulted therapeutic proteins are either detained in cytoplasmic matrix or migrate to periplasm zone. In addition, it

Outer membrane

DNA

Periplasm PROTEIN PROTEIN

Transcripon

Translaon

mRNA mRNA

CYTOPLASM

Fig. 2.3 Secretory protein production in Gram-negative bacteria

Inner membrane

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2 Management and Manufacturing Process of Biologics

cDNA from genomic library

E. coli with gene of interest

Selection of positive clone

Positive vector with gene of interest

Scale-up of transformants for Protein of interest Fig. 2.4 Heterologous protein expression in E. coli

has been a great challenge to separate the therapeutic proteins from numerous inclusion bodies like denatured proteins, nucleic acids, and other biomolecules. So, further tailoring the pathway of heterologous protein production, especially in Gram-negative bacteria would be helpful in getting high titer values of heterologous proteins. Protein tags technology has been commonly used for purification of recombinant proteins. Protein tags are peptide sequences genetically grafted onto a recombinant protein. During purification process, the protein tags are removed either by chemical agents or by enzymatic reaction. Generally, affinity tags are added to the target proteins to purify in conventional affinity chromatography technique. This technique is applicable to various types of proteins that include: chitin-binding protein (CBP), maltose-binding protein (MBP), Strep-tag, and glutathione-S-transferase (GST). Mostly, in separation of recombinant protein expressed in genetically engineered E. coli, solubilization tags are used. These include thioredoxin (TRX) and poly (NANP). For the separation and purification of heterologous protein from Gram-

2.3 Upstream Process for Recombinant Protein Production

73

negative bacteria, FLAsH and YebF are used as promising methods. More detail on recombinant protein purification is described in downstream process. Following important steps are involved in recombinant protein expression: • Screening/selection of competent E. coli from commercial source to captured DNA sequence of interest, • Integration of foreign DNA with bacterial genome/circularized the DNA with a plasmids, • Selection of transformed E. coli using selection marker (antibiotic commercially available), • Scale-up process of process, and • Isolation of targeted recombinant protein.

2.3.2.2

Yeast as Host Cells

Commonly, Saccharomyces cerevisiae and Pichia pastries are commercially used for the production of heterologous therapeutic protein production (mostly for human protein). The advantages with yeasts are their luxury growth with high titer values of target protein (Nielsen 2013; Huang et al. 2014). In addition, yeasts, especially S. cerevisiae and P. pastries, are genetically wellstudied host systems that can be easily handled by the tools and techniques related to recombinant DNA and protein engineering (Fig. 2.5). The growth media used for these yeasts culture is easy to procure with reasonable cost. Yeasts also follow the general eukaryotic post-translational modification pattern. Yeasts have been commercially used for the production of variety of biologic drugs (Jozala et al. 2016). P. pastoris has gained popularity due to commercial production of human insulin, human serum albumin, hepatitis B vaccine, interferon-alpha 2b, trypsin, and collagen (Pichia Produced Products 2018). This yeast is grown in methanol as carbon source, due to its obligate aerobic habit. Various recombinant proteins using S. cerevisiae as a host have been marketed. Nevertheless, in recent years, many proteins have become commercially available using other yeasts as hosts. Among them, P. pastoris has gained attention for marketing novel recombinant therapeutics such as human insulin, human serum albumin, hepatitis B vaccine, interferon-alpha 2b, trypsin, and collagen, among others. P. pastoris is an obligate aerobic yeast that can use methanol as a carbon source (Yanagita et al. 1992; Arakawa et al. 1998; Nykiforuk et al. 2006; Boyhan and Daniell 2011). Some of the salience features of heterologous protein expression are: • Selection of competent E. coli to take up cDNA (commercially available) selected from genomic library. • Integration of DNA into bacterial genome or circularization of the DNA sequence to exist as a plasmid. • Selection of competent E. coli (Commercially available) having DNA sequence of interest.

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2 Management and Manufacturing Process of Biologics

Transform cDNA library into E. coli E. coli with gene of interest

Selection and expression of positive clone

Yeast for scale up For protein of interest Plasmid /expression vector

Transformation of yeast Fig. 2.5 Heterologous protein expression in yeast system

• Selection of transformed E. coli using a selection marker (antibiotic). • Multiplication of E. coli (having target plasmid) in suitable media isolation of plasmid containing DNA of interest. • Isolation of plasmid. • Transformation into yeast. • Screening and isolation of yeast cloned with DNA of interest • Isolation of secreted target protein and further purification.

2.3.2.3

Insect Cells

Alike mammalian cells, insect cells are also used for high-level protein expression. The main advantages with mammalian cells are: ease of scale-up process, adaptable for high-density cell suspension culture, and occurrence of post-translation process as noticed in mammalian cells. The heterologous proteins produced in insect cells

2.3 Upstream Process for Recombinant Protein Production

75

are antigenically, immunogenetically, and functionally similar to the native mammalian proteins that are commonly expressed in yeast and other eukaryotic cells (Fig. 2.6). The cell lines derived from Spodoptera frugipperda, Sf21 and Sf9, are commonly used for recombinant protein expression. Baculovirus is a lytic, dsDNA

cDNA from genomic library

Transformaon Competent E.coli

Plasmid isolation

Plasmid with viral gene

Insect cells lines of Sf 21, sf9

Cotransfection of insect cells

Production of the insect cells with high titer recombinant virus stock for therapeutic

Fig. 2.6 Insect cell expression systems—Sf9 and Sf21

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2 Management and Manufacturing Process of Biologics

virus, commonly compatible in insect cells belonging to Lepidoptera family. It is non-infectious in vertebrates, and its promoters are inactive in mammalian cells. Generally, insect cells are very sensitive to environmental factors. So, it is necessary to optimise cell growth pattern before the practice for commercial production. Following are some of the important salient features for expression of therapeutic protein in insect cells by using baculovirus carrying DNA sequence of interest: • Selection of DNA (commercially available) to be inserted in E. coli. • Integration of the cDNA into bacterial genome or circularization of the cDNA sequence as a plasmid. • Selection of transformed E. coli using a selection marker (commercially available antibiotic). • Multiplication of E. coli in suitable media. • Isolation of cDNA or plasmid. • Preparation of a second plasmid containing viral genes required for multiplication and formation of virus particles. • Co-transfection of the expression plasmid and second plasmid into Sf9 or Sf21 insect cells. • Purification of recombinant viral stock. • Infection of the insect cells with high-titer recombinant virus stock. • Isolation of target therapeutic protein for further purification.

2.3.2.4

Mammalian Cells

Mammalian cells are also an ideal system for the expression of recombinant protein of interest. Mammalian cells express biologic drugs with proper folding and posttranslation with carbohydrate moiety or other necessary modification (Fig. 2.7). But, compared to insect cells, in mammalian cells the occurrence of glycosylation happens in relatively large quantity, which interferes in target protein isolation and purification process (Nettleship et al. 2010; Hartley 2012). Mammalian cell lines such as HEK 293, Chinese hamster ovary (CHO), and mouse myeloma cells, including NSO and Sp2/0 cells, have been commercially used for recombinant protein of interest (Griffin et al. 2007). Commonly two types of CHO cell lines, i.e. CVHO-K1 and CHO pro-3 are in use for commercial production of recombinant protein (Lee et al. 2010; Wurm and Hacker 2011). Following are some of the important steps in processing mammalian cells for therapeutic proteins: • cDNA integration into bacterial genome or circularization process for plasmid formation. • Selection of transformed E. coli using selection marker (commercially available). • Process for stability of newly developed clone. • Multiplication of stable E. coli in suitable culture media.

2.3 Upstream Process for Recombinant Protein Production

• • • • •

77

Isolation of target DNA/plasmid. Transfection of plasmid to mammalian cells. Selection of stable clone. Process for transient batch expression for 2–3 months. Isolation of recombinant protein for purification and crystallization.

cDNA from genomic library

Transformation of competent E. coli with cDNA of interest

Positive clone

Isolation of Plasmid

Isolated plasmid

Transfection of Mammalian cell Selection expression: Screening/multiplication of clone

Transient expression: Screening/ multiplication of clone for a short period

Fig. 2.7 Mammalian cell expression system—HEK 293 and CHO

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2 Management and Manufacturing Process of Biologics

2.3.2.5

Transgenic Plants as Host System

Foreign genes can be introduced in plant system through Agrobacterium tumefaciens and A. rhizogenes based vector-mediated gene transfer technique. When these bacteria are infected, a proportion of Agarobacterium Ti Plasmid is trans-located to the plant cell and integrated into the plant cell genome. The following advantages and disadvantages of these systems make them potentially attractive recombinant protein producer: Advantages • • • • •

Plant cultivation can be under low budget, and most suitable to farmers. Low-cost harvest equipment with easy way of handling and well-established. Easy way of isolating therapeutic proteins. Stable expression of proteins in seed proteins. Plant-based systems are free of human pathogens (e.g., HIV). Disadvantages

• In some cases, protein expression is low and variable. • Post-translational gene is in silencing mode (a sequence specific mRNA degradation mechanism). • Glycosylation process is different from that in human. • Plant growth is seasonal and geographical location based. Advantages and disadvantages of different types of host cells are given in Table 2.3.

2.4

Growth Medium for Different Types of Host Cells

Quality therapeutic proteins are resulted product of nature and nurture of host cells or microbes. Expression and production of secondary metabolites are primarily based on the availability of adequate quality nutrient, but not exclusively due to cell or microbe culture medium. So, necessity of maintenance of quality of culture medium is must for getting consistency in quality product, titers, and cell density. A typical culture medium is composed of complement of amino acids, vitamins, essential minerals, various form of carbohydrates, growth factor-like serum, and hormones (for animal/mammalian cells). The growth medium or culture medium is used in three forms, i.e. solid, liquid, or semisolid for variety of host cells like microorganisms, cells (plant, animal, mammalian tissues, and small plants like moss [Physcomitrella patens]). P. patens is used as a model organism for studies on plant evolution, development, and physiology. The compositions of growth medium vary on cell types and purpose of culture. Generally, special type of agar plate is used to initiate growth for bacteria and fungi. For conical flask culture or turnkey level growth nutrient broth is used based on the

2.4 Growth Medium for Different Types of Host Cells

79

Table 2.3 Advantages and disadvantages of different host cells for recombinant protein expression Type of host cells E. coli

S. cerevisiae

Baculovirus/ insect cells

Advantages Fast growth, high level of protein expression, relative genetic simplicity, and genetic stability Genetically well-studied host systems, which can be easily handled by the tools and techniques related to recombinant DNA and protein engineering Ease of scale-up process, adaptable for high density cell suspension culture, and occurrence of post-translation process as noticed in mammalian cells

Mammalian cells (transient expression)

Proper folding and post-translation with carbohydrate moiety or other necessary modification Moderately rapid expression. Work well for secrete proteins

Mammalian cells (stable expression) Transgenic plants

Work well for secrete proteins

• Plant cultivation can be under low budget, and most suitable to farmers • Low cost harvest equipment with easy way of handling, and wellestablished • Easy way of isolating therapeutic proteins • Stable expression of proteins in seeds Proteins • Plant-based systems are free of human pathogens (e.g. HIV)

Disadvantages

Characteristic N-linked glycan structures of proteins are different when compared to the typical mammalian proteins Insect cells are very sensitive to environmental factors. So, it is necessary to optimize cell growth pattern before the practice for commercial production Occurrence of glycosylation happens in relatively large quantity, which interferes in target protein isolation and purification process. Chances of infection with virus Expensive Difficult to scale up Expensive Difficult to scale up • In some cases protein expression is low and variable • Post-translational gene is in silencing mode (a sequence specific mRNA degradation mechanism) • Glycosylation process is different from that in human • Plant growth is seasonal and geographical location basis

need. Fastidious organisms (organisms need a specific complex nutritional requirement) require specialized environment and nutrients for growth. For example, viruses are obligate intracellular parasites and need growth medium containing living cells.

2.4.1

Medium for Bacteria

Carbon (C), oxygen (O), hydrogen (H), nitrogen (N), sulphur (S), potassium (K), calcium (Ca), magnesium (Mg), and iron (Fe) are required as essential elements for

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2 Management and Manufacturing Process of Biologics

the growth of microorganisms. The first six elements, as stated above, are used for the synthesis of carbohydrates, lipids, proteins, and nucleic acids. The remaining four elements play as cations for the synthesis of secondary metabolites or help in cellular transport process. Besides these macro elements, some micro elements such as manganese (Mn), zinc (Zn), cobalt (Co), molybdenum (Mo), nickel (Ni), and copper (Cu) in association with enzymes or cofactors play an important role in the biosynthesis of secondary metabolites. A variety of culture media are available for bacteria culture. Based on physical state and composition, the bacteria culture media can be discussed under two headings: 1. Culture medium based on physical state; 2. Culture medium based on the ingredients. 1. Culture medium based on physical state a. Solid medium Agar (1–2% aqueous sterilised) is used as a solid growth medium for the culture of microorganisms. Often, selective compounds (antibiotics) are added to promote growth in pure form. The formulations of agar used in plates may be defined or undefined form. The former type of formulation is made on the basis of the requirement of nutrients for the microbe, to be cultured or isolated, whereas in the latter type natural products such as yeast extract are used. Some commonly used agar types are detailed as follows. Blood Agar Blood agar plate (BAP) contains mammalian blood (usually sheep or horse), typically at a concentration of 5–10%. BAPs are enriched, differential media used to isolate fastidious organisms and detect haemolytic activity. Blood agar consists of a base containing a protein source (e.g. Tryptones), soybean protein digest, sodium chloride, agar, and 5-10% blood (as stated above). Chocolate Agar Chocolate agar (CHOC) or chocolate blood agar (CBA) is a nonselective, enriched growth medium used for isolation of pathogenic bacteria. It is an alternative of blood agar growth medium that contains red blood cells. These blood cells get lysed when heated at 80 0C. It is mainly used to grow fastidious respiratory bacteria. Horse Blood Agar Horse blood agar is a type of enriched medium mainly used in specific fastidious bacteria and also allows indication of haemolytic activity in these bacterial cultures. It contains pancreatic digest of casein and soy peptone, which provide essential carbon and nitrogen elements to support cell growth. Thayer-Martin Agar (Thyer-Martin Medium or VPN) It is an alternate for chocolate agar mainly used for isolation of Neisseria gonorrhoeae. It is a form of Mueller-Hinton agar with 5% chocolate sheep blood and antibiotic.

2.4 Growth Medium for Different Types of Host Cells

81

Thiosulfate-Citrate-Bile Salt-Sucrose Agar (TCBS Agar) It is a type of selective agar medium used for the isolation of Vibrio species. It contains high concentration of sodium thiosulfate and sodium citrate for the inhibition of Enterobacteriaceae. With the incorporation of ox gall (mixture of bile salts and sodium cholate), it inhabits the growth of Gram-positive bacteria. Sucrose is added as fermentable carbohydrate by Vibro species. Bile Esculin Agar (BEA) It is a selective differential agar used for the isolation of members of the genus Enterococcus. This medium contains esculin ferric citrate (as a source of ferric ions), and 4% oxbile to inhibit most other strains of non-group-D streptococci. Peptic digest of animal tissue and beef extract serves as the source of carbon, nitrogen, and essential growth factors. CLED Agar CLED agar (Cystine-Lactose-Electrolyte-Deficient agar) is a type of differential medium used for isolating urinary tract bacteria. It prevents the growth of Proteus species due to its lack of electrolytes. Granada Medium It is a selective and differential medium used for identification of Streptococcus sp., in clinical sample. It is composed of primarily proteose peptone starch agar supplemented with methotrexate and antibiotic. Hektoen Enteric (HE) Agar Hektoen enteric (HE) agar is selective medium used for the isolation of faecal bacteria of the family Enterobacteriaceae. It is mainly used for isolation of Salmonella and Shigella. It is composed of proteose peptone, yeast extract, sodium chloride, lactose, sucrose, salicin, bromothymol blue, acid fuchsin, sodium thiosulfate, iron (III) ammonium citrate, and bile salt with deionised water. MacConkey Agar It is a selective and differential medium used to differentiate between Gram-negative bacteria while inhibiting the growth of Gram-positive bacteria. It is composed of bile salt (for inhabitation of Gram-positive bacteria), crystal violet dye (inhabitation of certain Gram-positive bacteria), and neutral red dye. Mannitol Salt Agar It is a selective medium used for some specific pathogens. It is composed of high concentration (7.5–10%) of sodium chloride. Due to such high salt concentration, it is selective for specific types of Gram-positive and Gram-negative bacteria. It acts as differential medium for the mannitol-fermenting staphylococci due to the presence of mannitol-like carbohydrate. Mannitol salt agar contains beef extract and proteose peptone (rich in nitrogen, vitamins, minerals, and amino acids), which support the growth of specific Gram-positive bacteria.

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Nutrient Agar It is commonly used for growth of wide range of non-fastidious organisms. It is mainly composed of 0.5% peptone as a source of nitrogen, and 0.3% beef extract or yeast. Tryptic Soy Agar (TSA) It is mostly used for general purpose. It is produced by enzymatic digestion of soybean meal and casein. It is commonly used as base medium for other agar types. The common example is preparation of blood agar plates that consist of TSA with blood. It supports the growth of many semifastidious bacteria, including some species of Brucella, Corynebacterium, Listeria, Neisseria, and Vibro. Besides the above-stated agar media, there also some special types of agar media like Onoz agar, phenylethyl alcohol agar, R2A agar, xylose lysine deoxycholate, cetrimide agar that are used for some specific types of bacteria. b. Semisolid Media Semisolid media are prepared with the composition of agar at low concentration (0.5%). They are soft custard-like media used for the culture of microaerophilic or for determination of bacterial mobility. c. Liquid Media or Broth Media Liquid culture media are mainly used for sterility test, food and water quality monitoring as well as cell culture media. These media are composed of specific amount of nutrients without trace of gelling agents such as gelatine or agar. These media are used for turnkey level of bacteria production. 2. Culture medium based on the ingredients On the basis of composition, bacteria culture media can be divided into three classes: (a) synthetic or chemically defined medium, (b) non-synthetic or chemically undefined medium, and (c) bacterial media on the basis of functional use. a. Synthetic or chemically defined medium This medium is composed of chemically defined medium in which the exact composition of ingredients is known. For example, Davis and Mingioli medium is commonly used for bacteria growth (Table 2.4). Synthetic medium is also known as chemically defined medium or nutrient medium. In this medium, all the chemicals used are known; no yeast, animal, or plant tissue is present. Chemically defined media are of value in studying the minimal nutritional requirements of microorganisms, for enrichment cultures, and for a wide variety of physiological studies. Some examples of nutrient media include: plate count agar, nutrient agar, typically soy agar, etc. (Table 2.5). Defined media are usually composed of pure biochemicals off the shelf; complex media usually contain complex materials of biological origin such as blood or milk or yeast extract or beef extract, the exact chemical composition of which is obviously undetermined.

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Table 2.4 Davis and Mingioli medium for E. coli growth Substances Dipotassium hydrogen phosphate Potassium dihydrogen phosphate β-D-glucopyranose Ammonium sulfate Sodium citrate trihydrate Magnesium sulfate heptahydrate L-leucine L-methionine L-histidine Composition Constituents K+ Phosphate Ammonium β-D-glucopyranose Sulfate Citrate Mg2+ L-leucine L-methionine L-histidine

Concentration 7 g/l 3 g/l 2.5 g/l 1 g/l 0.5 g/l 100 mg/l 40 mg/l 20 mg/l 20 mg/l

Role Source of P Source of P Source of C Source of N, S Source of S

Concentration 101.8 mM 61.84 mM 15.13 mM 13.87 mM 7.97 mM 2.05 mM 405.73 μM 304.94 μM 134.04 μM 128.9 μM

Table 2.5 Tryptic soy broth (soybean casein digest medium) Name of ingredient Pancreatic digest of casein Sodium chloride Papaic digest of soybean meal Dextrose Dipotassium phosphate

Amount (in gram) per litre of deionized water (pH-7.3 at 25  C) 17.0 5.0 3.0 2.5 2.5

b. Non-synthetic or chemically undefined medium Non-synthetic medium is composed of at least one component that is neither purified nor completely characterized nor even completely consistent from batch to batch; for example, nutrient broth prepared from culture of yeast. Depending on the supplement ingredient incorporated, non-synthetic medium may be simple or complex in nature. The complex non-synthetic medium is mainly used for the growth of fastidious microorganisms.

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c. Bacterial media on the basis of functional use. For the isolation of bacteria on functional aspects, following are different categories of bacterial culture media in use: 1. General purpose media or basal media These are basically simple in composition to support the growth of most of the non-fastidious bacteria. For example, peptone water, nutrient broth, and nutrient agar (NA) are belonging to this category. 2. Enriched medium This type of medium is formulated by the addition of specific nutrients like blood, serum, egg yolk, etc. to a basal medium, on the specific requirement of microbe of interest. Various types of nutrient-enriched media are briefed in the earlier section (see Solid medium) 3. Selective media These types of culture media are prepared to grow a target microbe by eliminating unwanted bacteria. Selective media are agar based, which can be formulated by addition of certain inhibitor agents that do not affect the pathogen of interest. Various types of selective media are explained in earlier section (see Solid medium). Various ways to formulate selective media are prepared by the addition of antibiotics, dyes, chemicals, alteration of pH, or combination of these. Thayer Martin agar, MacConkey’s agar, potassium tellurite medium, pseudogel agar, crystal violet blood agar, Lowenstein–Jensen medium, and Eilson and Blair’s agar are some of the common selective media used for specific microbe.

2.4.2

Fungal Culture Media

Fungi are chemoheterotrophic in nature. They need various types of organic compounds to fulfil their energy requirement for the growth and development. Fungi absorb nutrients from plants or animals around them either in living condition or in dead form. The hyphae release extracellular enzymes that break down the food into substances that can be easily absorbed. Potato dextrose agar, malt extract agar, or commercially available agars are common components on which fungi can be grown. Commonly, fungal culture media are rich in carbohydrate, and the pH of the growth medium is maintained slight acidic with the range 5–6. Some fungi require minimal media (salts and glucose) with the addition of vitamin (thiamine or biotin), organic nitrogen, trace amount of iron as heme, and sulphur as in cysteine. Some fungi are unable to use nitrate or ammonium nitrogen. The simple carbohydrates (like glucose) diffuse through the membrane, directly (passive transport) or indirectly by active transport mechanism. The complex carbohydrates are broken into simple components by secreted enzymes before transported into cell. Fungi exhibit diauxic growth. In this process, the carbohydrates present in the medium are used in two consecutive phases. The first growth curve is performed rapidly by the consumption of immediately available sugar present in the medium. It is followed by a lag phase. During the lag phase, the cellular machinery used to metabolize the

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second sugar results in the increase of cellular growth. This process is often observed in filamentous fungi like Penicillium ochrochloron. Fungi do not have capacity to fix atmospheric nitrogen. The sources of nitrogen include: ammonia, urea, L-asparagine, and nitrate. But, ammonium is not a good component as it uses lower pH, thereby inhabiting growth. The nitrate is converted to ammonia by nitrate reductase or by nitrite reductase. Fungi utilize maximum amount of phosphorus, even though it is poorly available in the soil. The phosphate is stored in vacuole as polyphosphates. Iron plays an important role as electrons accepter or donor. The cellular requirement of iron in fungi is in very trace quantity.

2.4.3

Animal and Mammalian Cell Culture Media

In vitro cell culture media for human or animal cells are chemically defined media commonly comprised of basal medium supplemented with animal serum (such as foetal bovine serum, FBS) as a source of nutrients and other trace compounds. Serum is the amber fluid rich in protein that is isolated from coagulated blood. Often, FBS is used for research therapeutic and other clinical purposes. But serum is an undefined component and depends on the source of collection. Besides this, the serum is highly susceptible to pathogenic contamination. So, possibility of contamination and variability between serum batches may be primary factors for not getting consistency in quality production. Serum-free medium is formulated with undefined animal-derived products or synthetic ingredients to support the cellular growth. Serum-free medium is consistent between batches. Serum-free media contain substances that promote cell adherence, growth factors, enzyme inhibitors, binding proteins, and other trace elements. Trypsin, an enzyme, is used to separate cells from the surface of the culture dish for further propagation. On needs basis, trypsin is inactivated by protease inhibitors present in animal serum, so that the resulted free cells can be reattached to the dish surface during propagation. Commonly, trypsin used for such purpose is derived from plant-based soybean (Fang et al. 2017; Persistence Market Research 2017; IntechOpen n.d.). Mushroom extract has also been used to make serum-free medium (Gaydhane et al. 2018). Various companies involved in manufacturing and marketing serumfree media for animal or mammalian cells culture include: ThermoFisher Scientific, Athena Environmental Sciences, Inc., Pan Biotech UK Ltd., Bichrom, Irvine Scientific, Cellular Technology Ltd., Biological Industries, Sigma-Aldrich Co. LLC, Cell Genix GmbH, and HiMedia Laboratories. Chemically defined media for animal or mammalian cells culture are completely free from any animal-derived components that contain foetal bovine serum. These media are formulated with basal media (such as DMEM, F12, or RPM11640) supplanted with recombinant version of albumin, recombinant insulin, and growth factors. The basal media are comprised of amino acids, vitamins, inorganic salts,

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buffers, antioxidants, and energy sources. These are derived from rice or E. coli or synthetic chemical such as the polymer polyvinyl alcohol, which can functionally act as BSA or HSA.

2.5

Preservation Methods

The most important issue of a biopharma company is to maintain the potential continuity of different types of genetically modified host cells (microbial strain, mammalian cells, animal cells, and insect cells) to get product of interest consistently. Mainly two methods of preservation techniques: (1) Metabolically inactive preservation techniques (CABRI accepted methods) and (2) Metabolically active methods that are in practice. (3) Metabolic inactivation process involves in two processes: (a) cryopreservation and (b) drying.

2.5.1

Cryopreservation

Cryopreservation is the process of freezing biological materials at extreme temperatures; (typically 80  C using solid carbon dioxide or 196  C using liquid nitrogen, N2) (Fig. 2.8). At these low temperatures, all biological activity (enzymatic or biochemical activity) stops. There have also been documented cases of cross-contamination by

Fig. 2.8 Liquid nitrogen container (with courtesy from Stirling Cryogenics)

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virus pathogens via the liquid nitrogen medium. So, it is recommended to use vapour phase nitrogen. Modern designs of liquid nitrogen storage vessels are increasingly offering improved vapour phase storage technology. The cryopreservation method in theory makes it possible to store living cells as well as other biological material unchanged for centuries. The challenge of cryopreservation is to help cells to survive both cooling to extreme temperatures and thawing back to physiological conditions. During the process of cryopreservation, the extracellular solutes present in the suspended medium gets crystallized and damage the neighbouring cells. Besides this phenomenon, during the process of cooling at low temperature water migrates out of the cells and cause formation of ice crystal that may ultimately damage the cell membrane. Generally, in some host cells and tissue intracellular ice formation also causes the damage or death of cells under cryopreservation. The crucial elements to prevent this are the freezing rate (degrees per minute) and the composition of the freezing medium used. The cryo-protective additives (CPAs) used in the frozen storage of different host cells include a variety of simple and more complex chemical compounds, but only a few of them (dimethylsulfoxide [Me2SO], glycerol, blood serum or serum albumin, skimmed milk, peptone, yeast extract, saccharose, glucose, methanol, polyvinylpyrrolidone [PVP], sorbitol, and malt extract) have been used widely with satisfactory results. The main target of cryopreservation is to prolong the life of engineered host cells with consistency in quality production when revived. In addition, cryopreservation prevents the host cells from the possibility of microbial contamination, and crosscontamination with other cells, resulting in reducing risk of genetic drift and any phenotypic changes. The main challenge in cryopreservation method is the revival process of cryopreserved materials by slow freeze and quick thaw. There has been a large amount of developmental work undertaken to ensure successful cryopreservation and revival of a wide variety of cell lines of different cell types. The basic principle of successful cryopreservation and revival is a slow freeze and quick thaw in a cyclic manner suitable to host cells of interest. Generally, cooling rate of 1  C to 3  C/min and thawed quickly by incubation in a 37  C for 3–5 min are advisable in cryopreservation practice. For cryopreservation, the sample(s) of host cell lines or microbial cells should be collected from log phase of growth. The pure single line culture should be healthy and should have >90% viability. Mammalian cells or animal cells are cryopreserved to prevent from contamination, to minimize genetic changes in continuous cell lines, and to suppress ageing process. Commonly serum-containing medium (10% glycerol +10% DMSO, dimethyl sulfoxide), and basal medium are used for such formulation. Serum-free medium (50% fresh serum-free medium +7.5% DMSO) is also used for mammalian cells preservation.

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Drying

1. Preservation by shelf freeze-drying The term lyophilization or freeze-drying is application to a process in which water is frozen first and then removed from the sample by sequential follow-up: by sublimation (primary drying) and then by desorption (secondary drying). This drying process is applicable to manufacture of certain pharmaceuticals and biologics (proteins, microbes, pharmaceuticals, tissues, and plasma) that are thermo labile or otherwise unstable in aqueous solution for prolonged storage periods but that are stable in dry state. 2. Preservation by spin-freeze-drying Spin-freezing of any microbial suspension is an alternative freezing approach, which is evaluated as part of an innovative continuous pharmaceutical freeze-drying concept. In this process the microbial suspense is dried in a flask with spin-freezing arrangement in a cooling bath. The rotation spreads the liquid on the inner walls of the flask where it freezes. The freezing process produces a thinner layer and increases the potential area for sublimation, which considerably reduces the overall drying time.

2.5.3

Preservation by Liquid Drying (L-drying)

Liquid drying (L-drying) is a useful alternative method of vacuum-drying for the preservation of bacteria that are particularly sensitive to the initial freezing stage of the normal lyophilization process. The intrinsic feature of this process is that cultures are prevented from freezing; drying occurs direct from the liquid phase.

2.5.4

Preservation by Vacuum Drying

It is simplest process of drying the organisms suspended in culture fluid or resuspended in saline, serum, or blood in a desiccator in vacuum over dehydrating agents such as H2SO4, or P2O5. The suspensions are dried on sterile coverslips, filter paper, in small test tubes, or more conveniently in sterile ampoules, which can subsequently be sealed off in vacuum.

2.5.5

Metabolically Active Methods

a. Periodic transfer on agar or in liquid medium

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By this method the microbes are subjected to continuous transfer with a fix time period of interval to fresh culture medium. On the basis of microbial sample to be stored, the pH of the medium is fixed and incubated under specific temperature for a suitable time of interval. The time of incubation depends on specific growth rate of the microorganism. However, slow growth rate is desirable for preservation purpose. Nutrient agar is the most suitable for prolonged preservation of heterotrophs. Changes in character of strain due to the development of mutants and variants may cause the chances of loosing original strain. b. Keeping agar cultures under mineral oil/soil Storage in Sterile Oil This is a simple method of storing microbes under the liquid blanket of sterile oil. The microorganism slant grown on agar slant is covered with sterile oil with about 1-cm thick oil layer. It is easy to remove microbial sample with the help of sterile wire having small loop at the tip. With this method microbes can be preserved safely for 15–20 years. Storage in Sterile Soil Generally, spore-producing microbes are stored by this method at dormant stage for a very long period, about 70–80 years. The microbial seeds are mixed with sterile soil under perfect aseptic condition and dried under ambient condition by using desiccators. The microbial sample holding desiccators is stored in refrigerator for further use.

2.6

Bioreactor Used for Recombinant Protein Production

Bioreactor is a perfect shield vessel made of non-corrosive stainless steel or glass for the growth and development of variety of host cells (plant cells, animal cells, mammalian cells, and insect cells) or microbes (bacteria, fungi, and viruses) with the provision of ventilation of sterile oxygen (O2) or air, pH, and temperaturecontrolling devices. Besides these facilities, a bioreactor is having facilities of stirring and baffling the growth medium on requirement basis. The inlet and outlet of growth medium are well-shielded, thus one-way flow of growth medium is maintained. Figure 2.9 shows characteristic features of a typical bioreactor (stirred tank type). For laboratory purpose, the volume metric size of a bioreactor type of bioreactor (fermenter) is within the range of >1–300 l. The overall function of a bioreactor is to support best possible growth, biosynthetic conditions, and ease of manipulation for all operations associated with the production of therapeutic proteins of interest. For quality production in a consistency pattern, an ideal bioreactor should have the following technical provisions: (1) Fermenter body (vessel), (2) Agitator, (3) Coil, (4) Gas outlet, (5) Inoculation port, (6) Thermometer, and (7) Electrode. A good bioreactor (fermenter) should possess the following features:

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Fig. 2.9 A typical stirred tank bioreactor displaying different components

1. The volumetric size of a bioreactor should well enough to withstand pressure of culture medium while in operating condition 2. The bioreactor vessel should be made of non-corrosive stainless steel or pressure resistance glass 3. The bioreactor should be designed with the guarantee of no entry of any foreign pathogen or unwanted microbes 4. Good provision for infusion of sterile air/oxygen into the growth medium 5. Good shield bioreactor with no entry of any foreign microbes 6. Provision of CO2 release with one-way valve-controlling system 7. Provision of intermittent addition of antifoaming agents 8. Well-designed stirring system for the mixing of nutrients, oxygen, and antifoaming agents 9. Provision of temperature and pH controlling during the growth of host cells or microbes 10. Provision of aseptic sample collection valve 11. Provision of medium sterilization before inoculums of host cells or microbe 12. Provision of well drainage system, after finishing the operation of bioreactor 13. The bioreactor should be well-connected to access for cleaning, once the operation system is finished

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2.6.1

91

Bioreactor Vessel

Wide range of bioreactors is presently being used on the needs basis of host cells or microbes to be used. The operation condition of bioreactor is mainly dependent on mass transfer process of suspended host cells and product generated during fermentation process, mixing, sheer sensitivity, broth viscosity, oxygen demand, reliability of operational conditions, and the overall cost of design. On the needs basis, the volumetric size of bioreactor varies over several orders of magnitudes (Fig. 2.10). Figure 2.10a shows the scale-up process for biopharmaceutical production from slant to bioreactor, and Figure 2.10b shows the schematic diagram of scale-up process for turnkey level production. At the beginning stage the growth of host cells starts in a conical flask with the range of 100–1000 ml. Subsequently, it is transferred to laboratory-scale bioreactor of 1–50 L in size (pilot level). The volume of the broth can be increased to higher level, by the multiplication of tenfolds. Presently, for the production of therapeutic proteins, even 10–500 kL bioreactor is in commercially used. Following are the various components of a typical bioreactor: a. Bioreactor vessel A bioreactor is divided into two sections, i.e. (1) working volume and (2) headspace volume. The former space is about 70–80% of the total volume and is occupied with broth (culture medium). The remaining space left is known as head space. The overall geometric design of a commercial bioreactor is aimed to ensure sterility assurance, elimination of repeated cleaning, reduced capital investment, faster processing times with increase productivity, faster start-up, and other benefits. Bioreactors are made up of glass or stainless steel material with cylindrical appearance. The overall structural designed is made to withstand high-pressure steam for sterilization and mechanical stability. b. Cooling Jacket Industrial fermenters are made up of non-corrosive stainless steel. Generally, a commercial mega fermenter is cylindrical size closed at the top and the bottom having fitting of various pipes and valves to maintain required operational condition for the growth and development of host cells or microbes. The cooling space between inner jacket and outer body of the vessel is provided with cooling coils to maintain favourable temperature for supporting biomass growth. A cooling liquid flow through the jacket is maintained to collect heat energy from the outer surface of the reactor vessel and carrying it away as the cooling liquid exists at the jacket outlet (Fig. 2.11). c. Aeration Systems Aeration system into the bioreactor is a complex accessory through which sterile air or oxygen is passed through a series of filter system with desired pressure. Oxygen is sparingly soluble in water, which serves as growth-limiting factor in a

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Fig. 2.10 (a) Scale-up process for biopharmaceutical production from slant to bioreactor, (b) Schematic diagram of scale-up process for turnkey level production

bioreactor. Oxygen has low solubility in aqueous medium. In distilled water the solubility of oxygen is about 7 mg L1 at 30  C. This oxygen is quickly consumed in aerobic culture and must be constantly replaced by air sparging. Mainly the host cells or microbes used for recombinant protein production, therefore, require the facility for oxygen supply. On the basis of stoichiometry,

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Fig. 2.10 (continued)

Fig. 2.11 Schematic diagram of external cooling/heating coil circulated around a bioreactor

about 192 g of oxygen are needed for oxidizing 180 g of glucose. But the solubility of oxygen in glucose containing medium is about 6000-folds lesser than the oxygen solubility in distilled water. So, adequate amount of oxygen is to be passed through the medium, which can fulfil the requirement of complete oxidation of soluble carbohydrate, apart from the oxygen required for the growth of host cells.

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However, the intakes of oxygen by host cells or microbes (specific oxygen uptake) vary with the mass density of culture medium and nature of the culture. So, it is necessary to know air or oxygen solubility data in a specific culture medium having host cells of interest. This is mainly due to the interference of dissolve nutrients with partial pressure of oxygen or air. So, the value of oxygen solubility is not directly applicable to bioprocess system. Sparger Generally, aeration is carried out by sparging (bubbling) sterile air or oxygen into the culture broth with low pressure through small holes or pores present on the sparger tip. On the needs basis, three types of sparger systems are in use: (1) porous sparger, (2) orifice sparger (a perforated pipe), and (3) nozzle sparger (an open or partially closed pipe). Basically, the sparger system is used as laboratory-scale non-agitated fermenter. The sparger device is made of sintered glass, ceramic, or metal. Orifice sparger is used for small stirred fermenters below the impeller. Nozzle sparger system is attached with mechanically stirred fermenters and is used for laboratory as well as commercial purpose. This type of sparger is having single opening or partially closed pipe to provide stream of air bubble into the growth medium. Ideally the pipe should be positioned centrally below the impeller. The entaeration process is associated with four types of mechanical accessories that increase the efficacy of aeration process. The additional mechanical devises are (1) agitator, (2) stirrer, (3) baffle, and (4) sparger (Fig. 2.12). Agitators Agitation is a mechanical process for mixing nutrients and oxygen, which can be available in adequate quantity and favour the growth of host cells or microbes to obtain quality product in good quantity. Agitation process also helps in bringing homogeneity in suspended host cells or microbes throughout the culture broth. The size and structural getup of impeller mainly depend on the nature of host cells and the height of the vessel. A bigger size of bioreactor needs more than one impeller. Ideally, the impeller should be one-third of the fermenter diameter fitted above the base of the bioreactor. Mainly, the following types of impellers are in use for bioreactor (Fig. 2.13). Baffles Baffles are metal strips roughly cover one-tenth of a fermenter to prevent vortex and to improve aeration efficiency. Computer Accessory Attached to Bioreactor The efficacy in successful operation of a bioreactor is mainly dependent on the microchip-based instruments attached to regulate the operation process. Various physical factors like oxygen or air flow, pH and foaming controlling systems are controlled by computer.

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Fig. 2.12 Schematic view of a typical stirred tank bioreactor showing different controlling systems

2.6.2

Types of Bioreactor

A wide variety of bioreactors are presently in use for the production of therapeutic proteins and other biologics.

2.6.2.1

Stirred Tank Bioreactor

Stirred tank bioreactors are widely used for a variety of host cells as the most conventional bioreactor. On the basis of demand, a stirred tank bioreactor can also operate in continuous mode (Fig. 2.14). The key component of stirred tank bioreactor is the agitator or impeller (Fig. 2.14). The agitator is mainly responsible for aeration, heat transfer, and mass transfer process to maintain the homogeneity of the culture broth. Generally, two types of impellers, i.e. axial and radial flow are advisable for use to maintain desired fluid dynamic for specific host cells. Besides the impeller type, the other specifications for a stirred tank bioreactor are impeller off-bottom clearance, the impeller size, size of the baffles, sparger type, and geometric configuration such as the ratio of liquid height to tank diameter. Multiple impellers are used for a large size stirred tank bioreactor to maintain the order of hydrodynamic flow of cultured broth. For the shear-sensitive host cells such as animal and plant cells, conventional type of impeller is not used due to the generation of high shear. This is mainly due to the high sensitivity (fragile nature) of animal cells to shear and bubble, which causes damage to the host cells. In order to overcome such a problem, the biotechnology-based industry brings suitable amendments such as modification of oxygen sparging device, use of potential chemical or biochemical to provide support to membrane, and reducing shear by suitable baffles.

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Fig. 2.13 Different types of impellers used in bioreactor

Fig. 2.14 Line diagram of a continuous feed stirred tank bioreactor

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This could be possible by use of suitable oxygen device. Such as bubble-free aeration, gas basket, and cage-aeration, and use of additive like Pluronic 68 in the culture broth. Marine impeller is proven to be a better option to maintain the hydrodynamic nature of the broth, which can protect host cells from shear stress. The shear sensitivity of plant cells is lesser as compared to the animal cells. Ruston turbine and hydrofoil impellers are noticed to be better options for plan cells culture. The overall performance of stirred tank bioreactor is widely acceptable due to its several advantages for the cultivation of shear-sensitive cells on turnkey basis, easy maintenance, and easy of scale-up and control. So, the stirred tank bioreactor is widely used for monoclonal antibodies (Mabs) production.

2.6.2.2

Airlift Bioreactor

An airlift bioreactor consists of a cylindrical vessel divided into two interconnected zones by means of a baffle or draft tube. An airlift bioreactor consists of two separate channels for gas/liquid up-flow and in channel for gas/liquid down-flow. Both the chambers perform the function of gas/liquid circuit and also have the mechanism for output of gas/liquid. The latter provision is provided at the top of the bioreactor. Airlift bioreactors are used for animal or plant cells culture that is suspended in the culture broth in the form of pallet immobile with suitable gel. Mostly, an airlift bioreactor is having the provision of pneumatically gas inlet in order to create a current of agitation act in the liquid broth. The gas used for agitation can act to either introduce new molecules to the mixture inside of the bioreactor or remove specific metabolic molecules produced by microorganisms. The basic design of an airlift bioreactor is based on bubble column at the bottom of the bioreactor. The movement of air bubble is from bottom to the top of the bioreactor (Fig. 2.15). The pattern of fluid circulation inside the airlift bioreactor can be designed on the basis of customer’s requirement. Generally, two types of airlift bioreactors are in use: (1) baffled vessels (Fig. 2.16a, b) and (2) external loop type (Fig. 2.16c). External loop vessel is having distinct and separate conduits for circulation of gases and liquid. The baffle vessels are designed on the basis of spider diffraction techniques on the basis of host cells to be cultured.

2.6.2.3

Bubble Column Bioreactor

The bubble column bioreactor is a cylindrical vessel having the provision of gas sparging system at the bottom of the vessel. It is designed to monitor the rate of mass transfer and reaction between a gas (oxygen) and a liquid. For animal cell culture, large quantities of oxygen are fed continuously in a countercurrent or parallel flow style. Oxygen transfer, mixing, and other performance are mainly dependent on gas flow rate and rheological properties of the broth used for specific cells culture. On the

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Fig. 2.15 Line diagram of airlift bioreactor

Gas outlet

Air bubbles Gel bid contain host cells

Internal cylinder

Gas inlet

basis of gas flow and hydrodynamic nature of culture broth, the bubble column bioreactor is designed as per the customer specification (Figs. 2.17 and 2.18). The air sparger is a perforated pipe or plate or sintered glass. A variety of design is available for gas sparging system on the basis of customer’s requirement. The gas sparging process consumes lesser energy as compared to mechanical sparging process. Generally, a stationary bubble-swarm is used to aerate a mammalian cell culture bioreactor with slow gas flow rate. The reason being for monitoring such slow flow is to prolong the oxygen retention time of the gas bubbles within the medium to facilitate the oxygen availability to the animal host cells. By this method about 90% oxygen supply is made, even in high cell density condition.

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Fig. 2.16 Line diagram of airlift bioreactor: (a) draft-tube internal loop configuration, (b) a split cylinder device, and (c) an external loop device

2.6.2.4

Hollow Fibre Perfusion Bioreactor

Perfusion bioreactor is defined as bioreactor where both the retention of biomass and removal of waste medium occur simultaneously. During this process, the supply of fresh medium into the reactor vessel is balanced with the output of waste medium. The inner core of a perfusion bioreactor consists of bundle of hollow fibre. Hollow fibre membranes (HFMs) are extremely thin tubular bodies about 1 mm thick with numerous pores that allow water to flow freely through their fibrous walls (Fig. 2.19). Hollow fibre processing is a simple removal of wastewater in non-chemical way. It detains the essential minerals and other nutrients and only allows efflux of wastes growth medium. Hollow fibre membranes are fabricated with synthetic polymers nylon, polyethylene, polyester, teflon, and epoxy derived from petroleum products. Hollow fibre bioreactors are used to generate high concentrations of cell-derived products including monoclonal antibodies, recombinant protein, growth factors viruses, and virus-like particles.

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Air bubbles

Perforated circular l t Fig. 2.17 Line diagram of a bubble column bioreactor

Fig. 2.18 (a) Simple bubble column; (b) cascade bubble column with sieve trays; (c) packed bubble column; (d) multishaft bubble column; (e) bubble column with static mixer

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Feed inlet Hollow fiber (ECS) Flow

Flow

Schematic view of Perfusion

Extra capillary space (ECS) Feed outlet

Fig. 2.19 Hollow fibre perfusion bioreactor

2.6.2.5

Single-Use Bioreactor (Bag Bioreactor)

In this reactor, plastic culture vessel is either fitted with a cylindrical frame or placed on a metal platform having wave motion. These types of bioreactors are known as single-use bioreactor or bag bioreactor (Fig. 2.20). The disposable bag is made of three-layered plastic foil. The uppermost layer is made of polyethylene terephthalate or LDPE to provide mechanical support for the stability of the vessel. The middle layer is made of PVA or PVC and acts as gas barrier. The innermost layer that is in direct contact with culture broth is made of polyvinyl (PVA) or polypropylene (PP). Compared to stainless steel vessel, the single-use bioreactors are less expensive with minimum maintenance cost. Two different types of disposable reactor vessels are in use. Some single-use bioreactor is having provision of stirrer system that is integrated into the plastic bag. The closed bag and stirrer are pre-sterilized. The

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Fig. 2.20 (a) Single-use cylindrical bag bioreactor front view. (b) Waved motioned single-use horizontal type bioreactor

disposable bag is mounted in the bioreactor, and the stirrer is connected to a driver mechanically or magnetically. The other single-use bioreactors are agitated by a rocking motion. This type of bioreactors does not have any mechanical stirring device. The maximum geometric size of this type of bioreactor is 2000 L capacity. Single-use bioreactors are commercially used for mammalian cells and animal cells culture. Mostly, biopharmaceutical industries and biotechnology-based industries use single-use bioreactors for antibodies vaccines and stem cells production.

References Anné J, Maldonado B, Van Impe J, Van Mellaert L, Bernaerts K (2012) Recombinant protein production and streptomycetes. J Biotechnol 158:159–167

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Nykiforuk CL, Boothe JG, Murray EW, Keon RG, Goren HJ, Markley NA, Moloney MM (2006) Transgenic expression and recovery of biologically active recombinant human insulin from Arabidopsis thaliana seeds. Plant Biotechnol J 4:77–85 Öztürk S, Ḉalık P, Özdamar TH (2016a) Fed-batch biomolecule production by Bacillus subtilis: a state-of-the-art review. Trends Biotechnol 34:329–345 Öztürk S, Ḉalık P, Özdamar TH (2016b) Fed-batch biomolecule production by Bacillus subtilis: a state-of-the-art review. Trends Biotechnol 34:329–345 Park S, Schumann W (2015) Optimization of the secretion pathway for heterologous proteins in Bacillus subtilis. Biotechnol Bioprocess Eng 20:623–633 Persistence Market Research (2017) Serum-free media market: global industry trend analysis 2012 to 2017 and forecast 2017-2025. https://www.persistencemarketresearch.com/market-research/ serum-free-media-market.asp Pichia Produced Products (2018) Available online http://www.pichia.com/science-center/commer cialized-products/. Accessed 3 February 2018 Reed B, Chen R (2013a) Biotechnological applications of bacterial protein secretion: from therapeutics to biofuel production. Res Microbiol 164:675–682 Reed B, Chen R (2013b) Biotechnological applications of bacterial protein secretion: from therapeutics to biofuel production. Res Microbiol 164:675–682 Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:172 Wang A, Lu SD, Mark DF (1984) Site-specific mutagenesis of the human interleukin-2 gene: structure-function analysis of the cysteine residues. Science 224:1431–1433 Westers L, Westers H, Quax WJ (2004) Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochem Biophys Acta 1694:299–310 World Health Organization (1992) Annex 1 (WHO Technical Report Series, No. 822). http://www. who.int/biologicals/publications/trs/areas/vaccines/gmp/WHO_TRS_822_A1.pdf?ua¼1. Accessed 8 November 2015 Wurm FM, Hacker D (2011) First CHO genome. Nat Biotechnol 29(8):718–720 Yanagita M, Nakayama K, Takeuchi T (1992) Processing of mutated proinsulin with tetrabasic cleavage sites to bioactive insulin in the non-endocrine cell line, COS-7. FEBS Lett 311:55–59

Chapter 3

Downstream Processes

3.1

Introduction

The method of processing culture broth obtained after upstream process for the isolation and purification of therapeutic biomolecules is known as downstream process (Fig. 3.1). The therapeutic recombinant proteins or other biomolecules of interest are derived from biological sources such as animal cells, insects, plant cells, mammalian cells (Valderrama-Rincon et al. 2012; Rodríguez et al. 2014). The various sequential stages for downstream process include removal of insoluble and unwanted materials, purification of crude extract, and polishing of the product (Fig. 3.1). Downstream process is highly expensive process which plays a crucial role in final price fixation. In downstream process production of target protein is expressed in term of titre value. A biopharmaceutical product can be produced batch wise or in a continuous process. It has been experienced that a shift to continue process from batch process not only save time but also reduce the production cost. Followings are the detailed sequential process for biopharmaceutical (Fig. 3.2).

3.2

Conceptual Development of Biopharmaceutical

Generally, low molecular weight drugs are known as generic drugs (after the expiry of patent), derived by chemical or partially biochemical methods. Small molecular weight drugs have long history of origin and application (DiMasi et al. 2010, 2016; Paul et al. 2010; Cummings et al. 2014). The small size drugs are easy to deliver into the body either as tablet or in liquid form. Acceptability of small drugs in the body system is easy and rapid. Large molecule biopharmaceuticals are composed of more than 1300 amino acids and other molecules. Due to the high molecular weight, biologic drugs are directly delivered into blood stream through intravenous injection or muscular injection. © Springer Nature Singapore Pte Ltd. 2020 B. K. Behera et al., Competitive Strategies in Life Sciences, New Paradigms of Living Systems 1, https://doi.org/10.1007/978-981-15-7590-7_3

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Culture Broth Target product (intercellular /extracellular) + unwanted materials

Inter cellular

Extracellular Concentration

Host cells/ Microbes Disruption (Physical/chemical/ enzymatic process)

Concentrated broth With Solid and soluble materials

Process of rejection of untargeted material and solid liquid separation

Targeted product concentration (Evaporation/liquid-liquid separation/membrane filtration/precipitation/adsorption)

Product purification ( gel-filtration,ion-exchange, affinity, hydrophobic interaction)

Product polishing and formulation (Freeze drying/ crystallization)

Fig. 3.1 An outline of important steps involved in downstream process

The overall process for biologic drugs development and production is most complex, time consuming, and expensive (Fig. 3.3). The large molecule products include vaccines, blood, blood components therapeutic gene, and stem cells. Biologic drugs development is a challenging process with highly complex steps for isolation, separation, and purification of product of interest. Time taken for development to marketing of biopharmaceutical drugs is about 10–12 years of intense research and development. The larger size therapeutic biomolecules such

3.2 Conceptual Development of Biopharmaceutical

107

About 3-4 weeks

Fig. 3.2 Schematic representation of downstream process to obtain biopharmaceutical

as nucleic acids (DNA & RNA) (Deng et al. 2014; Wittrup and Lieberman 2015), amino acids (peptides and proteins), therapeutic gene, small interfering RNA (siRNA), and vaccines are very promising strategies (Crommelin et al. 2003), but with through clinical protocols (Cox et al. 2015; Chen et al. 2015). Proteins and peptides represent major class of biopharmaceuticals (Leader et al. 2008).

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Good Manufacturing Practice Good Laboratory Practice

Basic research rese re sear arch h on on drug drug design desi de sign ign Survey/ social demand Drug design R&D for drug synthesis In vivo pharmacology

Preclinical development development deve dev

Mutagenecity In vivo toxicity Metabolic stability Acute Pharmacology Genomic analysis In vitro toxicity

Good Clinical Practice

Clinical Cliinical trials Cl triaals tri

FDA FDA Filling/approval Filling/ap pproval

Ethic review Regulatory review Phase I to III trials

Fig. 3.3 Sequential processes involved in biologic drug development

3.3

Biologics, Biosimilar, and Biobetter

Biopharmaceutical drugs are available in the market under the categories biologics, biobetter, and biosimilar. It is interesting to note that by the introduction of a specific gene protein with specific amino acids sequence can be produced in different types of genetically modified host cells. But the same protein produced by different manufacture processing is having different characters. So, in order to have differentiation in biopharmaceuticals the basic therapeutic protein is referred as biologic drugs or reference medicine, whereas the following ones are denominated biosimilar. Biosimilars are different from biologic drug due to post-translational modifications (glycosylation, phosphorylation) and different manufacturing process. The term biobetter was recently used to refer to therapeutic macromolecules of the next generation having more efficient pharmacokinetic potential and structural stability (Beck 2011; Strohl 2015; Sandeep et al. 2016). The state of the art of separation of biopharmaceutical of interest is the most challenging process. The culture medium (broth) obtained after upstream process contains many impurities, besides the targeted therapeutic proteins. In the beginning of downstream process the unwanted biomolecules and other extracellular products are separated. The overall process for downstream is consisting of multiple steps in

3.4 Product Recovery

109

order to remove host cells related impurities (e.g. host cell protein, nucleic acids, DNA, etc.); process related impurities (e.g. buffers, leached ligands, antifoam, etc.), and product related impurities (e.g. aggregates, clipped species, etc.) (Azevedo et al. 2009; Rathore and Kapoor 2015). The overall downstream process is completed with three major steps: (1) initial recovery, (2) isolation of impurities, and (3) product purification and polishing (Rosa et al. 2010; Fields et al. 2016).

3.4

Product Recovery

The process of regaining product of interest from the biopharmaceutical processing broth (upstream process) is known as product recovery. The downstream process involves in recovery, isolation, and purification of the crude product obtained from host cells culture. Followings are some of the important sequential processes to obtain polished and formulated product of interest.

3.4.1

Initial Stages of Recovery

The first step in product recovery is the separation (harvesting) of whole cells (biomass) and other insoluble ingredients from the culture broth. In case of intercellular product location, the biomass is subjected to mechanical or enzymatic digestion for releasing the targeted product into the broth. Generally, filtration, centrifugation, sedimentation, precipitation, flocculation, electroprecipitation, and gravity setting methods are used for separation of biomass and other insoluble materials from the broth. In order to separate targeted product from solid biomass processes like grinding, homogenization, or leaching are in use. 1. Centrifugation The basic principle for centrifugation is based on density differences between the solid product to be separated from culture broth (i.e. liquid phase). The different types of centrifuges are depicted in Fig. 3.4a–d. Tubular Centrifuge This type of centrifuge is used for pilot plants (Fig. 3.4a). Tubular bowl centrifuge can be operated at a high speed and can be used for both batch and continuous mode of operation. The solid biomass is removed manually. Disc Centrifuge Disc centrifuge is used for separation of particle size with the range of 3–30 μm. This type of centrifuge can generate force from 4000 to 14,000 times gravitational force,

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Fig. 3.4 Different types of centrifuge used in dawn stream process

thus reducing separation time. These are most common type of centrifuge to separate microbial biomass in turnkey scale (Fig. 3.4b). The disk-type centrifuge consists of a vertical stack of thin disks in the shape of cones. The sedimentation process occurs in the radial direction in the space between adjacent cones. The angle of the cones is designed in such a way that upon reaching the inside surface of the cone the heavier material slides down along its surface in a manner that is similar to that of the 370 fixed-angle bottle centrifuge. Multi-Chamber Centrifuge It is a modified version of tubular bowl type centrifuge having the provision of several interconnected chambers which generate zigzag flow of the feeding liquid. The variation of centrifugal force in different tubes caused settlement of smallest particle on the bottom of the centrifuge tube.

3.4 Product Recovery

111

Scroll Centrifuge or Decanter It consists of rotating horizontal bowl taped at one end (Fig. 3.4b). Culture broth containing high density biomass is separated. A decanter centrifuge is versatile in nature, used in variety of industries for solid liquid separation. The basic operating principle of this system is based on separation via buoyancy. In contrast to chamber filter press, a solid scroll centrifuge or decanter can be operated continuously. High centrifugal force generated during operation is responsible for the separation of finely distributed non-soluble particles in the culture broth (Fig. 3.4c). Basket Centrifuge Basket centrifuge is also known as centrifugal filter or clarifier. It is consisting of perforated wall and cylindrical tubular rotor. Often, the outer boundary wall of a basket centrifuge consists of fine mesh screen with the mechanical support of heavier coarse screen which serves for deposit of particles of larger size. In basket centrifuge centrifugal force is generated for the separation of solid/ liquid. Feeding is provided through the rotating basket positioned on the top of the centrifuge. The solid materials are pulled radically from the liquid by centrifugal force and are collected along the inner wall of the basket. Provision of the outlet of clarified liquid is given on the top of the centrifuge. On the basis of customer requirement fully automatic or semi-automatic facility is provided (Fig. 3.4d).

3.4.2

Flotation

It is a simple process of removal of biomass from the culture broth. By the introduction of an inert gas into the culture medium the cells and other solid particles get absorbed on gas bubble. Rising bubbles to the foam layer can be collected and removed.

3.4.3

Flocculation

It is a process in which the suspended host cells are aggregated and separated through settling process. The process of flocculation depends on the nature of cells and ionic constituents of the medium. Addition of flocculating agents such as inorganic salt, organic polyelectrolytes, and mineral hydrocolloids are often necessary to achieve appropriate flocculation.

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3.4.4

Filtration

Filtration is the most commonly used technique for solid/liquid separation. Based on the nature of biomass, viscosity, and other insoluble materials the filtration can be customized. Different types of filtration methods are being practiced to remove solid materials or biomass from the culture broth (Cohn et al. 1946). Based on the mechanism, three types of filtration methods (Table 3.1) are commonly used in biopharmaceutical industries. Surface filtration, depth filtration, and ultrafiltration are the most common filtrations used in pharmaceutical industries. Materials that pass through the membrane of the filter are called “permeate materials”, while those that are held back and filtered out are called “retentate materials”.

3.4.4.1

Surface Filtration

Surface filtration process is defined by the retention of particles on the surface of the matter being filtered. These particles, at the initial stage, form a thin layer, but with the increase in process time for a thick deposit the filtration efficiency is about 100%. However, with the increase in the thickness of deposited layer, the rate of filtration gets reduced. In such situation the excess deposition is cleaned with a scraper attached to the filtration membrane.

3.4.4.2

Depth Filtration

In depth filter system the material to be separated is passed through a number of different filter layers before exiting the filter system. By this filtration process insoluble particle, soluble materials, and colloidal materials are isolated from the culture broth. At the beginning larger particles are first removed and followed by the removal of smaller size particles. It is a rapid filtration process in which small particles from culture medium can be separated effectively. Generally, depth filters used in biopharmaceutical industry are made of cellulose fibres and filter aids (e.g. diatomaceous earth) bound together by a polymeric resin which provides the necessary wet strength and imparts a cationic surface characteristic. The depth filters are composed of filamentous matrix such as Table 3.1 Major types of filtration with characteristic features No. 1 2

Type Microfiltration Ultrafiltration

3

Reverse osmosis

Size of particles to be separated (μm) 0.1–10 0.001–0.1 0.0001–0.001

Nature of particles Cell/cell fraction/viruses Compound with molecular weight greater than 1000 (e.g. enzymes) Compounds with molecular weight less than 1000 (lactose)

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Fig. 3.5 Filtration principle of dynamic body-feed filtration (DBF) with diatomaceous earth (DE) and conventional filtration

glass wool, asbestos, or filter paper. Wide range of depth filter are available for separation of biomass and other inclusive from the medium. Following are the few regularly used filter aids (Fig. 3.5): • • • • •

Positively charged are modified for separation of negatively charged particles. Silica is used for removal of lipids. Carbon is impregnated for specific adsorptive properties. Low pyrogenic for pyrogen-sensitive applications. The particles are trapped within the matrix and the fluid passes.

The use of diatomaceous earth (DE) is quite long back when people started using this filter aid for fractionation of human plasma (Cohn et al. 1946). In later stage, this technique was used to purify intravenous immunoglobulin, albumin, and clotting factor for commercial production (Curling et al. 2012; More et al. 1991; Stucki et al. 2008). This method is also widely used for separation of bacteria and CHO (TrexlerSchmidt et al. 2010).

3.4.4.3

Ultrafiltration

Ultrafiltration process is the most commonly used filter system in biopharmaceutical industries. In this system the filter pores are 80% water. So, it is necessary to capture the product of interest by concentration of filtrate broth. Evaporation, filtration, liquid–liquid separation, adsorption, and precipitation are common methods for concentring the filtrate. Selection of appropriate process mainly depends on the nature of the product to be separated.

3.6.1

Evaporation

The water content of filtrate is removed by simple evaporation process, but under controlled temperature and vacuum. On the basis of customers requirement variety of designed evaporators are available in the market. 1. Plate Evaporator Plate evaporator consists of series of heat exchange plates arranged in vertical manner with space- to- face having the passage of stream of evaporated filtrate. For temperature sensitive product the entire operation is continued under partial vacuum condition with low temperature. The temperature and vacuum can be arranged on an as-needed basis (Fig. 3.11). 2. Falling Film Evaporator Falling film evaporator is an equipment in which filtrate obtained after centrifugation or filtration is subjected to evaporation with a special type of device having the provision of vertically arrange heat exchanger tubes in which filtrate is passed down inside the surface of the tubes. The heating steam flows on the outside surface of the tubes, where it condenses and releases its latent heat to the falling film (Fig. 3.12). 3. Centrifugal Force Film Evaporators This type of equipment is based on evaporation of filtrate spread over the conical surface of the centrifuge in which centrifugal force is maintained on an as-needed basis. This process of concentration can be completed with shortest possible of residence time. This method of product concentration is suitable for separation of concentrated products like vitamins, DHA, and other heat sensitive biologically derived products.

3.6 Concentration

Fig. 3.11 Schematic line diagram of plate-heat exchanger evaporator

Fig. 3.12 Line diagram of a falling film evaporator

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3.6.2

3 Downstream Processes

Liquid–Liquid Extraction

This process is commonly being practised in biopharmaceutical industry for the separation of product of interest. The filtrate containing desired product is transferred from one liquid phase to another liquid phase, a phenomenon usually known as liquid–liquid extraction. Besides concentration this technique is also used for partial purification of a product. The process of liquid–liquid extraction may be broadly categorized as extraction of (a) low molecular weight product and (b) high molecular weight products. a. Extraction of Low Molecular Weight Products Generally, organic solvents are used for the separation of lipophilic compound. But it is difficult to separate hydrophilic compounds. Separation of lipophilic compound either can be easily extracted by physical method or can be isolated by dissociation extraction method. In case of the former method the compound gets itself distributed between two liquid phases based on the physical properties. This technique is used for the isolation of non-ionizing compound. In the latter method (dissociation extraction) ionizable compounds can be easily extracted. Sometimes, reactive extraction method is applicable to extract target product from the filtrate. In this method a carrier molecule (e.g. phosphorus compound, aliphatic amine) mixed in an organic solvent is used to separate the target product. This method works well when the product (e.g. organic acids) is highly soluble in water. b. Extraction of High Molecular Weight Compounds Proteins and antibodies are high molecular weight products. Organic solvents cannot be used for protein extraction, as they lose their biological activities. They are extracted by two methods: (1) aqueous two-phase systems (ATPS) and (2) reverse micelles formation (Fig. 3.13). In ATPS method polymer (e.g. polyethylene glycol) and salt solution (ammonium sulphate) are combined in suitable ration and used for extraction process. Water is the main component in ATPS, but the two phases are not miscible. Cells and other solids remain in one phase, while the proteins are transferred to other phase (Asenjo and Andrews 2011; Cascone et al. 1991). The distribution of the desired product is based on its surface and ionic character and the nature of the phased. The overall process of ATPS takes longer time. ATPS is the most convenient process for large scale protein recovery. The separation of protein mainly depends on the component of ATPS. Mostly, protein accumulates in top, hydrophobic, and less polar phase, usually PEG. By the addition of salt (NaCl), protein can be separated from one another. Reverse Micelle Method for Separation of Proteins Generally, micelle is an aggregate of molecules having both polar and non-polar regions. Reverse micelles are thermodynamically stable surfactants that hold water in their interior surrounded by organic phase. The reverse micelles are thermodynamically stable. When the filtrate is suspended in reverse micelles containing

3.7 Biopharmaceutical Purification by Chromatography Fig. 3.13 How the target product becomes concentrated in one of the phases and contaminants in the other. ATP aqueous two-phase system, PEG polyethylene glycol

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Desire protein

Contaminant

ATPS formation Sample loading and mixing

Product partitioning and recovery

suspension, the protein of interest is entrapped in micelle (Fig. 3.14) where other impurities remain in organic phase. This method is most suitable for the separation of proteins from the filtrate obtained after upstream process (Grilo et al. 2016; Van Berlo et al. 1998; Hatti-Kaul 2000, 2001; Ruiz-Ruiz et al. 2012; Asenjo and Andrews 2012; Molino et al. 2013).

3.7

Biopharmaceutical Purification by Chromatography

The crude form of biological product obtained after concentration contains closely related compounds, including the product of interest. On the basis of customer’s requirement variety of chromatography techniques are available. Mainly, the chromatography techniques are used for two purposes, i.e. (1) for analytical and preparative purpose, (2) for large scale separation of biomolecules present in a crude mixture. 1. For analytical and preparative purpose, mostly, paper chromatography or gas chromatography is used for analysis purpose (Cohn et al. 1946).

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Fig. 3.14 Reverse micelle method for separation of proteins

2. For large scale separation of biomolecules, the biopharmaceutical industries use the following chromatography techniques on a requirement basis.

3.7.1

Column Chromatography

During the 1930s column chromatography was accepted as a convenient technology for separation of a number of biologically important materials. Since that time, the technique has been developed rapidly. The beginning of 1950 has witnessed the popularity of column chromatography, even in large scale separation of biologically derived materials (Fig. 3.15). Column chromatography consists of two phases, i.e. stationary phase and mobile phase. The stationary phase is a porous solid matrix packed in a column onto which the mixture of compounds to be separated. The overall process of chromatography mainly depends on the physical nature of the targeted biomolecule to be separated from a crude product obtained after concentration. A variety of matrices are commercially available for chromatography separation method (Table 3.2). Agarose, cellulose, polyacrylamide, porous silica, cross-linked dextrin, and polystyrene are most commonly used solid phase. The main principle involved in column chromatography is adsorption of the solutes of a solution through a stationary phase and separates the mixture into individual components. In this method the mobile phase containing the mixture of products to be separated is loaded on the top of the column. The movement of

3.7 Biopharmaceutical Purification by Chromatography

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Fig. 3.15 Use of column chromatography during 1950s for scale up process of separation of biological materials

Table 3.2 Types of chromatography on the basis of physical–chemical nature of biomolecules No 1 2 3 4 5 6

Nature of chromatography Gel filtration Ion-exchange Chromatofocusing Affinity Hydrophobic interaction Immobilized meta-ion affinity

Principle Size and shape Net charge Net charge Biological affinity and molecular recognition Polarity (hydrophobicity of molecules) Metal ion binding

different molecules depends on the size and affinity to stationary phase. The molecules having low affinity move faster than the molecule having stronger affinity to stationary phase. The faster rate moved molecule is captured first, and slowly move molecules are capture subsequently, one by one (Fig. 3.16). The rate of the movement of the components is expressed as: Rf ¼ the distance travelled by the solute/ the distance travelled by solvent. Rt is the retardation factor. The column is packed in two ways: (1) dry packing/dry filing and (2) wet packing/wet filling In the former case the stationary phase (adsorbent) is loaded with fine dry powder and solvent with crude products are poured from the top of the column. The solvent is allowed to flow through the column till equilibrium is reached.

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Fig. 3.16 Separation of target biomolecules by column chromatography

In the latter case, stationary phase in the form of slurry is loaded from the top of the column. Care has to be taken for uniform filling of slurry without any air bubble. Sample mixture dissolved with minimum quantity of mobile phase is loaded on the top of the column. When the size of the column is in bigger form, slight pressure is developed on the top of the column by using some inert gas. After the loading of crude products sample, the mobile phase is allowed to pass through the column. By elution technique, the individual components are separated out from the column. The composition of the mobile phase may be kept constant (isocratic elution mode) or varied (gradient elution mode) during the chromatographic analysis. Isocratic elution holds good in the separation of sample components that are perfectly different in their affinity for the stationary phase. Gradient elution is the technique in which altering the composition of the mobile phase is made during the course of the chromatographic run. In case of coloured products, the separation can be simply monitored visually. In case of colourless products, the elutes are sequentially collected in small sample vials for identification of product by TLC. Column chromatography is commercially used for purification and separation of wide range of biologically derived products. For example, peptide hormones like pramlintide (analogue of Amylin) are isolated by using silica gel in the stationary phase. This hormone is used for treating type 1 and type 2 diabetics. Glycolipids, used for antiviral activity towards HSV-1 (herpes virus), are also separated by using silica gel as absorbent. Separation of DNA by silica adsorption is the most conventional method as compared to other biochemical methods. DNA of cells or tissues can be subjected to lysis by using enzymes or physical foresees (like osmotic pressure). The fluid obtained by such lysis method is known as lysate. It contains

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proteins, DNA, RNA, and even organelles. The lysate is passed through column where DNA is adsorbed on silica surface in the presence of buffer solution with a pH at or below the pKa of the surface silanol group. Once the DNA is adsorbed on silica surface, the other unwanted materials are eluted. The DNA is then washed to remove any excess waste particles from the sample and eluted from the column by using suitable buffer to release the adsorbed DNA molecules which can be further polished.

3.7.2

Gel Filtration Chromatography

It is used for separation of components based on the size of the molecule or molecular weight. It is the most convenient and easy method of separation of a biological mixture consisting of different molecules (Wilson and Walker 2018) (http://kirschner.med.harvard.edu/files/protocols/GE_gelfiltration.pdf; https://www. slideshare.net/asabuwangwa/gel-permeation-chromatography-gpc). The gel filtration medium is porous matrix in the form of spherical particles and acts as column packing materials. The gel particles are stable with adsorption properties. The packed gel is filled with suitable buffer which occupies the porous space of the gel beads. The liquid inside the pores is often referred to as stationary phase and the liquid present around the gel beds is in equilibrium and referred to as mobile phase (Fig. 3.17). On the basis of size, the smaller molecules move through the pores of the bed, while the bigger size molecules move slowly or detained in the bed. Followings are the some of the important purposes in which gel filtration chromatography is used. • Gel filtration is the most conventional and easy method for separation of enzymes, polysaccharides, nucleic acids, proteins, and other biological macromolecules. • Refolding of denaturized proteins can be resumed by careful control of changing the buffer condition of mobile phase. • Proteins fractionation can also be made by gel filtration technique. • Molecular weight of specific biomolecule can be determined by this technique. • This method is the easiest way of determining the quaternary structure of purified proteins.

3.7.3

Ion Exchange (IEX) Chromatography

It is a technique which is used to separate a mixture composed of differentially charged molecules based on differences in their net charge at a particular pH. In ion exchange the stationary phase is either negative charged functional group (cation

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Fig. 3.17 Separation of protein molecules on size basis by gel filtration column chromatography

exchange) to separate positively charged proteins or positively charged functional group (anion exchange) to separate negatively charged proteins. The basic principle of IEX chromatography involved is separation of charged molecules (as liquid phase) by using a column packed with stationary phase of specific charge (Fig. 3.18). Protein charge depends on the number and type of ionizable amino acids side chain group. In general protein is having an isoelectric point (pI), a pH at which the overall number of negative and positive charges is zero. When the liquid phase (buffer solution) is below the protein’s pI, the proteins become positively charged and bind to the negatively charged functional group of a cation exchange resin. In a buffered solution above the protein’s pI, the protein becomes negatively charged and will bind to the positively charged functional groups of an anion exchange resin (Fig. 3.19). It means, in principle, a protein binds to either a cation or anion exchange resin, but in practice, proteins are only stable within a narrow pH range and the choice of resin depends on the pH stability of the protein. Nature of Resins for Ion-Exchange Chromatography The stationary phase of ion-exchange chromatography consists of charged functional group. Cation exchange resins are negatively charged, and anion exchange resins are positively charged (Table 3.3). The ionization state of the functional groups of resins depends on the surrounding pH of mobile phase. So accordingly,

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127

Fig. 3.18 Basic principle of separation of a mixture of differently charges biomolecules by ion-exchange chromatography

ion-exchange resins are grouped as “weak” or “strong”. Weak ionized resins act over only a limited pH range, while strong exchangers show no variation in ion exchange capacity with changes in pH. The limiting factors that monitor the flow of elute are type of functional groups selected, the size of beads, and the porosity of beads. The first step involved in ion-exchange chromatography is determination of pI of protein of interest with the help of in silico software methods. Several online tools are also available for the determination of pI of protein of interest. Generally, the pI of a protein is determined by the aggregate charge of every amino acid in the protein chain. But, the theoretical pI is not exactly matched with actual pI of protein of interest. The success in eluting target protein mainly depends on the selection of suitable buffer, pH gradient, additives, and salt concentration. The elution of protein can be controlled by monitoring the concentration of salt in the buffer. In general, ion-exchange chromatography is an easy and convenient method which can be used at any stage of purification by using a wide range of resins, commercially available.

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1

2

3

4

5

Crude Mixture of proteins with MES

Caon exchanger column

Proteins are eluted with increasing salt gradient

Fig. 3.19 Separation of crude protein mixture by ion-exchange chromatography

3.7.4

Affinity Chromatography

It is a method of separation of biomolecules based on their specific interactions like antigen and antibody; enzyme and substrate; receptor and ligand (Hage and Ruhn 2005; Walters 1985; Cuatrecasas et al. 1968). This method is used both in laboratory for small scale separation and in biotechnology and biopharmaceutical industries for large scale production. Through

3.7 Biopharmaceutical Purification by Chromatography

129

Table 3.3 Nature of stationary phase of ion-exchange resins Resin DEAE ANX Q CM S

SP

Functional group Diethylaminoethyl [-N+(C2H5)2H+] Diethylaminopropyl [-N+(C2H5)2H+) Quaternary amine [-N+(CH3)3] Carboxymethyl [-O-CH2COO] Methyl sulfonate [O-CH2-CHOH-CH2-O-CH2-CHOH-CH2SO3] sulphonyl group [-CH2-CH2-CH2SO3]

Weak or strong Weak anion

Functional pH range 2–9

Weak anion

2–9

Strong anion

1–14

Weak cation

5–10

Strong cation

2–12

Strong cation

2–14

Fig. 3.20 Affinity chromatograph for separation of specifically interacting biomolecules like enzyme

hydrogen bonding, ionic interaction, disulfide bridges, hydrophobic interaction the biological macromolecules (enzymes and other proteins) interact with other molecules. In affinity chromatography these molecules interact with stationary phase (cellulose beads having specific substrate bind covalently) on the basis of specific interaction by separating from unwanted materials, which are eluted away (Fig. 3.20)

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(Ninfa et al. 2009a). The protein of interest binding on stationary phase (cellulose beads) gets released by suitable buffer of higher salt concentration. Relatively higher salt concentration buffer weakens the interactions between the enzyme and the immobilized substrate, and as a result the substrate binding enzyme gets released to buffer medium (Ninfa et al. 2009b). Types of Stationary Phase Several types of activated agarose gels are used for binding ligands. For an example CNBr-agarose is easy to use for the attachment of amines. But short space is left between the gel beads and the bound ligand, and as a result protein binding may be sterically hindered. Aminohexanoic acid-agarose (CH-agarose, for reaction with amines) and diaminohexane-agarose (AH-agarose, for reaction with carboxylic Acids) have relatively long methylene chains that keep the ligand a significant distance from the gel beads. An alternative reagent for attaching amines is carbonyldiimidazoleagarose, and epoxy-activated agarose is used for alcohols. Use of lectins as stationary phase has been becoming popular for the separation of carbohydrate-binding proteins (Hage et al. 2005; Liener et al. 1986). This method is commonly used for separation of glycoproteins, glycopeptides, glycolipids, and oligosaccharides (Hage et al. 2005; Yamamoto et al. 2002). The most common types of lectins that are used in affinity chromatography are concanavalin A, wheat germ agglutin (WGA), and jacalin (Hage et al. 2005; Hermanson et al. 1992; Bourne et al. 2002). Boronate is also used as stationary phase in affinity chromatography. In basic pH, boronate has the potential to target a molecule having cis-diol group, such as are present in many carbohydrate containing compounds (Middle et al. 1983). Boronate column commonly is used for quantitation of glycated haemoglobin for the diagnosis purpose of long-term diabetes (Mallia et al. 1981; Hage and Phillips 2005). Immobilized antibody or related agent is used as stationary phase for isolation and purification of hormones, enzymes, peptides, viruses, and other biologically relevant materials (Wilchek et al. 1984; Schiel et al. 2006) (Fig. 3.21). Like other affinity ligands, antibodies can be used on traditional affinity supporters like agarose or attached to supports that can be used in HPAC, such as silica or monolithic materials (Mallik and Hage 2006; Yoo and Hage 2010; Dong et al. 2009). In some affinity chromatography the stationary phase consists of an immobilized chelating ligand that forms complex with a metal ion. The chelating ligands (iminodiacetic acid, nitrilotriacetic acid, carboxymethylated-aspartic acid, and L-glutamic acid) are attached covalently to a support and are used to entrap metal ions (Ni2+, Zn2+, Cu2+, and Fe3+) via coordinate binding. Agarose was the first support utilized in such affinity chromatography which is commonly known as immobilized metal ion affinity chromatography (IMAC). But in later phase silica, cryogels, and polymethacrylate monolith have been in use in IMAC. This technique is widely used for analysing membrane proteins, histidine-tagged proteins, and phosphorylated proteins (Wang et al. 2009; Ye et al. 2010; Dong et al. 2009).

3.8 Biologic Drug Formulation

131

Fig. 3.21 Separation of specific antibody by affinity chromatography

IMAC has become a powerful tool for analysing membrane proteins, histidinetagged proteins, and phosphorylated proteins (Dong et al. 2009; Wang et al. 2009; Ye et al. 2010). This technique is also used for detecting drugs (tetracyclines, quinolones, macrolides, β-lactams, and aminoglycosides) (Takeda et al. 2010) in a given sample.

3.8 3.8.1

Biologic Drug Formulation Excipients

Biologic drugs are highly susceptible to variable temperature, pH extremes, freezing, light, agitation, sheer stress, and organic solvents that cause protein instability. Some strategies are suggested to improve protein stability by incorporating some compatible excipients with the original biologics. So, excipients are an integral part of any biologic drug formulation. The selection of excipients depends on the nature of active ingredient, route of administration, dosages form, and target population. Discovery of new therapeutic proteins and vaccines, it has been a great challenge for biopharmaceutical industry in selecting suitable and low-cost excipients for biologic drug formulation. Appropriate excipients enable development of novel therapies and highly stable pharmaceutical products. Excipients are inert substance which can support the structural integrity of biologics without any chemical or biological interaction. In addition, excipients add flavour to improve the palatability of medicines. Especially for therapeutic proteins and vaccines excipients help in enhancing half-life of the drugs, stabilize

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pH and tonicity, enhance pharmacokinetic properties, and prevent from aggregation or degradation of active ingredients. Some excipients used for vaccines formulation act as adjuvants (for enhancing efficacy to stimulate the ability of antigen). The excipients are belonging to organic or inorganic molecules. The selection of excipients also depends on the regulatory requirements in the respective market. In the USA, the FDA provides list of approved inactive ingredients (www.accessdata.fda. gov/scripts/cder/iig/index.cfm). Different countries on the basis of requirement provide list of excipients. But, Europe has not approved any excipients list for pharmaceutical or biopharmaceutical drug formulation. The formulation of low molecular weight products (solvent, organic acids) can be achieved by concentrating them with removal of water contain. Small biomolecules such as antibiotics, itric acid are crystallized by adding salts. Due to the instability nature of proteins, certain stabilizing agents like sugars (sucrose, lactose), polymers (polyethylene glycol), salts (sodium chloride, ammonium sulphate), and polyhydric alcohols (glycerol) are used while formulating biologic drugs.

3.8.2

Drying

Most of the biologically derived products are heat sensitive, and are subjected to drying or spray drying under low temperature and partial vacuum condition. The dehydrated biopharmaceuticals are easy to handle for packing and transport. The dehydrated biopharmaceutical can be preserved for a longer period without disturbing the structural stability of biological compounds. Freeze-drying is a common and easy method for storing biotherapeutics. Lyophilization process is also used for drying process. But, dehydration by lyophilization takes longer period. In this process, water is removed from a therapeutic molecule after it is frozen and placed under vacuum, allowing the ice to change directly from solid to vapour without passing through a liquid phase. Most of the biologically derived therapeutic molecules like proteins, peptides, oligonucleotide, enzymes, and mAbs are lyophilized for long-term storage and transport. Advantages • Processing liquid suspension is convenient and easy. • The stability of lyophilized sample is better than simple drying material. • Water molecules are removed without application of heat. Dissolution of reconstituted product is rapid. Disadvantages • The entire process is time consuming, and resulted in increase in cost. • Sterile diluent needed upon reconstitution. • Maintenance of equipment is expensive.

3.8 Biologic Drug Formulation

3.8.3

133

Spray Drying

Generally, this technique holds goods for handling large quantity of sample. In this method, small droplets of liquid containing the product are passed through a nozzle directing it over a stream of hot gas under the blanket of some inert gas. In such process the water evaporates and the solid particles are left behind (Fig. 3.22).

3.8.4

Integration of Different Processes

It is necessary to integrate the biopharmaceutical manufacturing, wherever there would be possibility of application. However, this has not been practicable for various reasons. But it is practicable to integrate certain stages in downstream process for isolation and purification of proteins. For example, protein concentration in two-phase systems combined with clarification and purification can be carried out simultaneously.

Fig. 3.22 Schematic diagram of spray drying process of biopharmaceuticals

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References Asenjo JA, Andrews BA (2011) Aqueous two-phase systems for protein separation: a perspective. J Chromatogr A 1218:8826–8835 Asenjo JA, Andrews BA (2012) Aqueous two-phase systems for protein separation: phase separation and applications. J Chromatogr A 1238:1–10 Azevedo AM, Rosa PAJ, Ferreira IF, Aires-Barros MR (2009) Chromatography-free recovery of biopharmaceuticals through aqueous two-phase processing. Trends Biotechnol 27(4):240–247 Beck A (2011) Biosimilar Biobetter and next generation therapeutic antibodies. MAbs 3 (2):107–110 Bourne Y, Astoul CH, Zamboni V, Peumans WJ, Menu-Bouaouiche L, Van Damme EJM, Barre A, Rouge P (2002) Structural basis for the unusual carbohydrate-binding specificity of jacalin towards galactose and mannose. J Biochem 364:173–180 Cascone O, Andrews B, Asenjo J (1991) Partitioning and purification of thaumatin in aqueous two-phase systems. Enzym Microb Technol 13:629–635 Chen HL, Chang JK, Tang RB (2015) Current recommendations for the Japanese encephalitis vaccine. J Chin Med Assoc 78(5):271–275 Cohn EJ et al (1946) Preparation and properties of serum and plasma proteins, IV: a system for separation into fractions of the protein and lipoprotein components of biological tissue and fluids. J Am Chem Soc 68:459–475 Cox DBT, Platt RJ, Zhang F (2015) Therapeutic genome editing: prospects and challenges. Nat Med 21(2):121–131 Crommelin DJ, Storm G, Verrijk R, De Leede L, Jiskoot W, Hennink WE (2003) Shifting paradigms: biopharmaceuticals versus low molecular weight drugs. Int J Pharm 266(1–2):3–16 Cuatrecasas P, Wilchek M, Anfinsen CB (1968) Selective enzyme purification by affinity chromatography. Proc Natl Acad Sci U S A 68:636–643 Cummings J, Morstof T, Zhong K (2014) Alzheimer’s disease drug development pipeline: few candidates, frequent failures. Alzheimer’s Res Ther 6:37–44 Curling J et al (2012) Production of plasma proteins for therapeutic use. Wiley-Blackwell, Hoboken Deng Y, Wang CC, Choy KW (2014) Therapeutic potentials of gene silencing by RNA interference: principles, challenges, and new strategies. Gene 538(2):217–227 DiMasi JA, Feldman L, Seckler A, Wilson A (2010) Trends in risks associated with new drug development: success rates for investigational drugs. Clin Pharmacol Ther 87:272–277 DiMasi JA, Grabowski HG, Hansen RW (2016) Innovation in the pharmaceutical industry: new estimates of R&D costs. J Health Econ 47:20–33 Dong XY, Chen LJ, Sun Y (2009) Refolding and purification of histidine-tagged protein by artificial chaperone-assisted metal affinity chromatography. J Chromatogr A 1216:5207–5213 Fields C, Li P, O’Mahony JJ, Lee GU (2016) Advances in affinity ligand-functionalized nanomaterials for biomagnetic separation. Biotechnol Bioeng 113(1):11–25 Grilo AL, Raquel Aires-Barros M, Azevedo AM (2016) Partitioning in aqueous two-phase systems: fundamentals, applications and trends. Sep Purif Rev 45:68–80 Hage DS, Phillips TM (2005) Immunoaffinity chromatography. In: Hage DS (ed) Handbook of affinity chromatography, 2nd edn. CRC Press, Boca Raton, pp 127–172 Hage DS, Ruhn PF (2005) An introduction to affinity chromatography. In: Hage DS (ed) Handbook of affinity chromatography. CRC Press, Boca Raton, pp 3–13 Hage DS, Bian M, Burks R, Karle E, Ohnmacht C, Wa C (2005) Bioaffinity chromatography. In: Hage DS (ed) Handbook of affinity chromatography, 2nd edn. CRC Press, Boca Raton, pp 107–135 Hatti-Kaul R (2000) Aqueous two-phase systems: methods and protocols. Springer, Berlin Hatti-Kaul R (2001) Aqueous two-phase systems. Mol Biotechnol 19:269–277 Hermanson GT, Mallia AK, Smith PK (1992) Immobilized affinity ligand techniques. Academic, New York

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Leader B, Baca QJ, Golan DE (2008) Protein therapeutics: a summary and pharmacological classification. Nat Rev Drug Discov 7(1):21–39 Liener IE, Sharon N, Goldstein IJ (1986) The lectins: properties, functions and applications in biology and medicine. Academic, Waltham Mallia AK, Hermanson GT, Krohn RI, Fujimoto EK, Smith PK (1981) Preparation and use of a boronic acid affinity support for the separation and quantitation of glycosylated haemoglobins. Anal Lett 14:649–661 Mallik R, Hage DS (2006) Affinity monolith chromatography. J Sep Sci 29:1686–1704 Middle FA, Bannister A, Bellingham AJ, Dean PDG (1983) Separation of glycosylated haemoglobins using immobilized phenylboronic acids. Biochem J 209:771–779 Molino JVD, Marques V, de Araújo D, Júnior AP, Mazzola PG, Gatti MSV (2013) Different types of aqueous two-phase systems for biomolecule and bioparticle extraction and purification. Biotechnol Prog 29:1343–1353 More J et al (1991) Purification of albumin from plasma. Blood separation and plasma fractionation. Wiley-Liss, Hoboken Ninfa AJ, Ballou DP, Benore M (2009a) Fundamental laboratory approaches for biochemistry and biotechnology, 2nd edn. Wiley, New York, p 133 Ninfa AJ, Ballou DP, Benroe M (2009b) Fundamental Approaches to Biochemistry and Biotechnology. Wiley, New York Paul SM, Mytelka DS, Dunwiddie CT, Persinger CC, Munos BH, Lindborg SR (2010) How to improve R&D productivity: the pharmaceutical industry’s grand challenge. Nat Rev Drug Discov 9:203–214 Rathore AS, Kapoor G (2015) Application of process analytical technology for downstream purification of biotherapeutics. J Chem Technol Biotechnol 90(2):228–236 Rodríguez V, Asenjo JA, Andrews BA (2014) Design and implementation of a high yield production system for recombinant expression of peptides. Microb Cell Factories 13:1–10 Rosa PAJ, Ferreira IF, Azevedo AM, Aires-Barros MR (2010) Aqueous two-phase systems: a viable platform in the manufacturing of biopharmaceuticals. J Chromatogr A 1217 (16):2296–2305 Ruiz-Ruiz F, Benavides J, Aguilar O, Rito-Palomares M (2012) Aqueous two-phase affinity partitioning systems: current applications and trends. J Chromatogr A 1244:1–13 Sandeep V, Parveen J, Chauhan P (2016) Biobetters: the better biologics and their regulatory overview. Int J Drug Regul Aff 4(1):13–20 Schiel JE, Mallik R, Soman S, Joseph KS, Hage DS (2006) Applications of silica supports in affinity chromatography. J Sep Sci 29:719–737 Strohl WR (2015) Fusion proteins for half-life extension of biologics as a strategy to make biobetters. BioDrugs 29(4):215–239 Stucki M et al (2008) Investigations of prion and virus safety of a new liquid IVIG product. Biologicals 36:239–247 Takeda N, Matsuoka T, Gotoh M (2010) Potentiality of IMAC as sample pretreatment tool in food analysis for veterinary drugs. Chromatographia 72:127–131 Trexler-Schmidt M et al (2010) Identification and prevention of antibody disulfide bond reduction during cell culture manufacturing. Biotechnol Bioeng 106(3):452 Valderrama-Rincon JD, Fisher AC, Merritt JH (2012) An engineered eukaryotic protein glycosylation pathway in Escherichia coli. Nat Chem Biol 8(5):434–436 Van Berlo M, Luyben KCA, van der Wielen LA (1998) Poly (ethylene glycol)-salt aqueous two-phase systems with easily recyclable volatile salts. J Chromatogr B 711:61–68 Walters RR (1985) Affinity chromatography. Anal Chem 57:1099A–1114A Wang CZ, Wang LL, Geng XD (2009) Optimization of refolding with simultaneous purification of recombinant human granulocyte colony-stimulating factor from Escherichia coli by immobilized metal ion affinity chromatography. Biochem Eng J 43:197–202 Wilchek M, Miron T, Kohn J (1984) Affinity chromatography. Methods Enzymol 104:3–55

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Wilson K, Walker J (2018) Principles and techniques of biochemistry and molecular biology, 8th edn. Cambridge University Press, New York Wittrup A, Lieberman J (2015) Knocking down disease: a progress report on siRNA therapeutics. Nat Rev Genet 16(9):543–552 Yamamoto K, Tsuji T, Osawa T (2002) Affinity chromatography of oligosaccharides and glycopeptides with immobilized lectins. In: Walker J (ed) The protein protocols handbook. Humana Press, Totowa, pp 917–931 Ye JY, Zhang XM, Young C, Zhao XL, Hao Q, Cheng L, Jensen ON (2010) Optimized IMAC protocol for phosphopeptide recovery from complex biological samples. J Proteome Res 9:3561–3573 Yoo MJ, Hage DS (2010) Affinity monolith chromatography: principles and recent developments. In: Wang P (ed) Monolithic chromatography and its modern applications. ILM Publications, London

Chapter 4

Bioprinting

4.1

Introduction

Bioprinting or three-dimensional (3D) is a process in which a biological liquid suspension consisting of human cells, growth factor, and other biomaterials supporting cell growth is deposited layer-by-layer method (additive method) with the help of 3D printing devisees to create a tissue-like structure, which can be used in medical and tissue engineering field (Fig. 4.1). This technique is also known as additive manufacturing (AM) or rapid prototyping (RP) or solid free-form technology (SFF). The liquid suspension of tissue (bioink) (Fig. 4.2) is composed of human cell, growth hormones, and other supporting material for tissue growth cell. Recently bioprinting technology has been used to print tissues and organs mainly for research purpose to find the possibility of application of this technique for clinical trials (Singh and Thomas 2018; Hinton et al. 2015). In addition, lot of preliminary initiatives have been taken to develop artificial tissues using 3D gel layer by layer (scaffolds) (Thomas 2016; Nakashima et al. 2017). The basic principle of 3-D bioprinting is similar to conventional 3D printing. In 3D bioprinting, special arrangement is made to maintain cell viability during additive process for forming layer-by-layer tissue. So, the entire operation is managed within sterilization condition. In this process complex live tissues (surgical model, moulds for titanium implants, prosthetic for amputees, dental crown and bridges, and cranial implants). Experimental trials are in progress to develop heart valves, bone, and even a functional heart (Aikawa et al. 2006; CBC News 2017; Burgess 2017; Wired 2016). The 3-D bioprint can generate on-demand production. For an example, bioprinting of crowns and brides is presently available in the market. In biologic drugs formulation, 3D printing will enable low-volume production and increased speed to market. The overall process for 3-D bioprinting is, sequentially, divided into three steps: i.e. (1) pre-bioprinting, (2) bioprinting, and (3) post-bioprinting (Fig. 4.3). © Springer Nature Singapore Pte Ltd. 2020 B. K. Behera et al., Competitive Strategies in Life Sciences, New Paradigms of Living Systems 1, https://doi.org/10.1007/978-981-15-7590-7_4

137

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3D bioprinter Dropping bio-ink layer-by-layer

Tissue growing on gel -scaffold

Fig. 4.1 3D bioprinter used for additive manufacturing of tissue layer-by-layer method

Fig. 4.2 Formulation of bioink from human cells collected from patient

4.2

Pre-bioprinting

Pre-bioprinting is first preparatory step for additive manufacturing of human tissue from biopsy cells isolated from a patient. Before fabricating an organ, a computeraided 3D design is to be developed. This should be computer compatible. Presently, varieties of software packages are available on anatomy, histological structure, composition, and topology of human organs. Advanced software packages on clinical bioimaging and ultrasound technology could able to design the gross anatomical in vivo characteristics of organs of a patient.

4.2 Pre-bioprinting

Prebioprinting

In vivo image formation by Computed tomography (CT) Magnetic resonance image (MRI) Tomographic reconstruction to 2D image

139

Bio-ink preparation & loading into cartridge

Composed of human cell, growth hormones and other supporting material for tissue growth cell in hydrogel

Post –processing

The multi layered tissues transfer to scaffold for further growth and development

Fig. 4.3 Schematic presentations of different stages in bioprinting process

A second approach is based on computer-aided reconstruction of a histological section. This method provides a high level of resolution and information about the size and shape of the organ, as well as details about its composition. Pre-bioprinting involves creating the digital model that the printer will produce. The digital model is prepared by computed tomography (CT) and magnetic resonance imaging (MRI) scans. Before the operation of bioprinting process, the tomographic reconstruction is done on the images to generate 2D images. For additive manufacturing bioink is filled in the bioprinting cartridge for further process (Ozbolat 2015; Chua and Yeong 2015).

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4.3

4 Bioprinting

Bioprinting Process

Bioprinting processing is an additive manufacturing process in which bioink forms layer-by-layer (about 0.5 mm or less in thickness) tissue-like structure, which is developed as natural tissue by growing in suitable scaffold. The delivery of smaller or larger deposits depends on the number of nozzles and the kind of tissue being printed. In 3D bioprinter, the cartridge is filled with bioink and has technical provision of moving in all direction as per requirement. This movement of cartridge can be mechanically controlled to move in a layer-by-layer manner to produce a 3D object of interest. The mechanical process of bioprinter works the same manner as a 3D printer but carries “bioink”. As already stated, bioink is a slurry of different types of living cells that behave much like liquid ink and can be loaded into a cartridge. In the process of layer-by-layer formation, the viscous liquid suspension containing desired cells gets solidified to hold its shape (Fig. 4.4). The process of blending and solidification is known as cross-linking and may be enhanced by UV light, specific chemicals, or heat (typically delivered via a UV light source.

Droplet of bioink holding human

Bioink spheroids printed into layers of

Hydrogel biopaper Formaon of ssue like structure aer the dissoluon of hydro-gel

Bioink spheroids Fig. 4.4 Schematic presentation of layer-by-layer spheroids formation into biopaper

4.4 Post-Processing

4.3.1

141

Bioink

Bioink formulation is the most crucial process in 3D bioprinting technology. The development of solidified tissue like structure, and its further differentiation depends on the formulation of bioink, meticulously. This is because of differentiation of extracellular fluid and in vivo biofluid surrounding the microenvironment of tissue or organelles (Gao et al. 2018; Hospodiuk et al. 2017; Zhang et al. 2018; Heinrich et al. 2019). The formulation of bionic is made on the basis of printability (rapid conversion from sol state to gel state), biocompatibility (ability of biomimicry with in vivo fluid), and other mechanical property to maintain the extrusion of bioink into biopaper (hydrogel paper) to withstand into scaffold material for further growth and development. Generally, bioink consists of hydrogel, decellularized matrix components, microcarriers, tissue spheroids, and cell pellet (Hospodiuk et al. 2017; Zhang et al. 2018). Hydrogel is one of the important gradients in bioink formulation. Alginate, fibrinogen, gelatine, collagen, silk fibrin, chitosan, pluronics, hyaluronic acid (HA), gelatin methacryloyl (GelMA, poly-ethylene glycol (PEG), and poly-ethylene oxide (PEO) are used as hydrogel in bioink formulation. Generally, these hydrogel agents are ion-sensitive, photosensitive, thermosensitive, enzyme sensitive, which can be easily change gel state to sol state with change in temperature (Heinrich et al. 2019). Mostly, a 3D bioprinter has provision of cartridges, which hold bioink and biopaper. The former cartridge holding bioink consists of desired human cells suspension with growth hormones, vitamins, and other essential supporting materials for the growth and development of mammalian cells to artificial tissue-like structure. The later cartridge holds hydro-gel (biopaper) to give mechanical support to the additive layers of cells till it gets solidified to tissue-like structure (Fig. 4.4). The release of bioink is controlled by the different types of mechanical devices attached to the cartridges head (Fig. 4.5). The entire process for development of tissue-like structure is fabricated with computer-aided design, which monitors the layer-by-layer deposition (also known as solid free-form fabrication) of cells or cell aggregates or cell-encapsulated hydrogels.

4.4

Post-Processing

In vitro manufacturing of tissues and organelles analogous to body parts, both in structure and function, has been a long challenge to get relief from legal litigations, ethical issues, and timely availability. On the basis of demand, there has been huge shortage of organs for transplantation. For instant, in 2016, there were 160,000 organ transplant recipients, but only 16,000 organ donors in USA (Dong et al. 2017). So, post-processing of bioprint tissues or organelles to make them analogous to in vivo parts of human body is in progress.

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Fig. 4.5 Type of cartridges used in bioprint technology

In reality, the traditional way of developing two-dimensional (2D) cell culture or animal experiments used for drug screening and other clinical tests have lot of drawbacks. So, it is necessary to develop in vitro models analogous to in vivo tissues or organelles of human body. Interestingly, 3D bioprinting is an upcoming technology, which will have a significant role in the construction of in vitro organ model in near future. Factually, 3D printed tissue development is made in an aqueous surrounding as in natural conditions. For this purpose, perfusion device such as bioreactors are used that allow the cells growth. The printed tissue obtained through this noble method should be analogous to natural occurring tissue, and having the character of rapidly self-assemble, mature, and differentiate to the target functional organ. At this stage, necessary care is taken for its proper development and perfect assimilation as per specific clinical defined characters. The most important aspect of bioprinting is formation of tissue-like structure, which can be compactable to body, after further process for human tissues and

4.5 Types of Bioprinter

143

organs. But, biological fabrication of artificial kidney or liver is lacking of blood vessel, tubules, and other biological supporting material to fulfil the physiological function of artificial organelles. So, it has been a great challenge to develop artificial organelles completely analogous to natural system. This is the most challenging process in drug testing and clinical trials. Another important aspect of bioprinting of human tissue and organelles would solve the longstanding moral and ethical problem of organ donation and transplantation (Cooper-White 2015; Harmon 2013; Murphy and Atala 2014).

4.5

Types of Bioprinter

Generally, bioprinters are categorized on the basis of cartridge use or without cartridge. In the former case nozzle-based technique like inkjet-based bioprinting or extrusion printing is used for biofabrication of additive tissue into a scaffold, and in latter case laser-based techniques like stereolithography or laser-assisted bioprinting are used for directly fabrication of additive tissue layer by layer on scaffold without any provision of cartridge.

4.5.1

Inkjet-Based Bioprinting

Inkjet bioprinter is basically analogous to a traditional type of inkjet printer, but having the technology to prevent the instantaneous drying of bioink, immediately after extrusion from cartridge (Li et al. 2016). Use of cells holding spheroids in a highly hydrated polymer is used for the layer-by-layer additive method. The hydrogel suspended spheroids are deposited on hydrogel biopaper in a precise manner (Nakamura et al. 2005) through thermal or piezoelectric processes (Fig. 4.6) (Murphy and Atala 2014; Zeng et al. 2018). Thermal-based inkjet printer has the provision of heating the bioink to nucleate bubbles, which build-up pressure within the bioink holder. The thermal element can generate temperature with the range of 100–300  C. But this high temperature is exposed to bioink for a few second, only (Li et al. 2016; Cui et al. 2012). The piezoelectric-based technology has provision of generating acoustic waves to eject bio ink. This mechanism holds good for highly concentrated and viscous bioinks (Murphy and Atala 2014; Tekin et al. 2008). The advantage of inkjet bioprint is with its low cost with high resolution and high cell viability (Boland et al. 2006; Cui et al. 2010; Xu et al. 2012).

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Fig. 4.6 Schematic of inkjet-based bioprinting. Thermal inkjet uses heat-induced bubble nucleation that propels the bioink through the micro-nozzle. Piezoelectric actuator produces acoustic waves that propel the bioink through the micro-nozzle

4.5.2

Extrusion-Based Bioprinting

This is also an inkjet type bioprinting device but based on pressure-driven technology. Extrusion of bioink is monitored through a nozzle, driven by either pneumatic or mechanical pressure, and deposited into a scaffold-holding accessory (Fig. 4.7) (Zeng et al. 2018). The main advantage of extrusion bioprinting is the ability to print with very high cell densities. But it has major drawbacks that are poor resolution (about 100 μm), and the pressure used can damage or alter the structure of tissues. So, it has limit application for certain soft tissue. However, still it has wider use in some hard tissues with the size about 10 mm.

4.5.3

Laser Bioprinting

Laser bioprinting (La BP) is based on computer-assisted bio-construction of living like solidified tissue. This technique could able to generate scaffold-free 3D tissue mass through a layer-by-layer additive technique. The laser bioprinter works on the basic principle of stereolithography (SLA) in which optical fabrication followed by photo-solidification is carried out to create tissue-like solidified biomass. The tissuelike biomass is further modified to artificial tissue analogous to in vivo tissues or

4.5 Types of Bioprinter

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Fig. 4.7 Schematic of extrusion-based bioprinting; from left, pneumatic-based and right, mechanical-based. Struts are extruded via pneumatic or mechanical pressure through micronozzles. Extrusion-based techniques can produce structures with great mechanical properties and print fidelity

organelles ready for use in clinical trials or serves as an ideal model for biologic drugs testing. Laser-induced forward transfer (LIFT) is presently being used, widely, for 2D or 3D bioprinting based on nozzle-free laser-assisted hydrogel microplate transfer. Laser bioprint holds bioink in a vat attached to the base platform of the printer. In addition, it has the provision of holding donor ribbons, and collector substances for LIFT bioprinter. In LIFT-based bioprinting technique, the laser beam of a LIFT bioprinter focuses on the donor-ribbon (a glass slide coated with an energy absorbing material), and a layer of bioink consisting mammalian cells suspension in hydrogel medium. When laser beam heats the surface of bio-ribbon, the energy absorbing layer gets evaporated evaporate with the generation of bubbles. Consequently, the gas bubbles propel jet from the hydrogel. The resulting jet falls on another glass slide having collector substrate, which is ultimately develop into tissue-like biomass (Fig. 4.8). LIFT technology is versatile in nature in which both the high and low cell density bioink can be used. Both cell in the form of spheroid and microorganisms can be handled easily. Mostly, laser wavelengths with the range of 193 and 1064 nm (short ultraviolet and near infrared ranges, respectably) are commonly used in La BP. Gold, titanium, and gelatine containing mixture are used as energy absorbing materials. The bioink used in La BP contains glycerol and methylcellulose or blood

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Fig. 4.8 Laser-based bioprinting showing orifice-free bioprinting

plasma to support cell growth. Hyaluronic acid, due to its high viscosity, is used in bioink formulation. LIFT technology has tremendous scope in tissue engineering for developing 2D or 3D artificial tissues, which can be used in clinical trials and biologic drugs diagnosis. Advantages: • It is a versatile bioprinting technique applicable to mammalian tissue fabrication and microbes. • The solidified tissue developed is heterogonous free from the hurdles faced in extrusion bioprint. • Single-cell resolution. • Spot-to-spot reproducibility. • No adverse effects to the oriented cells.

4.5.4

Laser-Assisted Bioprinting (LAB)

At the initial stage of development, LAB was mainly used to print viable embryonic chick spinal cord cells (Odde and Renn 2000). LAB consists of three compartments: a donor slide (or ribbon), a laser pulse, and a receiver slide. The ribbon is made of al layer of transparent glass, a thin layer of metal, and a layer of bioink. When laser pulse hits the ribbon surface, the metal layer gets evaporated due to increase in surface temperature, and the bioink is transferred from the ribbon onto the receiver slide, as shown in Fig. 4.9. This scaffold-free technique has very high cell viability (>95) and a resolution between 10 and 50 μm (Murphy and Atala 2014; Keriquel et al. 2017).

4.6 4D Bioprinting

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Fig. 4.9 Schematic of laser-assisted bioprinting: (a) transparent glass, (b) thin metal layer, and (c) vaporization-induced bubble. Bubble nucleation induced by laser energy propels droplets of bioink towards the substrate. This technique has minimal effect on cell viability. A receiver slide can be a biopaper, polymer sheet, or scaffold

4.6

4D Bioprinting

4D bioprint has been gaining wide popularity in tissue engineering and clinical diagnosis but has its limitation in use due to its static nature. Recently, “time” has been integrated with 3D bioprinting as the fourth dimension, the so-called 4D bioprinting (Gao et al. 2016; An et al. 2016), in which biofabricating tissue can be structurally and functionally changed with time with the application of external induction like temperature, pH, Ca2+, humidity, electric-field, light, and magnetic field (Ionov 2018; Morouço et al. 2017). As a result, its application is gaining appreciation in tissue engineering for the fabrication of structured biological materials, vascularized tissue, and functional human organelles more accurately and physiologically compactable with microenvironment of living tissues (Guermani et al. 2016; Rayate and Jain 2018). For an example, it could be possible to go for cellladen hydrogels for bioartificial pancreas (De Castro et al. 2005; Orive et al. 2018).

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Selection of Cell Lines for Bioprinting

Mostly, commercially available cell lines from animal or human sources are used in 3D bioprinting for developing tissue-like solidified biomass or organelles. The advantages of this practice are easily availability of cell lines and well-established protocols for culture and expansion. Primary cell lines are either isolated from animal cells or from volunteers or patients. But in practice, the isolated cells are mix population and require steps to isolate as pure cell lines with phenotype and genotype character. The pure cell lines obtained are used for bioprinting purpose. Primary cells from the same animal species are used for the cell expansion in bioprinting process. 3D bioprinting is an emerging technology for the development of artificial tissue or organelles, which may mimic the structure and function of organelles in “in vitro” micro-environment. But, inadequate data is available on to understand the impact of mechanical stress-like shear generated in bioink, and changes in magnetic field on cells present in bioink. It has been understood that shear and mechanical stress generated during flow of bioink onto receiver substrate has impact on endothelial and osteoblast (Zeng et al. 2018; Stolberg and McCloskey 2009). Besides this, mechanical stress can stimulate chondrogenic and osteogenic differentiation (Cui et al. 2012; Shav and Einav 2010). So, inkjet bioprinter and laser based bioprinter are better options for the biofabrication of solidified tissue like biomass without any problem of cell damage by shear or mechanical pressure, as noticed in extrusion bioprinting. But, as stated earlier, extrusion bioprinting works nicely in case of high cell density and heavy viscous biofluid. So, it is necessary to monitor the status of the stem cells by regulating the back pressure and composition of bioink (Li et al. 2016; Lee et al. 2015).

4.7.1

Bioprinting of Artificial Blood Vessels

Artificial blood vessels are mainly fabricated by using either cellular or acellular materials as the base for bioink formulation. Basically, three major steps are followed for fabrication of artificial vessels: (1) strategies for designing integrated bioink channels, (2) cross linking of spheroids containing cells to generate interconnected vessel systems, and (3) biofabrication of free-standing tubular structure onto scaffold or receiving substrate (Hoch et al. 2014). Bioink formulated based on acellular material works nicely with elevated temperature or organic solvent (Elomaa and Yang 2017). But, it is supposed to be challenging as compared to cellular-based bioink formulation and processing. In general, 3D bioprinting using cell-laden materials has many advantages. It works well with increase the initial cell loading density and maintains homogeneity in cells distribution pattern, irrespective of single or multiple cell types (Elomaa and Yang 2017). The present state of art of artificial vessels fabrication is resulted from the continuous efforts, which has been

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going on since the work of Kesari and colleagues for construction of tubular hydrogel structure (based on calcium chloride alginate formulation) through additive manufacturing process using inkjet bioprinting (Kesari et al. 2005). The main reason being used CaCl2 as ionic crosslink additive is to facilitate the passage of cells through printer nozzles. After a couple of year, in 2008–2009, it was demonstrated that by ejecting alginate droplets into a solution of CaCl2, the hydrogel spheroids contain cells get partially solidified, and facilitated the fabrication of tubular semisolid structure (Nakamura et al. 2008; Nishiyama et al. 2009). In the same year, Norotte et al. (2009), developed rapid, scalable, scaffold-free, extrusion-based technique. In this process the targeted vascular tissue is developed by biological self-assembly process. Various vascular cells types, such as fibroblasts and smooth muscle cells, are assembled into discrete units in the form of spheroids or cylinders of desired diameter, ranging from 300 to 5000 μm. These cells are fabricated by additive technology by layer by layer onto agarose rods as a moulding template, which are ultimately developed into single or double-layered vascular tubes (Norotte et al. 2009) and used in tissue engineering for the biofabrication of different shape and form of multi-layered vessels with a complex network like structure of interest (Chua and Yeong 2015). Immediately, after a year, electrospinning and fused deposition modelling techniques were developed biological fabrication of tubularshaped vascular tissue onto poly-L-lactide (PLLA) or poly-epsilon-caprolactone (PCL) scaffold (Centola et al. 2010) releasing heparin (Zeng et al. 2018; Centola et al. 2010). Heparin, releasing PLLA scaffolds are created by electrospinning in a tubular shape. Mostly, the tubes are then strengthened with single coil of PCL on the outer layer to improve mechanical properties. The scaffolds are seeded with human mesenchymal stem cells assed for their suitability from morphological, cell viability, and mechanical tensile strength point of views (Centola et al. 2010). In the same year, a group of scientists developed technique for direct printing of tubular vascular tissues with high viability through extrusion-based technology. In this process hyaluronan hydrogels cross-linked with polyethylene glycol were used as scaffold (Chua and Yeong 2015; Skardal et al. 2010). In 2015, 3D printing technology was developed for biofabrication of scaffold free tubular tissues. The structural and functional conformations of such vascular tissues were made by implanting into abdominal aortas of rats (Itoh et al. 2015). Still, research on fabrication methodologies are in progress to apply in tissue engineering investigations (Hoch et al. 2014; Elomaa and Yang 2017; Pinnock et al. 2017).

4.7.2

Artificial Liver Through 3D Bioprinting

Liver is the largest organ in the human body and operates many metabolic, endocrine, and exocrine functions to have control over the body and sustaining life. In spite of its excellent regenerative capacity, damage caused by chronic liver diseases or viral infection may cause to permanent loss of liver function. About 2 million deaths per year are due to the liver failure (Asrani et al. 2019).

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Mostly, liver transplantation is performed on a patient when a patient seriously suffers from completely damage of liver. But, its success mainly depends on donor’s health condition and status of liver just before transplantation with the risk of rejection. So, in order to overcome with such problem, tissue engineering is the most needed area of development of artificial fabrication of in vitro liver with lot of challenges (Dhawan et al. 2010). Generally, the 3D bioprinting of liver is processed through the followings sequential steps: (1) development of monolayer additive tissues by self-aggregation of spheroids or biological extrusion on receiver substrate, (2) strategies for designing hollow fibre, (3) bioink holding vat/cartridge, and (4) perfusion beds.

4.7.2.1

Micropatterned 2D and 3D Liver Model

Micropatterning process is a technique to have in vitro control over cell and tissue architecture. It has been emerged as powerful platforms for modelling tissue microenvironments at different scales and complexities. This technique helps in controlling cell shape, position, and multiphase tissue architecture. This technique is presently used in liver tissue engineering to construct in vitro tissue model using primary hepatocytes, hepatic cell lines isolated from tumours or liver slices, and stem cell-derived hepatic cells (Khetani and Bhatia 2008; Hewitt et al. 2007). Tissue engineering technique could able to develop monolayer culture, organoid culture, and co-culture platforms by using culture plates (Griffith et al. 1997; Takayama et al. 2013), commercially available as well (Gaskell et al. 2016), dielectrophoretic micropatterning (Takayama et al. 2001), and physical mask-based additive photopatterning method (Gaskell et al. 2016). But, it has been observed that hepatocytes cultured by such techniques are functionally viable for few weeks (Takayama et al. 2013; Ho et al. 2013), only.

4.7.2.2

3D Bioprinting for Liver Models

3D bioprinting is also applicable for fabrication of artificial liver model development, but with the use of modified conventional bionk. Mostly, laser-based bioprinting, ink-jet bioprinting, and extrusion bioprinting are applicable for liver tissue fabrication. Detailed basic information of these techniques has already been described earlier. Besides, special techniques like “liver-on-chip” and microplatform-based bioreactor are emerging technologies for fabricating 2D and 3D model for liver, which can provide well-defined microenvironment. Microtechnology is a challenging field of research in tissue engineering and liver system development, which can compete with in vivo liver microenvironment and microlevel ultrastructure by using a small number of human cells under 2D and 3D culture condition. Liver-on-Chip Platform Perfusion systems or cell microfluidic platforms have many advantages over static 3D printing model. Through, microfluidic platforms

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one can have control over several conditions such as culture medium, pH, temperature, fluid pressures, cell shear stress, nutrient supply, and waste removal. Through microfluidics technique manipulation and controlling fluids, usually in the range of microliters (10 6) to picolitres (Chua and Yeong 2015; Gao et al. 2018; Hospodiuk et al. 2017) in networks of channels with dimensions from tens to hundreds of micrometres, can be managed. This technique can be used in engineering liver tissues (Kwon et al. 2010; Chung et al. 2005) for mimicking the native and dynamic cellular environment compared to static cell culture system (Andersson and Van Den Berg 2004; Takayama et al. 2001). Through this technique, hepatocytes can easily interact with mesenchymal cells, stellate cells, Kupffer cells, macrophages, and lymphocytes (Saxena et al. 2015; Geerts 2001), with the enhancement of liver function (Prot et al. 2011). Variety of biocompatible polymers has been used as starting materials for fabrication of stable matrices (scaffolds) for the growth and proliferation of primary hepatic cell lines. The scaffolds are used as matrix for the cell culture of the differentiated hepatocyte-derived carcinoma cell line. Now it could be possible to fabricate artificial liver-like solidified tissues, which can mimic the in vitro liver from both structure and functional point of views. Recently, Human Genome and Stem cell Research Center (HUG_CELL), University of Sao Paulo (USP), developed hepatic organoids (mini-liver) that can perform all of the liver’s typical functions, such as vital protein metabolism, acting as storage house for vitamins, and secretary bile. This tissue was developed by using blood cells (Goulart et al. 2019). The in vivo liver-like structure can be produced in the laboratory within a period of 90 days. At the beginning, the blood cells subjected to induce pluripotency characteristic of stem cells. At this stage the blood cells are known as induced pluripotent stem cells (iPSCs). This technique was developed by Shinya Yamanaka for which he was awarded Nobel Prize for medicine in 2012. In the subsequent step, the spheroids are then mixed with bioink, and process for post-printing. The maturation of tissue onto scaffold takes about 18 days.

4.7.3

3D Bioprinting of Artificial Bone

By means of 3D technology, it could be possible to fabricate bone-like porous structure customized to the patient and specific clinical conditions. This is designed by layer-by-layer additive manufacturing technology guided by computer-aided design (CAD). The bone graft or scaffolds with complex shapes identified in patients via medical imaging techniques such as computed tomography (CT) and magnetic resonance imaging (MRI) can be manufactured (Reichert et al. 2011; Mota et al. 2015). Generally, polymers like chitosan, collagen, and polycaprolactone are combined with bioceramic particles and used for bone scaffolds fabrication with good osteoinduction properties. Besides, 3D bone construction should have also provision

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of blood vessels to support new bone formation. The scaffold obtained through 3D printing can be used in biomolecular applications ranging from customized medical implant design to tissue engineering (Sun et al. 2004; Ciocca et al. 2009). The scaffolds or artificial constructs can be designed with predefined micro- as well as macrostructure (Yang et al. 2002). Mainly, pore size, orientation, and surface chemistry are regulated through microstructure constructs in order to print personalized bone grafts customize to the patient and specific clinical condition.

4.7.4

Artificial 3D Dental Tissues

Presently, nonbiological dental implants are the most common practice to replace damaged tooth within short period. The artificial tooth gives natural appearance and to some extend manage the regular function of tooth for chewing solid or semisolid or soft food materials. But, this short of clinical practice (differences in physical and physiological properties) brings peri-implantitis with alveolar bone loss (Jung et al. 2008; Cassetta et al. 2015). About 64% of donors underwent alveolar bone loss within 2 months of dental implant surgery (Cassetta et al. 2015). In order to overcome such problem, 3D bioprinting technologies have emerged as new tissue engineering for biofabrication of alveolar bone and periodontal ligaments (Nakao et al. 2007; Zhang et al. 2009). Scaffold-based approaches are also in progress for artificial regeneration with tooth cells (Yang et al. 2015; AbdulQader et al. 2015). The main problem being faced by tissue engineers is to mimic in vivo dental tissues characters (specific shape, dentin, and enamel) of specific requirement for patient specification (Smith et al. 2017; Chalisserry et al. 2017). Factually, scaffold-based approaches have limitation in controlling the size and shape of tooth. Besides, scaffolds-based approaches cannot solve the problem of dental tissue regeneration as in “in vivo” system (Obregon et al. 2015).

4.7.5

Artificial Ovary Through 3D Bioprint

Biofabrication of artificial ovary is an emerging trend in tissue engineering field. Artificial ovary is implantable in place of defective in vivo ovary to regenerate fertility potential in women to become pregnant (Liverani et al. 2019). Recently, electrospinning technique has been applied for developing porous scaffolds to support of ovarian follicles growth. The basic principle of this technique is based on to generate high electric potential between two electrodes with opposing polarity. This phenomenon develops surface tension of a polymer solution and thus enables the solution to vaporize completely and form a fibrous structure. Electrospinning is a simple, feasible, and efficient technique employed to obtain nanofibres for fabricating scaffolds. The process

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Fig. 4.10 Application of electrospinning technique for developing nano-porous scaffold for artificial ovary

does not require the use of coagulation chemistry or high temperatures to produce solid threads from solution (Fig. 4.10). In vivo restoration of endocrine function in artificial ovary or scaffold is most challenging aspect of reproductive tissue engineering field. Still, it is needed to develop more optimized scaffold, better transplantation techniques to prevent postsurgical ischaemia, and genetic safety are essential for clinical application with safety and security. Besides, better understanding of mechanical and biochemical properties of the ovary and folliculogenesis after cryopreservation, transplantation with or without scaffold, and development of sophisticated in vivo imaging techniques of transplanted artificial ovary are needed for successful clinical trials.

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Ozbolat IT (2015) Bioprinting scale-up tissue and organ constructs for transplantation. Trends Biotechnol. 33(7):395–400 Pinnock CB, Xu Z, Lam MT (2017) Scaling of engineered vascular grafts using 3d printed guides and the ring stacking method. J Vis Exp 2017:121 Prot JM, Aninat C, Griscom L, Razan F, Guillouzo CG, Corlu A, Brochot C, Legallais C, Leclerc E (2011) Improvement of HepG2/C3a cell functions in a microfluidic biochip. Biotechnol Bioeng 108:1704–1715 Rayate A, Jain PK (2018) A review on 4D printing material composites and their applications. Mater Today Proc 5:20474–20484 Reichert JC, Wullschleger ME, Cipitria A et al (2011) Custom-made composite scaffolds for segmental defect repair in long bones. Int Orthop 35:1229–1236 Saxena V, Nyberg L, Pauly M, Dasgupta A, Nyberg A, Piasecki B, Winston B, Redd J, Ready J, Terrault NA (2015) Safety and efficacy of simeprevir/sofosbuvir in Hepatitis C-infected patients with compensated and decompensated cirrhosis. Hepatology 62:715–725 Shav D, Einav S (2010) The effect of mechanical loads in the differentiation of precursor cells into mature cells. Ann N Y Acad Sci 1188:25–31 Singh D, Thomas D (2018) Advances in medical polymer technology towards the panacea of complex 3D tissue and organ manufacture. Am J Surg 217(4):807–808 Skardal A, Zhang J, Prestwich GD (2010) Bioprinting vessel-like constructs using hyaluronan hydrogels crosslinked with tetrahedral polyethylene glycol tetracrylates. Biomaterials 31:6173–6181 Smith EE, Zhang W, Schiele NR (2017) Developing a biomimetic tooth bud model. J Tissue Eng Regener Med 11(12):3326–3336 Stolberg S, McCloskey KE (2009) Can shear stress direct stem cell fate? Biotechnol Progr 25:10–19 Asrani SK et al (2019) Burdern of liver diseases in the world. J Hapatol 70(1):151–171 Sun W, Darling A, Starly B, Nam J (2004) Computer-aided tissue engineering: overview, scope and challenges. Biotechnol Appl Biochem 39:29–47 Takayama S, Ostuni E, LeDuc P, Naruse K, Ingber DE, Whitesides GM (2001) Subcellular positioning of small molecules. Nature 66:1016 Takayama K, Kawabata K, Nagamoto Y et al (2013) 3D spheroid culture of hESC/hiPSC-derived hepatocyte-like cells for drug toxicity testing. Biomaterials 34:1781–1789 Tekin E, Smith PJ, Schubert US (2008) Inkjet printing as a deposition and patterning tool for polymers and inorganic particles. Soft Matter 4:703 Thomas DJ (2016) Could 3D bioprinted tissues offer future hope for microtia treatment? Int J Surg 32:43–44 Wired (2016). http://www.wired.co.uk/article/heart-chip-harvard-sensors Xu T, Binder KW, Albanna MZ, Dice D, Zhao W, Yoo JJ, Atala A (2012) Hybrid printing of mechanically and biologically improved constructs for cartilage tissue engineering applications. Biofabrication 5:015001 Yang S, Leong K, Du Z, Chua C (2002) The design of scaffolds for use in tissue engineering. Part II. Rapid prototyping techniques. Tissue Eng 2002; 8:1–11 Yang JW, Zhang YF, Sun ZY (2015) Dental pulp tissue engineering with bFGF-incorporated silk fibroin scaffolds. J Biomater 30(2):221–229 Zeng M, Jin S, Ye K (2018) Tissue and organ 3D bioprinting. SLAS Technol Transl Life Sci Innov. https://doi.org/10.1177/2472630318760515 Zhang W, Abukawa H, Troulis MJ (2009) Tissue engineered hybrid tooth-bone constructs. Methods 47(2):122–128 Zhang Z, Jin Y, Yin J, Xu C, Xiong R, Christensen K et al (2018) Evaluation of bioink printability for bioprinting applications. Appl Phys Rev 5(4):041304

Chapter 5

Biopharmaceuticals: New Frontier

5.1

Introduction

Biopharma is the application of living organism or active ingredient extracted from biological system from its original form or genetically modified form to prevent, relief, or treat diseases. Biopharmaceutical companies or life science industries manufacture drugs from biological components of substances, known as biologics or biological medications, that are developed from blood, proteins, viruses, and living organisms. Variety of genetically modified host cells for the extraction of biotherapeutic molecules are derived from living sources such as plants, animals, mammals, insects, and microbes. The first FDA approved biotherapeutic molecule was recombinant human insulin (rHI, trade name Humulin) was developed by Eli Lily in 1982. Introduction of biopharmaceuticals has brought revolution in drug therapy by saving the lives of thousands patients suffering from cancer, diabetes, cardiovascular disease, immune diseases, and other health problems which cure would have been question through conventional pharmaceutical drugs. Biopharmaceuticals are analogous to natural compound present in most of the animate system. Botox, Humira, Lantus, Enbrel, etc. are the most popular biopharmaceutical products dominating the global medicine market. Some of the important biologic drugs have been used widely are briefed below: Lantus (Insulin Glargine) It is a long-acting form of human insulin for controlling type 2 diabetes, and applicable to both adult and children of six year above. It is an aqueous soluble drug at neutral pH. Insulin glargine is a recombinant human insulin analogous. After injection into the subcutaneous tissue, it gets precipitate by mixing with body fluid at neutral pH. The microprecipitate form of insulin glargine releases in sustainable pattern, up to 24 h. Lantus is produced by recombinant DNA technology. Non-pathogenic laboratory strain of Escherichia coli (K12) is used as host system. Insulin glargine differs from human insulin in that the amino acid asparagine at position A21 is replaced by © Springer Nature Singapore Pte Ltd. 2020 B. K. Behera et al., Competitive Strategies in Life Sciences, New Paradigms of Living Systems 1, https://doi.org/10.1007/978-981-15-7590-7_5

157

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5 Biopharmaceuticals: New Frontier

glycine and two arginines are added to the C-terminus of the B-chain. Lantus Solo STAR is manufactured by Sanofi, located in Yizhuang, Beijing. Humira (Adalimumab) It is clinically used for rheumatoid arthritis (RA) and other autoimmune diseases like psoriasis and Crohn’s disease. Besides, Humira (adalimumab), Remicade (Infliximab), Rituxan (rituximab), and Enbrel (etanercept) are also in use for RA. The drugs ending with “-mab” are from monoclonal antibodies. Humira is a tumor necrosis factor-α blocker or TNF blocker. Humira is manufactured by Puerto Rico (Abbott Laboratories’ new Barceloneta). Herceptin (Trastuzumab) It is mainly used for treatment of HER-2-positive breast cancer and HER-2-positive metastatic stomach cancer. Basically, it is a monoclonal antibody. The active substance trastuzumab is produced in recombinant Chinese hamster ovary cells using a serum free medium. Herceptin is marketed by Genentech, USA; in Japan by Chugai and internationally by Roche. Avastin (Bevacizumab) Avastin (bevacizumab) and Lucentis (ranibizumab) are widely used in ophthalmology, specifically, to cure age-related macular degeneration. Lucentis is a medication solely for the eye; whereas Avastin can also be used in certain breast, colorectal, kidney, lung, brain, and ovarian cancers. Both drugs are belonging from monoclonal antibody. Botox (Onabotulinumtoxina) Botox is a neurotoxic protein produced by a bacterium known as Clostridium botulinum. On the basis of diseases caused and application, botulinum toxin can be grouped into seven types (generally known as A-G). Types A, B, E, and F are pathogenic in nature; types C-G are rare in nature. Botulinum toxin types A and B are used in medicine to treat various muscle spasms. Botox is most popular in cosmetic surgery for reducing facial wrinkles. Botox is also used to treat neck spasms (cervical dystonia), excessive underarm sweating (hyperhidrosis), migraines, and loss of bladder control due to nerve damage caused by multiple sclerosis (MS) or spinal cord injury. Etanercept (ETN) Etanercept (brand name-Enbrel) is an expensive drug used for the treatment of rheumatoid arthritis. It is also used to treat psoriatic arthritis, ankylosing spondylitis, and psoriasis. It is a soluble protein that inhibits tumor necrosis factor (TNF), a proinflammatory cytokine. ETN is synthesized in Chinese hamster ovary (CHO) by recombinant DNA technology as a fusion protein, with a fully human TNFRII ectodomain linked to the Fc portion of human Ig G1. The active substance is manufactured at Boehringer Ingelheim Pharma, at Germany or at Amgen Rhode Island, USA and Amgen and Wyeth Pharmaceuticals, a division of Wyeth, market Enbrel in North America.

5.2 Biological Product Definitions

159

Table 5.1 The top 20 best selling biopharmaceuticals of 2020 No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Trade name Humira Revlimid Opdivo Harvoni Prevnar 13 Avastin Herceptin Soliris Tecfidera Orkambi Entresto Rituxan Enbrel Remicade Xtandi Januvia Keytruda Eliquis Eylea Triumeq

Drug Adalimumab Lenalidomide Nivolumab Ledipasvir and sofosbuvir Sterile suspension of saccharides of the capsular antigens of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F Bevacizumab Trastuzumab Eculizumab Dimethyl fumarate Lumacaftor/ivacaftor Sacubitril/valsartan Rituximab Etanercept Infliximab Enzalutamide Sitagliptin Pembrolizumab Apixaban Aflibercept Abacavir/dolutegravir/lamivudine

The growth of biopharmaceutical companies is in expansion state. In 2015, the world market for biopharmaceuticals was about $176.9 billion, and within a span of a year it was raised to $192.2 million. It is forecasted that by the end of 2021 it will capture market with the value of $291 billion (https://gaeu.com/artiklar/ biopharmaceuticals-new-frontier/). Efficacy of biologic drugs is more as compared to traditional pharmaceuticals. Patients suffering from cancer, diabetes, cardiovascular disease, immune diseases, and other chronic diseases have been getting relief without many side effects. Biopharmaceutical drugs are designed for target attack to give permanent relief to patients. So, biopharmaceutical drugs are grabbing global market in an exponential manner. Many biopharmaceuticals have been gaining recognition as blockbusters having sales more than $ billion per annum (Table 5.1).

5.2

Biological Product Definitions

What Is Biological Product? Biological therapeutic products are derived from living sources (plants, animals, cells, mammalian cells, insects, and microbes) either from its original form or genetically modified form. The FDA approved (or any other international regulatory body) and are used to diagnose, prevent, treat, and cure

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disease and medical conditions. The manufacturing process of biologics is highly complex and expensive.

5.2.1

Reference Product and Biosimilar

A reference biologic drug is approved by FDA, against which a proposed biosimilar product is compared. On the basis of fully safety and effectiveness data, a biosimilar is compared to and evaluated against reference product to ensure that the product is clinically similar to a reference biologic.

5.2.2

Highly Similar

Often a word “highly similar” is used to indicate that a biosimilar is structurally and functional basis to the reference product by extensive analysis.

5.2.3

Interchangeable Product

It is a biosimilar product that meets additional requirements outlined by Biologic Price Competition and Innovation Act. This type of biosimilar shows the character of interchangeable with same clinical result as the reference product in any given patient. A manufacturer dealing with an interchangeable product is supposed to provide additional information to show that an interchangeable product is expected to produce the same clinical result as the reference product in any given patient.

5.2.4

Biosimilar vs. Generic Drugs

Biosimilar drugs and generic drugs are very different, mainly because while generic drugs are similar to the original in chemical composition, whereas biosimilar drugs are highly similar to a reference biologic drug. For example, the active ingredients of generic drugs are similar to the branded drugs. Generic drugs are bioequivalent to the brand name drug. By construct, biosimilar manufacturers must demonstrate that the biosimilar is highly similar to the reference product, except for minor differences in clinically inactive components.

5.3 Discovery Process and Development

5.3

161

Discovery Process and Development

Highly skilled research and survey are necessary for the discovery and development of biologic drugs. Biologic drugs are heterogeneous therapeutic biomolecules which include a wide variety of products derived from human, animal, or microorganisms by using biotechnology. Biologics are clinically used for variety of diseases which are behind the control of traditional pharmaceuticals. Since the last two decades biologic drugs have been capturing global market due to their high efficacy in curing diseases (Tables 5.2 and 5.3). The biopharmaceuticals are highly cost oriented, and take prolong time for design and discovery. Lot of differentiations are noticed between biopharmaceutical and traditional pharmaceuticals, both in structure and function. Basically, biopharmaceuticals are high molecular weight complex compounds as compared to traditional generic drugs. The best example to understand biopharmaceutical structural complexity is to compare it with a fighter plane having thousands of parts with manifold functions. Whereas traditional generic drug is like a plastic plane with limited function and structural simplicity (Bui et al. 2015). The developmental process of new biologic drugs, at initial stage, is associated with many problems like availability of volunteers in different geographical location, side effect of drug under trial, fewer hospitalization, improvement of life standard, competitive marketing, etc. In order to keep pace with time line development biotechnologists, biopharmacologist and life scientists have been involved in advanced research and development to find out novel and risk-free biologic drugs for saving life from the diseases not curable by traditional generic medicines. Today, biologic drugs have made great challenge in developing better option for treatment of lifethreatening diseases like Dengue fever, Leishmaniasis, and Malaria like highly infectious diseases. Presently, more than 200 biologic drugs and vaccines are in use for giving relief to patients, worldwide. Under the new regulatory acts, the process for developing complex innovative biopharmaceuticals is more challenging and complex than ever. It requires intensive survey and research, heavy budget, high risks, and approval from world regulatory agency (like FDA), before launching in competitive global market (Fig. 5.1). It has been roughly calculated that the average cost for developing biologic drug is about $26 billion (Joseph et al. 2016). On the basis of recent market survey, it has been understood that a biologic drug design and development need minimum budgetary provision of about $2.6 billion. This includes the cost of failures of hundreds of therapeutic molecules that may be screened and assessed early in the R&D process (Joseph et al. 2016). This estimation was based on Tuft’s analysis by taking final budgetary expenses of 10 pharmaceutical companies for the development and marketing of 106 randomly selected drugs during the period of 1995–2007 (Joseph et al. 2016). About a period of a decade is needed to develop and market a biologic drug.

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Table 5.2 List of some popular biologic drugs on global market S. No. 1

Brand name Humira

Generic name Adalimumab

2

Rituxan

Rituximab

3

Enbrel

Etanercept

4

Herceptin

Trastuzumab

5

Avastatin

Bevacizumab

6

Remicade

Infliximab

Brief information Rheumatoid arthritis, plaque psoriasis, Crohn’s disease, ulcerative colitis, ankylosing spondylitis, psoriatic arthritis, polyarticular juvenile idiopathic arthritis Manufacturer: AbbVie, an Abbott Laboratories spinoff Global Sales in 2017: $18.4 billion Launch date: 2002 It was developed by IDEC Pharmaceuticals under the name IDEC-C2B8. Rituxan is currently co-marketed in the USA by Biogen Idec and Roche subsidiary Genentech Indication: Non-Hodgkin, lymphoma, chronic lymphocytic leukaemia, rheumatoid arthritis Manufacturer: Roche Global sales in 2017: $9.2 billion Generic name: Rituximab Launch date: 1997 It was developed by researchers at Immunex. Today the drug is co-marketed in North America by Amgen and Pfizer, by Takeda Pharmaceuticals in Japan, and by Wyeth in the rest of the world Indication: Rheumatoid arthritis, plaque psoriasis, psoriatic arthritis Manufacturer: Pfizer/Amgen Global sales in 2017: $7.9 billion Launch date: 1998 It was developed by Genentech, now a Roche subsidiary, and UCLA’s Jonsson Comprehensive Cancer Center Indication: HER2+ breast cancer Manufacturer: Roche Global Sales in 2017: $7.4 billion Launch date: 1998 It was launched in 2004, Genentech’s Avastin was one of the most expensive drugs on the market Indication: Breast, colorectal, kidney, non-smallcell lung, glioblastoma, ovarian cancers Manufacturer: Roche Global sales in 2017: $7.1 billion Launch date: 2004 It was originally developed by Centocor Ortho Biotech, which is now Janssen Biotech, a Johnson & Johnson subsidiary Indications: Rheumatoid arthritis, Crohn's disease, ankylosing spondylitis, psoriatic arthritis, plaque psoriasis, ulcerative colitis Manufacturer: Johnson & Johnson/Merck & Co. (continued)

5.3 Discovery Process and Development

163

Table 5.2 (continued) S. No.

Brand name

Generic name

7

Lantus

Insulin glargine [rDNA origin] injection

8

Neutropenia

Pegfilgrastim

9

Avonex

Interferon beta-1a

10

Lucentis

Ranibizumab

Opdivo

Nivolumab

Keytruda

Pembrolizumab

Eylea

Aflibercept

Xarelto

Rivaroxaban

Brief information Global sales in 2017: $7.1 billion Launch dates: 1998 It was developed at Sanofi-Aventis's biotechnology research center in Frankfurt-Höchst, Germany Indication: Diabetes Manufacturer: Sanofi Global sales in 2017: $5.7 billion Launch date: 2000 It is related to cancer chemotherapy Manufacturer: Amgen Global Sales in 2017: $4.7 billion Launch date: 2002 It is marketed in the USA by Biogen Idec and by Merck under the brand name Rebif Germany’s Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB and CinnaGen Company cloned Interferon beta-1a and since 2006 the drug has been sold as CinnoVex, a biosimilar, in Iran Indication: Multiple sclerosis (MS) Manufacturer: Biogen Idec Global sales in 2017: $2.1 billion Launch date: 1996 Developed by Genentech, ranibizumab, an injectable, is marketed in the USA by Genentech and by Novartis outside the USA Indication: Age-related macular degeneration Manufacturer: Roche, Novartis Global sales in 2017: $1.5 billion Launch date: 2006 Bristol Myers Squibb, Ono Pharmaceutical. It is a prescription medicine used to treat advanced stage lung cancer (called non-small cell lung cancer). Nivolumab is a human monoclonal antibody that blocks the interaction between PD-1, PD-L1, and PD-L2 Merck & Co, Pembrolizumab is a highly selective humanized monoclonal IgG4 antibody directed against the PD-1 receptor on the cell surface Regeneron, Eylea (aflibercept) is used to treat with neovascular (wet) age-related macular degeneration Bayer, Johnson & Johnson; It blocks the activity of certain clotting substances in the blood. Xarelto is used to prevent or treat a type of blood clot called deep vein thrombosis (DVT), which can lead to blood clots in the lungs (pulmonary embolism)

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5 Biopharmaceuticals: New Frontier

Table 5.3 List of FDA approved biologic drugs in 2019 S. No 1

Brand name Ubrelvy

2

Enhertu

3

Dayvigo Caplyta

4

TissueBlue

5

Padcev

6

8

Vyondys 53 Oxbryta Xcopri Givlaari

Voxelotor Cenobamate Givosiran

9

Adakveo

crizanlizumab-tmca

10

Fetroja

Cefiderocol

11

Brukinsa

Zanubrutinib

12

Reblozyl

Luspatercept-aamt

13

ExEm Foam

Air polymer-type A

14

Trikafta

15

Reyvow

Elexacaftor/ ivacaftor/tezacaftor Lasmiditan

16

Scenesse

Afamelanotide

17

Beovu

Brolucizumab-dbll

18 19

Aklief Ibsrela

Trifarotene Tenapanor

20

Nourianz

Istradefylline

7

Generic name Ubrogepant fam-trastuzumab deruxtecan-nxki Lemborexant Lumateperone tosylate Brilliant blue G ophthalmic solution Enfortumab vedotinejfv Golodirsen

Indication To treat acute treatment of migraine with or without aura in adults To treat metastatic breast cancer To treat insomnia To treat schizophrenia Dye used in eye surgery To treat refractory bladder cancer To treat certain patients with Duchenne muscular dystrophy To treat sickle cell disease To treat partial onset seizures To treat acute hepatic porphyria, a rare blood disorder To treat patients with painful complication of sickle cell disease To treat patients with complicated urinary tract infections who have limited or no alternative treatment options To treat certain patients with mantle cell lymphoma, a form of blood cancer For the treatment of anaemia in adult patients with beta thalassemia who require regular red blood cell transfusions A diagnostic agent used to assess fallopian tube patency (openness) in women with known or suspected infertility

For the acute treatment of migraine with or without aura, in adults To increase pain-free light exposure in adult patients with a history of phototoxic reactions (damage to skin) from erythropoietic protoporphyria Treatment of wet age-related macular degeneration To treat irritable bowel syndrome with constipation in adults To treat adult patients with Parkinson’s disease experiencing “off” episodes (continued)

5.3 Discovery Process and Development

165

Table 5.3 (continued) S. No 21

Brand name Ga-68DOTATOC

Generic name Ga-68-DOTATOC

22

Xenleta

Lefamulin

23

Rinvoq

Upadacitinib

24

Inrebic

Fedratinib

25

Rozlytrek

Entrectinib

26

Wakix

Pitolisant

27

Pretomanid

28

Turalio

Pexidartinib

29

Nubeqa

Darolutamide

30 31

Accrufer Recarbrio

32

Xpovio

Ferric maltol Imipenem, cilastatin, and relebactam Selinexor

33

Vyleesi

Bremelanotide

34

Polivy

35 36

Piqray Vyndaqel

polatuzumab vedotin-piiq Alpelisib tafamidis meglumine

37

Skyrizi

Risankizumab-rzaa

38

Balversa

Erdafitinib

39

Evenity

Romosozumab-aqqg

40

Mayzent

Siponimod

41

Sunosi

Solriamfetol

Indication For use with positron emission tomography (PET) for localization of somatostatin receptor positive neuroendocrine tumours (NETs) To treat adults with community-acquired bacterial pneumonia To treat adults with moderately to severely active rheumatoid arthritis To treat adult patients with intermediate-2 or high-risk primary or secondary myelofibrosis To treat adult patients with metastatic non-small cell lung cancer (NSCLC) whose tumours are ROS1-positive To treat excessive daytime sleepiness (EDS) in adult patients with narcolepsy For treatment-resistant forms of tuberculosis that affects the lungs To treat adult patients with symptomatic tenosynovial giant cell tumour To treat adult patients with non-metastatic castration resistant prostate cancer To treat iron deficiency anaemia in adults To treat complicated urinary tract and complicated intra-abdominal infections To treat adult patients with relapsed or refractory multiple myeloma (RRMM) To treat hypoactive sexual desire disorder in premenopausal women To treat adult patients with relapsed or refractory diffuse large B-cell lymphoma To treat breast cancer To treat heart disease (cardiomyopathy) caused by transthyretin mediated amyloidosis (ATTR-CM) in adults To treat moderate-to-severe plaque psoriasis in adults who are candidates for systemic therapy or phototherapy To treat adult patients with locally advanced or metastatic bladder cancer To treat osteoporosis in postmenopausal women at high risk of fracture To treat adults with relapsing forms of multiple sclerosis To treat excessive sleepiness in adult patients with narcolepsy or obstructive sleep apnea (continued)

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5 Biopharmaceuticals: New Frontier

Table 5.3 (continued) S. No 42

Brand name Zulresso

Generic name Brexanolone

43

Egaten

Triclabendazole

44

Cablivi

Caplacizumab-yhdp

45

Jeuveau

prabotulinumtoxinAxvfs

BASICRES EARCH

DRUG DISCOVERY

PRECLINICAL TRIALS

Indication To treat postpartum depression (PPD) in adult women To treat fascioliasis, a parasitic infestation caused by two species of flatworms or trematodes that mainly the affect the liver, sometimes referred to as “liver flukes” To treat adult patients with acquired thrombotic thrombocytopenic purpura (aTTP) For the temporary improvement in the appearance of moderate to severe glabellar lines associated with corrugator and/or procerus muscle activity in adult patients

CLINICAL TRIALS PHASE I PHASEII

PHASE III

FDA REVIEWA PPROVAL

POST R&D Phase IV

FDA APPROVED

New

biologic

Potential New

Biologic Drugs

10

100

> 1000

Number of volunteers

Fig. 5.1 Schematic presentation of biologic drug discovery, development, and application

5.3.1

Earlier Process (Preclinical Trials)

Armed with an idea, biotechnologists investigate to find out potential medicine with minimum or zero side effect to patients under clinical investigation. Initially, the investigators conduct experiments in cells, tissues, and animal models (minimum of two variety) to understand the possibility of impact of newly designed medicine on target disease. Target validation is a critical information on drug interaction with pathogenic living moiety, before going for advanced clinical phases trials. Mostly, the beginning phase for a discovery of biopharmaceutical is dedicated for intensive survey to understand the need of therapeutic biomolecule at different geographical location and population density for whom the biologic is to be served.

5.3 Discovery Process and Development

167

This short survey may need a minimum period of three years and is followed by laboratory investigation to perform initial test on animal systems. Thanks to the latest development of molecular medicine and powerful tools to enhance computational capacity which could catalyse the better and quick understanding and finding of human disease at the molecular level. Mostly, biopharmaceutical companies conduct basic research either indecently or in collaboration of educational institute, with the funds of venture capitalist or subsidiary from government. On the whole, the process of biologic drug development is highly integrated and complex, and involved variety of diversified activities (Fig. 5.2). The preclinical research is helpful for generating data information sheet on various aspects of disease under investigation, and leads the biotechnologists to narrow the field of further identification of the therapeutic biomolecule which would be helpful and bring confidence for clinical trials on phase basis. The overall efforts involved in such trials are based on timely identifying the compounds found in nature by means of advanced genetic engineering technique, computational biotechnology data analysis, and protein engineering skill. Even at early stage, the investigators also keep in mind the possible best way of delivering the biologic drugs with body friendly pharmacokinetic drug interaction processes (Fig. 5.3). Once the investigational compound meets criteria for further processing, the company files an investigational new drug application with FDA to pursue clinical testing in humans. At this stage the company initiates patent filing with the U.S. Patent and Trademark Office.

5.3.2

Clinical Research Organization

Clinical research organizations (CROs) play significance role in development of a biopharmaceutical drug. CROs support biopharmaceutical companies for R&D activities during clinical research phases and also find out way and means for trial purpose both in laboratory and on field trials. Laboratory Corporation of America Laboratory Corporation of America is renowned for its worldwide network for providing services on clinical research and development from early-stage research to post-regulatory approval, especially in biopharmaceuticals and life sciences. It has 31 units around the world, well equipped with modern laboratory facilities. In the year the company had about $10.44 billion revenue turnover. One of the recent examples about the successful clinical research is development of a new therapy for complicated Urinary Infection (cUTI) therapy as joint collaboration project with the Chinese Food and Drug Administration (CFDA) and an Indian Pharmaceutical company. Iqvia It is a US-based clinical research organization developed with the merged of Quintiles and IMS Health in 2016. In 2017, its turnover was $9.74 billion. Iqvia

168

5 Biopharmaceuticals: New Frontier

PHASE ITRIALS

PHASE II TRIALS

PHASE III TRIALS FORWARDED FOR

PRECLINICAL TRIALS

PRODUCT APPROVAL

Earlier process for survey

Clinical trial organisatio n (CRO)

Biopharmaceutical manufacturing company

Internaonal Regulatory approval body FDA/ EMA

Partially Funding Agency State government Venture capital

PHASE IV Affiliaon or Approval

R & D Joint collaboration Research institute University

GLOBAL MARKETING Post-Approval R &D monitoring for further improvement

Fig. 5.2 Biologic drug development process of a biopharmaceutical company

has been serving worldwide by covering 92 units. Iqvia dedicates to serve biopharmaceutical and pharmaceutical companies in the areas: clinical development, commercialization, and consulting. Syneos Health The current structural getup and functional diversity of Syneos Health are resulted from merging of INC Research and Ventiv Health, based on USA. Syneos provides

5.3 Discovery Process and Development Fig. 5.3 In vivo interaction of biologic drug in body

169

In vivo Biologic drug interaction

Pharmacokinetic absorption, distribution, metabolism, and excretion (ADME). Bioavailability of biologic

Pharmacodianamic Biochemical and physiologic effects of biologic drugs

biopharmaceutical services in three areas: clinical development, commercialization, and consulting. Besides, this company provides support to biopharmaceutical companies in perusing various phases of clinical trials till the transform of specific biologic drug for FDA approval. Syneos Health also extends services for biostatistics, pharmacovigilance, and patient recruitment. Syneos Health revenue for 2017 was $2.13 billion for the first half of the year. Parexel International Corporation This esteem research funding agency is located in Massachusetts (US) owned by Pamplona Capital Management since 2017. At the beginning stage the company is used to be concentrated on supporting German and Japanese pharmaceutical companies. But, at later phase has extended services worldwide in more than 100 countries. The company mainly focuses on clinical study design and capacity by allowing the linking of healthcare data from different sources. PRA Health Sciences PRA Health Sciences is a US-based agency actively engaged in giving guideline on quality operation of manufacturing process and designing therapeutic biomolecules through integrated research and development process. It has 80 global offices for conducting both early- and late-stage clinical trial processes, as well as the fields of consultancy, technology, strategy, and bio-analysis. Pharmaceutical Product Development It is a private organization based on North Carolina for providing consultancy on pharmaceutical product development (PPD) having 44 units worldwide. The company undertakes contract research on drug development, laboratory, and lifecycle management services. Besides, pharmaceutical product development, the company manages to manufacturing and development of medical devices, coordinate R&D at academic organization level. In addition, the company undertakes the responsibility of new patient enrolment model known as Patient Advantage. The company takes responsibility of reducing the time and cost of conducting clinical trials to identify eligibility of patients, quickly. Charles River Laboratories It is a 71 years-old public limited US company focuses on processing for drugs approval by FDA. The company undertakes works on entire R&D process from

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5 Biopharmaceuticals: New Frontier

basic research to preclinical testing to manufacturing and commercialization under the regulatory acts for Good Laboratory Practice (GLP) and non-GLP. Icon Icon is based in Ireland and offers consulting, process developing, and commercialization services in 37 countries with special emphasis on the Latin America and Asia-Pacific regions. Mainly, Icon covers the areas related to pharmaceutical industry and health care organization. WuXi Apptec It is a China based private organization focuses on pharmaceutical and medical device product development process within shortest possible time. It supports in R&D process for the development and clinical trials of wide ranges of therapeutic molecules, biologics, stem cells culture, and genomic and proteomic aspects of biologic drugs. In addition, WuXi Apptec helps in undertaking R&D projects of biotechnology and pharmaceutical companies. Medpace Holdings It is US based public sector undertaking consulting agency offers full-service on clinical trial of biologic drugs in different phases of development. Filing Application and Clinical Trial Planning In order to ensure safety for clinical trial on volunteers, the biopharmaceutical company serves the preclinical data sheet on investigational new drug (IND) to FDA for the permission of clinical phase trials. The application provides the results of the preclinical work, genomic and proteomic of biologic, detail on how the investigational medicine is thought to work in the body, side effect and possible risks, and overall manufacturing information. The IND also submit detailed clinical trial plan narrating all about the sites selected for trials and authenticity the scientist or technical personalities to under whom the trials will be conducted. All IND are submitted to the FDA and proceed after 30 days if there is no additional feedback or restriction given from the agency. It is also mandatory that in IND application, all clinical trials must be examined by the institutional review board (IRB) or ethics committee (EC) at the institutions where the trials will be conducted. The IRB/EC has the right to reject or suggest for further modification before approving for clinical trials in phase wise. The clinical trial research team consists of doctors, nurses, and clinical investigators to continually monitor trials and provide data sheet to the company for further action plan. In case of any severe clinical causality happen with volunteer under trial, the company has right to seize the further investigation, with prior notice to FDA. Biopharmaceutical companies also ensure that the trials are conducted correctly with authentic data which can be disclosed in public domain at the appropriate time.

5.3 Discovery Process and Development

5.3.3

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Phase I Clinical Trial

This trial is conducted in people for the first time with a small group of healthy volunteers having number