Protein Purification. From Molecular Mechanisms to Large-Scale Processes 9780841217904, 9780841212831, 0-8412-1790-4

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ACS SYMPOSIUM SERIES 427

Protein Purification

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.fw001

From Molecular Mechanisms to Large-Scale Processes Michael R.Ladisch,EDITOR Purdue University

Richard C. Willson, EDITOR University of Houston

Chih-duen C. Painton, EDITOR MallinckrodtMedical,Inc.

Stuart E. Builder, EDITOR Genentech, Inc.

Developed from a symposium sponsored by the Division of Biochemical Technology at the 198th National Meeting of the American Chemical Society, Miami Beach, Florida, September 10-15, 1989

American Chemical Society, Washington, DC 1990

Library of Congress Cataloging-in-Publication Data

Proteinpurification:from molecularmechanismsto large-scale processes MichaelR.Ladisch,editor . . . [et al.]. p.

cm.—(ACS symposium series, ISSN 0097-6156; 427)

"Developed from a symposium sponsored by the Division of Biochemical Technology at the 198th National Meeting of the American Chemical Society, Miami Beach, Florida, September 10-15, 1989." Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.fw001

Includesbibliographicalreferences. ISBN 0-8412-1790-4 1. Proteins—Purification—Congresses. 2. ProteinsBiotechnology—Congresses. I. Ladisch, Michael R., 1950- . II. American Chemical Society. Division of Biochemical Technology. III. Series TP248.65.P76P765 1990 660'.63—dc20

90-35551 CIP

The paper used in this publication meets the minimum requirements of American National Standard for Information Sciences—Permanence of Paper for Printed Library Materials, ANSI Z39.48-1984. Copyright © 1990 American Chemical Society All Rights Reserved. Hie appearance of the code at the bottom of the first page of each chapter in this volume indicates the copyright owner's consent that reprographic copies of the chapter may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per-copy fee through the Copyright Clearance Center, Inc., 27 Congress Street, Salem, MA 01970, for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating a new collective work, for resale, or for information storage and retrieval systems. The copying fee for each chapter is indicated in the code at the bottom of the first page of the chapter. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. PRINTED IN THE UNTIED STATES OF AMERICA

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ACS Symposium Series M. JoanComstock,Series Editor

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.fw001

1990 ACS Books Advisory Board Paul S. Anderson Merck Sharp & Dohme Research Laboratories V. Dean Adams Tennessee Technological University

Michael R. Ladisch Purdue University JohnL.Massingill Dow Chemical Company Robert McGorrin Kraft General Foods

Alexis T. Bell University of CaliforniaBerkeley

Daniel M. Quinn University of Iowa

Malcolm H. Chisholm Indiana University

Eisa Reichmanis AT&T Bell Laboratories

Natalie Foster Lehigh University

C. M. Roland U.S. Naval Research Laboratory

G. Wayne Ivie U.S. Department of Agriculture, Agricultural Research Service

Stephen A. Szabo Conoco Inc.

Mary A. Kaiser Ε. I. du Pont de Nemours and Company

Wendy A. Warr Imperial Chemical Industries Robert A. Weiss University of Connecticut

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.fw001

Foreword The A C S S Y M P O S I U M S E R I E S was founded in 1974 to provide a medium for publishing symposia quickly in book form. The format of the Series parallels that of the continuing A D V A N C E S I N C H E M I S T R Y S E R I E S except that, in order to save time, the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. Papers are reviewed under the supervision of the Editors with the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, verbatim reproductions of previously published papers are not accepted. Both reviews and reports of research are acceptable, because symposia may embrace both types of presentation.

Preface

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.pr001

IROTEIN PURIFICATION: From Molecular Mechanisms to Large-Scale

Processes had its genesis during informal discussions in Houston, Texas, in April 1989, based upon Richard Willson's suggestion of this project The thought was that a relevant cross-disciplinary treatment of largescale protein purification could be possible given the rapid progression of several recombinant protein products from laboratory to large-scale, and the willingness of industry to present some of the fundamental aspects of parameters that have an impact on practical considerations of protein purification. This was reflected, in part, by the papers which had already been submitted and organized as part of the Miami Beach program (Jim Swartz, Genentech, Program Chairman) of the Division of Microbial and Biochemical Technology, now known as the Division of Biochemical Technology. Specifically, two sessions on separations: Large-Scale Protein Purification (M. R. Ladisch and C-D. Painton, chairpersons) and New Advances in Protein Purification (R. G Willson

and S. E. Builder, chairpersons) included approximately 50% of the papers from industrial contributors. Attendance at these sessions reached as many as 250 people. Upon embarking on this project, Dr. Chih-duen Painton (of Mallinckrodt) and Dr. Stuart Builder (of Genentech) were quickly enlisted to assist in the development of this book. The cooperation of all the contributors was tremendous, the response of the reviewers impressive, and the assistance of the ACS Books Department outstanding. The result is that we can bring this volume to you in a timely fashion. MICHAEL R. LADISCH

Purdue University West Lafayette, IN 47907 February 12, 1990

vii

Chapter 1

Large-Scale Protein Purification Introduction 1

2

Richard C. Willson and Michael R. Ladisch 1

Department of Chemical Engineering, University of Houston, Houston, TX 77204 Laboratory of Renewable Resources Engineering and Department of Agricultural Engineering, Purdue University, West Lafayette, IN 47907 Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch001

2

Large scale protein purification is thefinalproduction step, prior to product packaging, in the manufacture of therapeutic proteins, specialty enzymes, and diagnostic products. The art and science of protein purification evolves from laboratory scale techniques which are often adapted and scaled up to satisfy the need for larger amounts of extremely pure test quantities of the product for analysis, characterization, testing of efficacy, clinical orfieldtrials, and, finally, full scale commercialization. Development of appropriate strategies for proteinrecoveryand purification differs from development of separation techniques for more traditional chemical or agricultural processing technologies by the broadness of cross-disciplinary interactions required to achieve scale-up. The uncompromising standards for product quality, as wellasrigorousquality control of manufacturing practices embodied in current good manufacturing practices(cGMP's),provide further challenges to the scale-up of protein purification. Analysis ofelectrokinetic,chromatographic,adsorptive,and membrane separation techniques suggests that if yieldrecoveryisparamount,documented purity is critical, and both must ultimately be attained within certain cost constraints. Examples of purification of insulin andproinsulin,IgM,recombinantinterferon-alpha, interleukins,histidinecontaining peptides,lutenizinghormonereleasinghormone, and bovine growth hormone illustrate conceptual approaches used in successful industrial processes. Bio-separation processes have a significant impact on the economics of producing proteins for animal and human health care products (i). Therecoverysequence for isolating product from a fermentation broth has changed little over the last ten years and consists essentially of (2): 1. removal of insolubles; 2. primary product isolation; 3. purification; and 4. final product isolation. 0097-6156/90/0427-O001$06.00/0 © 1990 American Chemical Society

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch001

2

PROTEIN PURIFICATION

The advent of manufacture of recombinant proteins and peptides for pharmaceutical, diagnostic, and agricultural applications has, however, changed the way in which separation techniques are chosen, purification strategies are developed, and economics applied to purification scale-up (see Chapters 2, 3, and 14). Purification costs are particularly important determinants in production costs of diagnostic reagents, enzymes and animal care products. Even the most promising human pharmaceuticals are ultimately subject to cost constraints. Within this context, purity and product activity are still the primary goal. Removal of trace contaminants that are difficult to detect is becoming a key issue as new recombinant and cell culture production technologies are phased in (Chapter 2). Examples of such contaminants include pyrogens, viruses, and transforming DNA, inaccurately translated or glycosylated forms of the protein, degradation and oxidation products, aggregates and conformational isomers which are similar to the desired product. Process validation is therefore quite complex and requires many different types of analytical procedures as shown in Table I. The detection of trace contaminants also presents many challenges (see, for example, Chapters 2,11, 14, and 15). It is particularly important that anyfractionation-basedanalytical method used in product characterization employ a separation mechanism different from those already used in the purification process. Otherwise, a contaminant which has copurified with the product through the preparative process could escape detection. This concept represents an orthogonal protein separation strategy, also used in large scale processes where several purification steps based on different principles would be used (7). For example, a purification sequence might include ion exchange, hydrophobic interaction chromatography (HIQ, and affinity chromatography. Technical issues for each of these steps are the effect of overload on protein retention (Chapter 7) attaining high throughput at reasonable pressure drop (Chapter 8), prediction of protein retention in HIC as a function of salt type (chaotrope vs. kosmotrope) and salt concentration (Chapter 6), and selection of appropriate affinity chromatography techniques for attaining high final protein purity (Chapters 10 and 11). Novel affinity methods can reduce the number of separation steps by enabling highly selective separation from a relatively impure starting materials. Recent developments in this context include chelating peptide-immobilized metal affinity chromatography for fusion proteins (Chapter 12) and immobilized metal chelates attached to water soluble polymers for use in two phase extraction (Chapter 10). Purification Strategies The primary factors determining a preferred separation method depend on parameters of size, ionic charge, solubility and density as illustrated in Figure 1 (from reference J). This applies to both small molecule and protein separations. Recovery and separation of proteins covers this entire range: i.e., microbial cells (ca. 1 to 5 microns), inclusion bodies (ca. 0.1 to 0.5 microns), protein aggregates (ca. 10 nm to 200 nm), as well as proteins and peptides themselves (less than ca. 20 nm). This book emphasizes protein properties and purification and consequently focusses on chromatography and partitioning in liquid systems. Analogies between traditional chemical separation principles and those applicable to proteins are apparent (Chapter 3). However, the structure and function of proteins results in product molecules which differ from variants by as little as one amino acid out of 200. Separations also need to accomplish removal of other macromolecules (such as DNA and RNA) which could compromise product efficacy at trace levels. This requires a long list of special analytical techniques, many of which are based on use of recombinant technology, to validate product purity and process operation (Table I) (3,4).

Microns(yu)

—

Angstroms (A) — 1

Density

Surface Activity

Solubility

Ionic Charge Vapour Temperature Pressure

Diffusivity

Sise

Primary Factor Affecting Separation

Figure 1.

Macromolecular Range

1000-

Micron Particle Range

Fine M • Particle Range

Liquid Cyclones ' • Gravity Sedimentation -

"Centrifuges -

• Foam and Bubble Fractionation ·

100 ·

1000 — Coarse • Particle Range

-Cloth and Fibre Filters^ Screens ^ *nd Strainers

Downstream processing unit operations as a function of size, and physical properties (reprinted with permission, from reference 5, copyright of The Nature Press, MacMillan Publishers, Ltd., allrightsreserved).

Ionic Range""

100-

-4

• Ultracentrifuges -

^

• Solvent Extraction

> Distillation/Freese Concentration-

• Ion Exchange"

—Electrodialysisn.

Dialysis —

- Reverse Osmosis —

-Gel Chromatography -

• Ultrafiltration -

Microfilters ·

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch001

Detect contaminating proteins

Detect contaminating proteins

Detect host proteins

Detect contaminants (i.e., antibodies leached from affinity columns); small epitope size makes antibodies poor for variants ("see" small area). Determine terminal sequence of a newly produced protein for comparison against a standard

Liquid chromatography

ELISA

Immunoassays

Amino acid sequence, composition

Objective

Electrophoresis

ANALYTICAL

Methods

Carry out controlled hydrolysis of protein. Analyze for amino acids or peptides using appropriate liquid chromatography techniques. (See Chapter 12 for discussion of lutenizing hormone releasing hormone (LHRG) and His-Trp proinsulin.)

Based on spécifie antigen-antibody reactions (discussed in Chapters 2,11, and 14).

Enzyme Linked Immunosorbent Assay. Competitive binding assay (see ref. 3 for monoclonal antibodies; Chapter 14 for IgM (human) monoclonal antibodies).

Select appropriate stationary phase (recall orthogonal separation principle), inject samples look for more than one peak (examples for insulin, insulin A, insulin Β in Chapter 7; IgM in Chapter 14).

Carry out electrophoresis (examples for bovine growth hormone in Chapter 2, human serum albumin and hemoglobin in Chapter 10, recombinant IFN and IL-1, IL-2 in Chapter 11, signal peptide in Chapter 14; principles discussed in Chapters 15 and 16). Use Coomassie blue. For more sensitive detection follow with destaining, and silver staining to detect protein bands. Also combine silver staining and immunoblotting.

Procedure

Table I. Example: Assessment of Product Purity and Processing Conditions

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch001

Binding of complementary nucleic acid sequences to find specific sequences of DNA or RNA (see reference 4 for background on techniques). These cover a wide range of analytical techniques discussed in biochemistry and biology textbooks (see, for example, ref 4).

Standard Assay. Check for production of anti-virus antibodies by pathogen free mice immunized with product samples.

Detect transforming DNA sequences

Characterization of biophysical properties, and state of aggregation

Detect presence of pyrogens

Detect viral contamination

Hybridization

NMR, MS, Light Scattering, Analytical Ultracentrifugation Size Exclusion Chromatography

Rabbit pyrogen test

Antibody Production Test

Host organism, not producing the recombinant products, put through purification process at full production scale, followed by isolation of contaminants (if any) not eliminated by the purification process. Add radiolabeled contaminants, viruses to crude product to demonstrate their elimination after the product is processed through the purification train. Run product through the purification sequence many times,

Detect proteins, elutingfromcolumn, based on activity, protein content using methods based on different detection principles (see Chapter 7 for example with β-gal/BSA separation).

Detect contaminants due to host

Demonstrate elimination of certain contaminants

Detect if changes occur during purification due to product/stationary phase interactions and other purification operations

Verify that protein recovery is complete

Blank Runs

Tracer Studies

Repeated Processing of product

Protein and activity material balance of eluting peaks from chromatography system

PROCESS

Protease digestion. Reverse phase chromatography of resulting peptides (See Chapter 13 for discussion of proteases used for site-specific cleavage applied to fusion proteins).

Map protein based on analysis and identification of peptide fragments

Tryptic mapping

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch001

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch001

6

PROTEIN PURIFICATION

Process validation is another key consideration in developing a purification strategy. Process validation refers to establishing documented evidence which assures that a specific process will consistently produce a product meeting its predetermined specifications and is based on FDA guidelines (D. Julien, Triad Industries, at Purdue University Workshop on Chromatographic Separations and Scale-Up, October 3,1989). The industrial perspective appears to be to keep the separation strategy as simple as possible (Chapter 2). Genetic engineering can simplify purification by increasing product titer and providing a molecular structure which is in a proactive form or is otherwise modified in vivo to enhance purification efficiency (Chapter 11). Process development should thus include a cross-disciplinary approach which considers the engineering of the organism*s traits to fit scale-up constraints in fermentation or cell culture, as well as in purification. Similarly, purification conditions should be developed, if possible, to help overcome limitations in the organism's protein production and transport mechanisms with particular emphasis on traits which are difficult to alter by genetic manipulation. Some aspects of purification development are so product specific that the necessary skills are best assembled in an industrial setting. Individual purification steps need to be addressed in a generic context so that a fundamental, mechanistic knowledge base for each type of separation technique will ultimately be developed. This type of research is also cross-disciplinary, by definition, given the large number of factors other than the absence of contaminating molecules which impact the definition of purity. Examples are: protein refolding from inclusion bodies; protein secretion and expression in novel host organisms; operational aspects of immunoaffinity chromatography and preparative chromatography of complex mixtures; post-translational modifications and immunogenicity; and process integration and validation. In addition to biotechnology production companies, equipment suppliers and instrumentation companies also benefit from such a knowledge base. These companies have a critical function in developing new separations apparatus, chromatographic adsorbents, analytical instrumentation, and process monitoring and control equipment. New Approaches Through Cross-disciplinary Collaboration Product quality requirements for part-per-million impurity levels has led to new emphasis on high-resolution methods capable of removing subtly-altered forms of the desired product. At the sametime,the prospect of gram- and even kilogram-scale production is driving the application of refined forms of classical large-scale methods to new problems. The need for practical solutions is helping to focus efforts of investigators from many disciplines on complex problems of protein purification. Polymer chemists and biochemists address the longstanding need for materials possessing hydrophilic, protein-compatible surfaces, which are sufficiently rugged to be useful as adsorbents and filtration media. Advances in the chemistry of separations materials, which frequently derive from parallel interests in biomedical device technology, have continually allowed the development of new separation methods. Fundamental studies on understanding mechanisms of protein interactions with their environment have fostered development of new separation schemes and/or improved operating conditions. Two examples given in this book are on the effect of amino acid sequence on peptide and protein partitioning in two phase aqueous systems (Chapter 4) and protein-polyelectrolyte complexation (Chapter 5). The first tool used in development of a modern protein purification method is frequently not a centrifuge orfiltrationapparatus, but a DNA synthesizer. As illustrated by Chapters 12 and 13 of this volume, molecular geneticists and microbial physiologists can help to define the nature of the purification problem. The potential influence of genetic and culture manipulations has rapidly expanded to include not only host-related factors such as expression level

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch001

1. WILLSON AND LADISCH Introduction

7

and cellular location, but also characteristics of the protein molecule which influence its purification behavior (see Table I for illustrations). Increasing attention to trace contaminants has driven the introduction of increasingly sophisticated instruments and bioassay techniques for the monitoring of protein purification processes. Analytical chemists also identify intractable contaminants for potential elimina­ tion from the host genome. Chemists, together with physical biochemists and chemical engineers, are promoting advances in both the understanding and application of electrophoretic and electrokinetic separation techniques (see Chapters 15 and 16). Biochemists, biol­ ogists, and biochemical engineers are the groups most directly involved the development of protein purification methods. As they jointly develop new methods, each group applies its unique collection of skills and experience. Engineers contribute the quantitative simulation and optimization of processes, and understanding of economics, and familiarity with largerscale operations and automated process control (Chapters 2, 3, and 6-10). Biochemists know the sometimes unforgiving properties of proteins and biological materials, possess the accu­ mulated experience of decades of research-scale purifications (Chapter 11 and 14), while biologists understand the utility of biological approaches to what appear at first to be engineering problems (Chapters 12 and 13). Overview of This Volume The book is divided into several distinct sections. Chapters 2 and 3 give overviews of separa­ tion strategies. The subsequent chapters present research results on phase equilibrium behavior in aqueous two phase systems (Chapters 4 and 5). New engineering approaches to analysis of mass transfer and chromatography (Chapters 6-9); affinity based separations (Chapters 10-13); a case study on IgM human monoclonal antibodies (Chapter 14) and electr­ ically driven separations (Chapters 15-16). Strategies for Large Scale Protein Purification. Chapter 2 by S. V. Ho describes the impact of the composition of the process stream as it passes from initial composition tofinalpurity on the efficiency with which different classes of contaminants can be removed by various methods. This results in the division of the overall process of purification into several gen­ eral stages. This division serves to limit the complexity of the design process, as each possi­ ble unit operation is normally useful in only a limited number of stages. Based on his experience in large-scale protein purification, the author highlights some practical considerations which are illustrated with case studies. The case studies illustrate the surprising effectiveness of scaleable, classical methods such as precipitation and extraction when cleverly applied and carefully optimized. This is a theme which is further illustrated in later chapter, which has important implications for the development of truly large-scale processes. The Challenge of Separations in Biotechnology. In Chapter 3 by Ε. N . Lightfoot, the initial concentration step is presented as dominating processing costs, with many methods of initial concentration become progressively less economic at lower product concentration. The operating costs of these processes are proportional to the increasing volume of inerts pro­ cessed. Adsorptive separation processes can escape this unfavorable trend. The author addresses the balance between the essential mass transfer functions of adsorp­ tive separation equipment, and the closely-related momentum transfer processes which govern drag and pressure drop. Differences between mass and momentum transfer can be used to optimize the former without unnecessarily magnifying the latter. The design of adsorptive protein separation processes is also discussed.

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch001

8

PROTEIN PURIFICATION

The Effect of Amino Acid Sequence on Peptide and Protein Partitioning in Aqueous TwoPhase Systems. Chapter 4 by A. D. Diamond, K. Hu, and J. T. Hsu presents a structural approach for the prediction of partition coefficients of peptides and proteins in aqueous twophase systems. By analyzing the partition behavior of many pairs of dipeptides of the same composition but opposite sequences, the authors regress a set of parameters characteristic of the influence of each residue type of partition behavior. These parameters can be used in a group-contribution equation (with corrections for the effects of N - and C-termini) to predict the partition coefficient of a peptide from its sequence alone. The method performs well for di- and tripeptides similar to those from which the parameters were regressed. It also predicts qualitatively the dependence of partition coefficient on structure for larger molecules. While the method in its current state cannot accommodate the effects of secondary and tertiary structure, it represents an initial approach to prediction and correlation of partition data on a structural basis. Further development of such predictive methods will be essential if the design of protein separations is to be put on the rational, predictive basis characteristic of more established processes. Protein Separation via Poly electrolyte Complexation. Chapter 5 by M . A. Strege, P. A. Dubin, J. S. West, and C. D. Flinta analyzes the complexation and coacervation of proteins by the polycation poly(dimethylallylammonium chloride. The authors demonstrate the existence of stable, soluble intrapolymer complexes, and estimate the number of protein molecules bound per polymer chain as a function of pH andfreeprotein concentration. They also point out that the selectivity of precipitation by polyelectrolyte coacervation can be surprisingly high. Finally, they demonstrate that the process appears to be sensitive to the nonuniform distribution of charges on a protein's surface, rather than simply to its net charge. Polyelectrolyte coacervation, therefore, may allow the initial steps of a process to achieve a much greater selectivity than has traditionally been expected. Mechanisms of Protein Retention in Hydrophobic Interaction Chromatography. Chapter 6 by B. F. Roettger, J. A. Myers, F. E. Régnier, and M . R. Ladisch discusses hydrophobic interaction chromatography (HIC) which separates proteins and other biological molecules based on surface hydrophobicity. Adsorption and desorption is influenced by the type of salt employed in the mobile phase, as well as its concentration. HIC differs from reversed phase chromatography in that proteins separated at HIC conditions elute in their active conformation due to mild elution conditions and use of salts which stabilize the proteins. Elution occurs in a decreasing gradient. In comparison, proteins in reversed phase chromatography are adsorbed to a more strongly hydrophobic stationary phase and increasing gradients of organic solvents are required for elution. Conformational changes of the proteins may occur, and can account for different elution orders. This chapter described experimental results which give preferential interactions of ammonium salts with HIC supports as determined by densimetric techniques. Preferential interactions of the ammonium salts of SOJ, C2H3O2, CI" and Γ with the supports and pro­ teins were found to explain adsorption behavior. A predictive equation which relates the capacity factor for a polymeric sorbent to the lyotropic number (i.e., reflects salt type) and salt molality is reported. The result suggests that protein retention can be estimated as a function of salt type and concentration. Separation and Sorption Characteristics of an Anion Exchange Stationary Phase for βGalactosidase, Bovine Serum Albumin, and Insulin. Chapter 7 by M . R. Ladisch, K. L. Kohlmann, and R. L. Hendrickson discuss anion exchange chromatography. Anion exchange media are widely used in the chromatography of proteins, with many examples given throughout this book. The theory for anion exchange chromatography at low concen­ trations is well established. This chapter addresses the impact of adsorption of one protein on

1. WILLSON AND LADISCH Introduction

9

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch001

the retention of another. This occurs at mass overload conditions, which results from operat­ ing in the nonlinear region of competitive adsorption isotherms of at least one of the com­ ponents involved. The Craig distribution model, which has previously been reported in the literature, was found to be a useful starting point in this work. Batch equilibrium studies, carried out with BSA and β-galactosidase show the polymeric, derivatized adsorbent used by the authors to have a relatively high loading capacity (200 mg protein/g dry weight), and that adsorption of β-galactosidase could affect subsequent adsorption of the BSA. The Craig distribution con­ cept would thus suggest that altered peak retention could result for BSA, if the concentration of the β-gal were high enough. Dynamic Studies on Radial-Flow Affinity Chromatography for Trypsin Purification. Chapter 8 by W. C. Lee, G. J. Tsai, and G. T. Tsao discuss a new engineering approach to analysis of mass transfer in radial flow chromatography. The development and application of mathemat­ ical modeling for simulation of radial-flow affinity chromatography is demonstrated. When combined with experiment, the model allows the estimation of parameters governing the pro­ cess, and identification ofrate-determiningsteps. This analysis will be useful in scale-up, and in the development of other separations using this technology. Affinity chromatography may be particularly amenable to improvement by changes in the geometry of solid/liquid contacting devices. This is because the strong, specific protein/adsorbent interactions involved can often achieve a high degree of purification in the equivalent of a single theoretical plate. Even very short liquid paths through the adsorbent bed, therefore, may allow effective separations. The viability of this notion is further illus­ trated by the recent commercialization of membrane-based affinity separations. Impact of Continuous Affinity-Recycle Extraction (CARE) in Downstream Processing. Chapter 9 by N . F. Gordon and C. F. Cooney describes further development and simulation of the Continuous Affinity-Recycle Extraction (CARE) process recently developed in their laboratory (7). Based on solid/liquid contacting in well mixed vessels, this method allows adsorptive purification to be used at an earlier process stage than possible with conventional chromatography, because of its tolerance for particulates and viscous cell debris. Distribution of the adsorbent among several vessels allows adsorption efficiency to approach that of a column of equivalent size. In the present work, the authors describe the extension of CARE to separations based on ion-exchange adsorption, and directly compare the method with column chromatography for the purification of β-galactosidase from crude E. coli lysates. Numerical simulation of the CARE process is used to evaluate the trade-offs among perfor­ mance measures such as degree of product concentration and purification, yield, and throughput. Novel Metal Affinity Protein Separations. Chapter 10 by S. S. Suh, M . E. Van Dam, G. E. Menschell, S. Plunket, and F. H. Arnold discusses two methods on metal-affinity separation recently introduced by the authors. These are metal-affinity aqueous two-phase extraction and metal-affinity precipitation. Both methods can be implemented using the metal chelator iminodiacetic acid (IDA) covalently attached to polyethylene glycol (PEG), but they depend on different mechanisms to achieve separation. Metal-affinity partitioning in aqueous two-phase systems involves the use of PEG molecules singly derivatized with IDA. In a PEG-based aqueous two-phase system, this molecule partitions strongly into the PEG-rich phase. In the presence of metal ions such as Cu(II), selective interactions of IDA-bound copper atoms with proteins containing exposed surface histidine or cysteine residues enhance the partitioning of these proteins into the PEGrich phase. Metal-affinity partitioning is based on similar interactions, but uses bis-chelates

10

PROTEIN PURIFICATION

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch001

chelating two copper atoms at the ends of the PEG chain. Interaction of the chain ends with two different protein molecules produces a crosslink between them leading to precipitation by mechanisms functionally similar to the immunoprecipitation of multivalent antigens with bivalent antibodies. Recovery of Recombinant Proteins by Immunoaffinity Chromatography. Chapter 11 by P. Bailon and S. K. Roy covers practical applications of affinity chromatography. Immunoaffinity chromatography is described as a predecessor to affinity chromatography, with the first well characterized immuno-adsorbent prepared by chemically bonding the antigen ovalbumin to a solid matrix for use in isolating antibodies to ovalbumin (9). This chapter presents an overview, as well as results from experimentation, on the preparation, use, and stability of immunoadsorbents. There appears to be considerable scientific background which is required to obtain a working immunoaffinity column. First the monoclonal antibodies, which bind the target protein, must be selected. However, it is noted that often antibodies which show high affinity in solid phase immunoassays exhibit little or no affinity when immobilized on a stationary phase and vice versa. The practical approach of binding the antibody on a small scale, followed by directly testing the immobilized antibody is suggested. The chapter presents a most useful survey and discussion of procedures for preparing the immunoaffinity supports and gives reference to published procedures and commercially available materials which can be used for this purpose. Residual immunoreactivity, effect of coupling pH, activated group and antibody coupling density, detection and prevention of antibody leaching, stabilization, and even solubilization and renaturation of recombinant proteins are covered in a concise, yet complete manner. The practical matter of the FDA's stringent regulations on testing monoclonal antibodies for polynucleotides, retroviruses, and ecotropic murine leukemia virus is also mentioned. These descriptions, backed up with demonstrated separations of recombinant IFN-alpha, IFN-gamma, IL-l, and IL-2, give insight into an industrial perspective of immunoaffinity chromatography. Chelating Peptide-Immobilized Metal Ion Affinity Chromatography. Chapter 12 by M . C. Smith, J. A. Cook, T. C. Furman, P. D. Gesellchen, D. P. Smith, and H. Hsiung is on the use of genetic manipulation to alter the retention properties of proteins in immobilized metal affinity chromatography (IMAC), by the N-terminal addition of chelating peptides (CP) with high metal affinity is describe is described. Development of the method, which they term chelating peptide immobilized metal affinity chromatography (CP-IMAQ first required the identification of small peptides with the necessary high metal affinity. This was done by screening approximately fifty candidate peptides for retention behavior on IMAC columns, resulting in the selection of one di- and two tripeptides for further study. As E. coli expression often results in addition of an N-terminal methionine residue which could inhibit CP interaction with IMAC supports, the IMAC retention behavior of N-methionyl analogs of the candidate peptides was also studied. Although CP affinity for Co(II) was abolished in the methionyl analogs, they retained nearly full affinity for Ni(II) and (in one case), Cu(II). These results establish the applicability of CP-IMAC to proteins expressed in E. coli. CP-IMAC has been applied to purification of recombinant human insulin-like growth factor-II (IGF-II). A synthetic DNA sequence was used to extend the N-terminus of the protein to include a CP sequence, connected to IGF-II via a specific proteolytic cleavage site. During purification from a crude E. coli lysate, this chimeric protein bound strongly to a Cu(II) IMAC column, along with only a small minority of the contaminating host proteins. After elution by pH change, native IGF-II was liberated from the chimeric protein by enzymatic cleavage to remove the chelating peptide. A second cycle of Cu(II) IMAC efficiently removed the contaminants which had been retained along with the CP-protein in the first

1. WILLSON AND LADISCH Introduction

11

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step. The large change in retention behavior induced in the desired protein by CP removal illustrates a major advantage of the use of removable affinity handles. Site-Specific Proteolysis of Fusion Proteins. Chapter 13 by P. Carter presents a generic issue in the use of gene fusions to assist protein purification-removal of the affinity handle to recover the native sequence. As illustrated by Smith et al. (Chapter 12), affinity handles added by gene fusion can greatly facilitate purification. Even if not exploited as part of the purification strategy (to discriminate against contaminants of constant retention behavior), removal of affinity handles is normally required since foreign sequences may result in immunogenicity. This chapter reviews the state of the art in selective removal of affinity handles by chemi­ cal and enzymatic means. The difficulties which can result from inaccessibility of the cleavage site are described. These range from adventitious cleavage by host proteases to misfolding. Highly-specific proteolytic enzymes which have been employed for selective protein cleavage, noting commercial sources and practical aspects of their use, are surveyed. An example is given by the serine protease subtilisin BPS from Bacillus amyloliquifaciens. The substrate specificity of this enzyme is too broad to be useful for selective removal of affinity handles. However, a mutant enzyme in which the histidine in the catalytic triad was replaced by alanine (H64A) is highly specific for histidine-containing substrates, apparently because the substrate histidine can partially substitute for the missing catalytic group (8). This behavior has been called "substrate-assisted catalysis." The H64A subtilisin mutants are available for research purposes upon request to the authors. Purification Alternatives for IgM (Human) Monoclonal Antibodies. Chapter 14 by G. B. Dove, G. Mitra, G. Roldan, M . A. Shearer, and M . S. Cho gives a case study on purification of monoclonal IgM's from tissue culture of human Β lymphocyte cell lines. The process described gave a purification sequence in which final protein purity was greater than 99%, DNA clearance was greater than 1,000,000 and virus clearance was 100,000 times. Contam­ inants which must be removed from the IgM's include the residual media components (albu­ min, transferrin, insulin and other serum proteins) as well as nucleic acids, viruses, and cellu­ lar products. DNA removal was achieved by passing the DNA containing product stream over an immobilized enzyme column in which DNA hydrolyzing enzymes decrease the size of the DNA from a molecular weight of 1,000,000 to 100,000 to 10,000. This procedure alone increased DNA clearance from lOx (without DNAse digestion) to 10,000x when the treated stream was fractionated over a size exclusion chromatography column. This is but one example of the separation techniques which are discussed in a purification sequence of filtration, precipitation, and size exclusion, anion, cation, hydroxylapitite, and immunoaffinity chromatography. This chapter provides fascinating insights into purification development for a large protein. Analytical, Preparative and Large-Scale Zone Electrophoresis. Chapter 15 by C. F. Ivory, W. A. Gobie, and T. P. Adhi is a comprehensive and readable summary of electrophoretic techniques which integrates key theoretical considerations with clear diagrams and descrip­ tions of basic analytical, preparative, and large-scale electrophoretic systems which separate proteins on the basis of differences in molecular weights, mobilities and/or isoelectric points. Numerous illustrations of these separation mechanisms are given. According to the authors, a convincing demonstration that zone electrophoresis provides high resolution on a large scale will open the way to full scale bioprocessing applications. Samples in the 1 μg to 100 mg range might be handled by electrochromatography while zone electrophoresis may offer significant advantages over other electrokinetic methods at load­ ings of greater than 1 gm. Capillary zone electrophoresis is shown to be able to attain

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PROTEIN PURIFICATION

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efficiencies in excess of 500,000 plates/meter but, unfortunately is limited to microgram size protein samples. This chapter gives the reader a sense of the rapid progress being made in practical large scale applications of electrophoretic separations. This is illustrated by recycle isoelectric focusing and recycle continuous flow electrophoresis, which are techniques that have the potential to process proteins in the 100 g/hr range. The authors make a convincing case that this area of technology has an exciting future. In the meantime, this chapter and the succeeding one bring the reader clear descriptions of the state of the art. Applied Electric Fields for Downstream Processing. Chapter 16 by S. Rudge and P. Todd gives clear descriptions which illustrate principles of how electric fields may be applied to drive or enhance rate processes in downstream processing. These include consideration of thermodynamics at charged interfaces; the mathematics and physical chemistry of surface charge and the double layer, and the electrokinetics in transport processes. The authors present relevant scaling rules and use these rules to delineate physical processes which can occur in a closed system to cause backmixing. Their analysis shows heating is the single most important limitation to electrokinetic scale-up. Approaches to overcome heating and mixing effects are discussed. The scaling of mass transfer in electrophoretic systems compared to chromatographic systems is a particularly interesting part of this chapter. The authors explain why the transport rate in electrophoresis is 1,000 to 10,000 times greater than ordinary liquid chromatography while attaining the same relative equilibrium associated with chromatography. This observation is drawn from comparison of electrophoretic and chromatographic Peclet numbers which reflect the ratios of transport velocity to the rate of diffusive mass transfer. This type of analysis is also incorporated in subsequent discussions of processing applications including demixing of emulsions, cell separations, density gradient column electrophoresis, continuous flow electrophoresis, and analytical applications of electrokinetics for process monitoring. Conclusions Large scale protein purification protocols are moving from the developmental laboratory to the pilot plant and to commercial production. While purity, regardless of cost, may be the goal during the early phases of the product discovery and development process, production economics are a necessary consideration as scale-up is pursued. For chromatographic separations, a preliminary cost estimate must consider stationary and mobile phase costs as well as the impact of throughput and support stability on these costs (JO). Since purity at the commercial scale must usually be the same, if not better, than that initially obtained at the laboratory scale, the economic element becomes a key constraint in choosing large scale purification strategies and optimizing their operational conditions, while maintaining uncompromising standards of product purity. The chapters in this volume present insights, examples, and engineering approaches from industry, and fundamental models and engineering analysis from university researchers with both discussing many novel approaches and exciting new ideas for obtaining high purity products with large scale separations. A cknowledgments One of the authors (ML) acknowledges the support of NSF Grant BCS-8912150 which supported parts of the material in this work. The authors thank A. Velayudhan and G. J. Tsai for their helpful suggestions and comments during the preparation of this manuscript.

1. WILLSON AND LADISCH Introduction Literature Cited 1. 2. 3. 4.

5.

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6. 7. 8. 9. 10.

Knight, P. Bio/Technology 1989, 8, 777. Belter, P. Α.; Cussler, E. L., Hu, W-S. Bioseparations; J. Wiley & Sons: New York, NY, 1988. MacMillan, J. D.; Velez, D.; Miller, L. In Large Scale Cell Culture Technology; Lydersen, B. J., Ed.; Hansen Munich, 1987. Alberts, B.; Bray, D.; Lewis, J.; Raff, M.; Robert, K.; Watson, J. D. Molecular Biology of the Cell, 2nd edition; Garland Publishing: New York, NY, 1989. Atkinson, B.; Mavituna, F. Biochemical Engineering and Biotechnology Handbook; MacMillan Publishers, Inc.: Surrey, England, 1983. Kroeff, E. P.; Owens, R. Α.; Campbell, E. L.; Johnson, R. D.; Marks, Η. I. J. Chromatogr,1989,461,45-61. Pungor, E.; Afeyan, W. G.; Gordon, N. F.; Cooney, C. L. Bio/Technology 1987, 5(6), 604-608. Carter, P.; Wells, J. A. Science 1987, 237, 394. Campbell, D. H.; Lusher, E.; Lerman, L. S. Proc. Nat'l. Acad. Sci. USA 1951, 37, 575-8. Ladisch, M. R. In Advanced Biochemical Engineering; Bungay, H. R. and Belfort, G., Eds.; J. Wiley and Sons: New York, NY, 1987; pp. 219-327.

RECEIVED

March 1, 1990

Chapter 2

Strategies for Large-Scale Protein Purification

Sa V. Ho

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch002

Bio-Products Division, Eastman Kodak Company, Rochester,NY14652-3605

The development of economical processes for purifying proteins from recombinant sources requires the integration of many isolation and purification methods as well as healthy cross-exchange among molecular biology, fermentation, purification and analytical groups. With regards to purification, two schools of thought seem to have emerged. One approach is to start with a highly specific method early on to achieve the required purification in a single step. While aesthetically appealing, in practice this approach lacks a truly high resolution method that is also economical and scalable. The second approach utilizes a cascade of conventional methods to achieve the required protein purity. Here, what methods to use and in what order represent a major task in the process development effort. Based on the author's industrial experience and process information from the literature, some general guidelines for developing optimal purification processes could be established. Examples showing the applicability of this approach will be discussed. The development o f p u r i f i c a t i o n p r o c e s s e s f o r l a r g e - s c a l e manufacture o f p r o t e i n s i s a v e r y c h a l l e n g i n g a c t i v i t y . While p r o t e i n p u r i f i c a t i o n i t s e l f i s a l r e a d y complex, the r e q u i r e m e n t o f " l a r g e s c a l e " imposes a d d i t i o n a l i m p l i c a t i o n s such as economy, s c a l a b i l i t y , and r e p r o d u c i b i l i t y , which s e v e r e l y c o n s t r a i n what can and have t o be done. The f o c u s i n t h i s paper i s not on o p t i m i z i n g ( o r a d v o c a t i n g ) any p a r t i c u l a r p u r i f i c a t i o n method, f o r which one c o u l d c o n s u l t e x p e r t s i n t h e f i e l d o r draw on the w e a l t h o f literature available. R a t h e r , we w i l l t r y t o t a c k l e the c h a l l e n g i n g t a s k o f how t o c o n v e r t a f e r m e n t a t i o n b r o t h o r crude s o l u t i o n i n t o the p u r i f i e d p r o d u c t t h a t s a t i s f i e s a l l the r e q u i r e m e n t s ( c o s t , purity, efficacy, etc.). Only r e c e n t l y have s e v e r a l e x c e l l e n t p u b l i c a t i o n s appeared a d d r e s s i n g v a r i o u s a s p e c t s o f p r o c e s s development f o r l a r g e - s c a l e p r o t e i n p u r i f i c a t i o n ( 1 - 3 ) .

0097-6156/90/0427-0014$06.25/0 © 1990 American Chemical Society

2. HO

Strategies for Large-Scale Protein Purification

15

STRATEGY DISCUSSION

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P u r i f i c a t i o n p r o c e s s development, e s p e c i a l l y f o r r D N A - d e r i v e d products, i s a m u l t i f a c e t e d a c t i v i t y that requires close p a r t i c i p a t i o n o f many s c i e n t i f i c d i s c i p l i n e s as i l l u s t r a t e d i n Figure 1. The s i g n i f i c a n c e and the i n t e r - r e l a t i o n s h i p o f these elements a r e the f o c u s o f t h i s d i s c u s s i o n . PURIFICATION PROCESS. U n l i k e f e r m e n t a t i o n development which b a s i c a l l y i n v o l v e s o p t i m i z i n g the o p e r a t i n g p a r a m e t e r s i n a more o r l e s s s t a n d a r d v e s s e l , t h e r e i s not a s i n g l e t e c h n i q u e o r equipment t h a t i s c a p a b l e o f d e l i v e r i n g p u r i f i e d p r o t e i n i n i t s f i n a l form d i r e c t l y from the f e r m e n t a t i o n b r o t h . So the f i r s t major problem c o n f r o n t i n g a p u r i f i c a t i o n development p e r s o n i s , among the f a i r l y l a r g e number o f t e c h n i q u e s and equipment a v a i l a b l e , n o t o n l y what t e c h n i q u e s s h o u l d be used, but a l s o i n what o r d e r (sequence) as w e l l as how each one i s o p e r a t e d . These t h r e e a s p e c t s a r e i n t e r r e l a t e d and a r e d e t e r m i n e d by the f o l l o w i n g two main f a c t o r s . The f i r s t one i s the n a t u r e o f the s t a r t i n g s o l u t i o n , which can come from many d i f f e r e n t s o u r c e s ( m i c r o b e s , t i s s u e c u l t u r e , s y n t h e s i s ) , w i t h the p r o d u c t i n d i f f e r e n t forms ( s o l u b l e o r as i n c l u s i o n b o d i e s ) and at d i f f e r e n t l o c a t i o n s ( c y t o p l a s m , p e r i p l a s m , i n the b r o t h ) . Each s i t u a t i o n imposes d i f f e r e n t c o n s t r a i n t s and c h a l l e n g e s t o the development e f f o r t . The second key f a c t o r r e l a t e s to the p r o d u c t . A s p e c t s such as p u r i t y , form, i m p u r i t y p r o f i l e , e t c . ( p r o d u c t s p e c i f i c a t i o n ) and c o s t s t r o n g l y d i c t a t e what an a c c e p t a b l e p u r i f i c a t i o n p r o c e s s would be l i k e . F o r both c o s t and p u r i t y r e q u i r e m e n t the spectrum spans from human t h e r a p e u t i c s such as t-PA, i n s u l i n , hGH ( h i g h c o s t , u l t r a p u r i t y ) t o a n i m a l growth hormones (medium c o s t and h i g h p u r i t y ) to i n d u s t r i a l enzymes where the c o s t i s low but p u r i t y i s not so critical. THE ROLE OF FERMENTATION. F e r m e n t a t i o n c o n d i t i o n s and p r o t o c o l s determine the q u a n t i t y as w e l l as the q u a l i t y o f the s t a r t i n g material (concentration, conformation, p u r i t y , impurity p r o f i l e ) , which g r e a t l y impacts downstream p r o c e s s i n g . The o p t i m i z a t i o n ( o p e r a t i n g c o n d i t i o n s , raw m a t e r i a l s used, e t c . ) , t h e r e f o r e , s h o u l d not be based s o l e l y on performance a t the f e r m e n t a t i o n s t a g e . Due to the complex n a t u r e o f the b r o t h , h i g h e r t i t e r measured by HPLC o r SDS-PAGE, f o r example, may not be d i r e c t l y r e l a t e d t o the f i n a l amount o f p u r i f i e d , a c t i v e p r o d u c t o b t a i n e d . A peak on the HPLC o r a band on the g e l may c o n t a i n more than one component, a r i s i n g from s m a l l d i f f e r e n c e s i n amino a c i d r e s i d u e s o r d i f f e r e n t conformations. A l e s s well-known impact o f f e r m e n t a t i o n on p u r i f i c a t i o n i s t h a t s u b t l e m o d i f i c a t i o n s o f the p r o d u c t s r e s u l t i n g i n a m i x t u r e o f c l o s e l y - r e l a t e d compounds t h a t can be e x t r e m e l y d i f f i c u l t t o s e p a r a t e may be r e s o l v e d a t the f e r m e n t a t i o n s t a g e . An e x c e l l e n t example o f t h i s i s the work done by Amgen s c i e n t i s t s on n o r l e u c i n e misincorporation i n i n t e r l e u k i n - 2 ( 6 , 7 ) · They found t h a t the i n c o r p o r a t i o n o f n o r l e u c i n e i n s t e a d o f m e t h i o n i n e i n the p r o d u c t , which r e s u l t s i n p r o d u c t h e t e r o g e n e i t y w i t h u n p r e d i c t a b l e immunogenic consequences, c o u l d be m i n i m i z e d o r even e l i m i n a t e d by

Proteins

nature of p r o t e i n

ANALYTICAL

Process Economics

(conditions)

(sequence)

How

(techniques)

What

FORMULATION

purification

. endotoxins - Activity

. nucleic acids

. proteins

- Contaminants

- Purity

Specifications

PURIFIED PROTEIN

(Porath et al)

Where

Secretion

- Metal Chelation -

. Peptide S y n t h e s i s

. T i s s u e Culture

Engineering

- PolyArg tail (Searle) - Fusion

Figure 1. The m u l t i f a c e t e d p r o c e s s development.

extracellular

. insoluble

. soluble

intracellular

Microbial:

STARTING MIXTURE

FERMENTATION

Genetic

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ο 2

1

a

3 2

3

2. HO

17

Strategies for Large-Scale Protein Purification

s i m p l y adding l e u c i n e and/or methionine f e r m e n t a t i o n medium.

a t low l e v e l s

to the

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch002

FORMULATION CONSTRAINTS. F o r m u l a t i o n c o u l d impose f u r t h e r c o n s t r a i n t s on p r o d u c t c h a r a c t e r i s t i c s s i n c e the p r o d u c t i n i t s d e s i r e d d e l i v e r a b l e form ( s t a b i l i t y , a c t i v i t y , r e l e a s e r a t e , c o l o r , e t c . ) i s t r u l y the f i n a l g o a l . I t i s possible that a p r e f e r r e d p u r i f i c a t i o n p r o c e s s p u r e l y from a p r o c e s s i n g s t a n d p o i n t may produce a l e s s d e s i r a b l e p r o d u c t from a f o r m u l a t i o n s t a n d p o i n t (such as s t a b i l i t y , impurity p r o f i l e , delivery rate). So a p u r i f i e d p r o d u c t s h o u l d be r a p i d l y c a r r i e d through f o r m u l a t i o n and t e s t i n g t o ensure that i t i s acceptable before a p u r i f i c a t i o n process i s locked i n . THE IMPACT OF GENETIC ENGINEERING. The advent o f g e n e t i c e n g i n e e r i n g has p r o b a b l y e x e r t e d the b i g g e s t impact on p u r i f i c a t i o n p r a c t i c e , i n t r o d u c i n g new c h a l l e n g e s as w e l l as o f f e r i n g new solutions. I t i s p o s s i b l e to not o n l y a c h i e v e h i g h p r o d u c t i o n o f a p r o t e i n p r o d u c t but a l s o modify i t f o r improved s e p a r a t i o n and/or s t a b i l i t y ( e . g . , p o l y a r g i n i n e t a i l ( 8 ) , metal a f f i n i t y s i t e , f u s i o n p r o t e i n s ) or f o r determining i t s eventual residence (cytoplasmic, periplasmic or e x t r a c e l l u l a r ) . In a d d i t i o n , f o r some p r o t e i n s the number o f c y s t e i n e r e s i d u e s c o u l d be changed f o r improved r e f o l d efficiency ( 9 ) . THE ROLE OF ANALYTICAL. While not o b v i o u s , a n a l y t i c a l p l a y s a v e r y c r i t i c a l r o l e i n p r o c e s s development f o r rDNA-derived p r o d u c t s . P r o v i d i n g a q u a n t i t a t i v e assay f o r the p r o d u c t i s o n l y a s m a l l p a r t o f a n a l y t i c a l development. The a b i l i t y to i d e n t i f y contaminants t h a t not o n l y a r e p r e s e n t a t v e r y low c o n c e n t r a t i o n s but a l s o d i f f e r v e r y s l i g h t l y from the main p r o d u c t ( e . g . due to m i s i n c o r p o r a t i o n , p r o c e s s - r e l a t e d chemical m o d i f i c a t i o n or aggregation) i s a b s o l u t e l y e s s e n t i a l i n the o v e r a l l e f f o r t o f d e v e l o p i n g an o p t i m a l p u r i f i c a t i o n process. In summary, the o p t i m i z a t i o n o f a p u r i f i c a t i o n p r o c e s s r e q u i r e s c l o s e i n t e r a c t i o n s among a l l o f the above f u n c t i o n s , c a r e f u l p l a n n i n g as w e l l as a l l o w i n g room f o r i t e r a t i o n s . This i s , however, an i d e a l s i t u a t i o n . In r e a l i t y , the p r e s s u r e o f e a r l y market i n t r o d u c t i o n and slow r e g u l a t o r y c l e a r a n c e u s u a l l y f o r c e s one to l o c k i n w i t h an " i n f e r i o r " p r o c e s s , which c o u l d be f r u s t r a t i n g but n e c e s s a r y . J u s t b e i n g aware o f a l l these i s s u e s i s i n i t s e l f an important element o f p r o c e s s development. Then e a r l y p l a n n i n g i n combination w i t h a sound s t r a t e g y based on e x p e r i e n c e i s p r o b a b l y the b e s t one can do under the c i r c u m s t a n c e s . STRATEGY DEVELOPMENT The q u e s t i o n here i s how t o c o n v e r t an impure p r o t e i n s o l u t i o n such as a f e r m e n t a t i o n b r o t h i n t o a p u r i f i e d p r o d u c t t h a t meets a l l the requirements. I s t h e r e a methodology t h a t would o f f e r some guidelines? There seems t o be two g e n e r a l approaches. In one, a h i g h l y s p e c i f i c method such as i m m u n o a f f i n i t y i s u t i l i z e d e a r l y i n the p r o c e s s to a c h i e v e the maximum degree o f p u r i f i c a t i o n ( e . g .

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PROTEIN PURIFICATION

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch002

a l p h a - i n t e r f e r o n , Hoffmann La Roche). T h i s approach i s not g e n e r i c , c o u l d be c o s t l y , and, i f a n t i b o d i e s are used as l i g a n d s , may have o t h e r o p e r a t i n g c o m p l i c a t i o n s such as s i g n i f i c a n t l o s s o f b i n d i n g c a p a c i t y upon i m m o b i l i z a t i o n , and l e a c h i n g o f the a n t i b o d i e s i n t o the p r o d u c t stream. A n t i b o d i e s may a l s o b i n d to the d e n a t u r e d , u n f o l d e d , m i s p a i r e d , and a g g r e g a t e d forms o f the p r o d u c t and i t s analogs. The o t h e r approach i s to use a s e r i e s o f " t r a d i t i o n a l " methods i n a c o n c e r t e d way to a c h i e v e the d e s i r e d g o a l ( p u r i t y , y i e l d , cost, s c a l a b i l i t y ) . The l a t t e r approach appears to be q u i t e e f f e c t i v e and has been used c o m m e r c i a l l y f o r p u r i f y i n g i n s u l i n (E. L i l l y ) and human growth hormone (Genentech) and i s p r o b a b l y used i n development stage f o r o t h e r p r o t e i n p r o d u c t s such as f i b r o b l a s t i n t e r f e r o n , i n t e r l e u k i n - 1 and animal growth hormones. A c t u a l l y the d i v i s i o n between the two approaches can be arbitrary. S i n c e no s i n g l e t e c h n i q u e c o u l d d e l i v e r the f i n a l p u r i f i e d p r o d u c t d i r e c t l y from the f e r m e n t a t i o n b r o t h o r crude e x t r a c t , h i g h l y s p e c i f i c methods such as i m m u n o a f f i n i t y still r e q u i r e the s u p p o r t o f o t h e r methods to a c c o m p l i s h the t a s k , e s p e c i a l l y i n l i g h t o f some o f i t s "unexpected" drawbacks d i s c u s s e d above. Thus, i n e i t h e r case p r o c e s s o p t i m i z a t i o n i s s t i l l required. The key d i f f e r e n c e i n the two approaches i s i n the i n i t i a l focus. In g e n e r a l , u n l e s s a h i g h l y s p e c i f i c and unique a f f i n i t y method has a l r e a d y been i d e n t i f i e d , we recommend t h a t the approach d e v e l o p e d below be used. B o n n e r j e a e t a l ( 2 ) a n a l y z e d 1 0 0 p u b l i c a t i o n s on l a b - s c a l e p r o t e i n p u r i f i c a t i o n procedures. They p l o t t e d the r e s u l t s ( F i g u r e 2 ) showing the number o f s t e p s used i n the p u r i f i c a t i o n scheme as a f u n c t i o n o f the f r e q u e n c y a method i s used i n each s t e p . A pattern seems to emerge. Homogenization i s the most f r e q u e n t l y used f i r s t s t e p , p r o b a b l y because most p r o t e i n p r o d u c t s are i n t r a c e l l u l a r , hence the need to break the c e l l s open. I t i s i n t e r e s t i n g that p r e c i p i t a t i o n i s p o p u l a r as the f i r s t p u r i f i c a t i o n s t e p , f o l l o w e d by more r e s o l v i n g methods such as ion-exchange and a f f i n i t y methods, and f i n i s h e d w i t h g e l f i l t r a t i o n . While these e x a c t methods are c e r t a i n l y not g e n e r a l l y a p p l i c a b l e , they seem to suggest a f a i r l y s e n s i b l e sequence o f e v e n t s . Going beyond these s p e c i f i c t e c h n i q u e s and combining our e x p e r i e n c e w i t h o t h e r s i n the l i t e r a t u r e r e g a r d i n g p u r i f y i n g p r o t e i n s from recombinant s o u r c e s , we propose the g e n e r a l p u r i f i c a t i o n scheme shown i n F i g u r e 3 . These are b l o c k s o f a c t i v i t i e s t h a t may r e q u i r e more than one s t e p . The essence o f the proposed scheme i s the d e l i b e r a t e b r e a k i n g down o f the development a c t i v i t y f o r p r o t e i n p u r i f i c a t i o n i n t o two s e p a r a t e b l o c k s : g r o s s p u r i f i c a t i o n and h i g h - r e s o l u t i o n purification. In g r o s s p u r i f i c a t i o n , the f o c u s i s to u t i l i z e a simple y e t e f f e c t i v e s t e p ( s ) t o s i g n i f i c a n t l y c l e a n up the s o l u t i o n i n such a way t h a t i t f l o w s n a t u r a l l y i n t o the next b l o c k where h i g h r e s o l u t i o n methods are used f o r the f i n a l p u r i f i c a t i o n . T h i s way, i t i s the g r o s s p u r i f i c a t i o n t h a t d e t e r m i n e s what and how h i g h - r e s o l u t i o n methods s h o u l d be used. There are s e v e r a l advantages w i t h t h i s approach. For one, t h e r e always e x i s t a l a r g e number o f i m p u r i t i e s i n the crude s o l u t i o n t h a t have extreme p r o p e r t i e s ( h i g h l y charged, e i t h e r v e r y l a r g e o r v e r y s m a l l , h i g h l y h y d r o p h o b i c , e t c . ) and t h a t c o u l d be removed e a s i l y i n a simple s t e p i f the a p p r o p r i a t e method i s used. Only a f t e r t h i s treatment i s i t

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch002

HO

Strategies for Large-Scale Protein Purification

I

2

3

4

5

6

7

Stage in purification scheme Figure 2 . A n a l y s i s o f the p u r i f i c a t i o n methods used at s u c c e s s i v e s t e p s i n the p u r i f i c a t i o n schemes (Reproduced w i t h p e r m i s s i o n from Ref. 2 C o p y r i g h t 1 9 8 6 Nature P u b l i s h i n g Company.)

20

PROTEIN PURIFICATION

. Fermentation Synthesis

. Tissue Culture

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch002

. Peptide Synthesis

. Homogenization .

. Centrifugation

Isolation &

. Filtration

Concentration

. L i q / L i q Ext. Refold . Adsorption

Gross

. Precipitation

Purification

. L i q / L i q Ext.

Hi-resolution

Chromatography

Purification

Concentration (Buffer Ex.)

Affinity

.

Interactions

Ultrafiltration

. Gel Filtration

Sterile Filt. Drying

— Figure

3-

Proposed

ι

—

general

purification

scheme.

2. HO

21

Strategies for Large-Scale Protein Purification

c l e a r what contaminants are l e f t . These tend to have s i m i l a r p r o p e r t i e s to the p r o d u c t o f i n t e r e s t . F o c u s i n g on s e p a r a t i o n o f s i m i l a r compounds, f r e e from o t h e r i n t e r f e r i n g contaminants, will r e s u l t i n h i g h e r c a p a c i t y , b e t t e r r e s o l u t i o n and l o n g e r l i f e f o r the more complex and more c o s t l y h i g h r e s o l u t i o n s t e p . The above scheme a u t o m a t i c a l l y o r g a n i z e s the l a r g e number o f i s o l a t i o n / p u r i f i c a t i o n t e c h n i q u e s i n t o a few manageable c a t e g o r i e s as shown i n F i g u r e 3 a l o n g w i t h the flow diagram and expanded i n T a b l e s I-IV. These b l o c k a c t i v i t i e s are c o n s i d e r e d i n d e t a i l below.

Table

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch002

a)

b)

Cell

I.

Isolation

Rupture ο Homogenization ο E x t r a c t i o n (Bio/Chemical

S o l i d s / L i q u i d Separation ο Centrifugation ο Filtration ο Cross-Flow UF/MF ο Aqueous Two-Phase

Table

Methods

Treatment)

Partitioning

II.

Refold

a)

Dissolution ο Dénaturants ( u r e a , guanidine.HC1, SDS, e t c . ) and/or extreme pH

b)

Oxidation ο Complex, y i e l d l o s s due to a g g r e g r a t i o n ο Key parameters: pH, T, time con. o f dénaturants additives ο Genetic Engineering: 3 S-H • 2 S-H ( b e t a - i n t e r f e r o n )

Table

III.

Bulk

charged,

Purification

ο

Adsorption:

hydrophobic,

ο

Precipitation - pH - Temperature - Salts - Polymers (Neutral/Charged) - Organic S o l v e n t s * Affinity?

ο

Liquid/Liquid Extraction - Organic/Aqueous - Aqueous/Aqueous

Methods

affinity

adsorbents

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PROTEIN PURIFICATION

Table

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch002

ο

IV.

High

R e s o l u t i o n Methods

Chromatography - Ion-Exchange ( c h a r g e ) - Hydrophobic I n t e r a c t i o n s - Reverse Phase - S i z e - E x c l u s i o n ( s i z e &. shape) - Chromatofocusing (pi) A f f i n i t y Interactions - Antibody - - Antigen - Hormone - - R e c e p t o r - Enzyme - - S u b s t r a t e / A n a l o g / I n h i b i t o r - Metal ion - - Ligand - Dye - - L i g a n d Characteristics - can be c o s t l y - powerful f o r Dilute/Low solutions

Purity

ISOLATION / RECOVERY. The g o a l here i s to c o n c e n t r a t e the s t a r t i n g s o l u t i o n ( e . g . , f e r m e n t a t i o n b r o t h ) , p r e f e r a b l y w i t h some degree o f purification. T h i s may or may not i n v o l v e c e l l breakage s i n c e the p r o d u c t s may be s e c r e t e d . H o m o g e n i z a t i o n has been found e f f e c t i v e f o r i n t r a c e l l u l a r p r o d u c t s , e s p e c i a l l y i n c l u s i o n b o d i e s , even though e x t r a c t i o n by b i o / c h e m i c a l means i s a f e a s i b l e o p t i o n . An advantage w i t h i n c l u s i o n b o d i e s i s t h a t they appear to be q u i t e s t u r d y and can withstand r a t h e r severe o p e r a t i n g c o n d i t i o n s . For s o l u b l e products (enzymes, p e p t i d e s , e t c . ) , however, c a u t i o n has to be taken to a v o i d d e n a t u r a t i o n and p r o t e o l y t i c c l i p p i n g s . Here speed and s o l u t i o n c o n d i t i o n s f o r minimizing p r o t e o l y t i c a c t i v i t y (temperature, pH, i o n i c s t r e n g t h , i n h i b i t o r s , e t c . ) are o f the e s s e n c e . Product s e c r e t i o n by g e n e t i c e n g i n e e r i n g means i s an a t t r a c t i v e approach due to the p o t e n t i a l p r o c e s s s i m p l i f i c a t i o n ( VO), p r o v i d e d t h a t comparable p r o d u c t i v i t y can be a c h i e v e d . Depending upon the n a t u r e o f the p r o d u c t a number o f l i q u i d / s o l i d s e p a r a t i o n methods can be used. For s e p a r a t i o n o f c e l l d e b r i s and p r o d u c t s i n s o l i d form ( e . g . , i n c l u s i o n b o d i e s , p r e c i p i t a t e d p r o d u c t s ) c e n t r i f u g a t i o n has emerged as the method o f c h o i c e i n which s i z e and d e n s i t y d i f f e r e n c e s between c e l l d e b r i s and p r o t e i n p a r t i c l e s are e x p l o i t e d (_11). The drawbacks here are h i g h c a p i t a l and o p e r a t i n g c o s t s . For some s i t u a t i o n s c r o s s - f l o w f i l t r a t i o n (UF/MF) may be a f e a s i b l e a l t e r n a t i v e . Aqueous two-phase e x t r a c t i o n , i n p a r t i c u l a r P E G / s a l t systems, has been s u c c e s s f u l l y a p p l i e d to c o n c e n t r a t i n g c e l l s and/or c e l l d e b r i s i n one phase and e x t r a c t i n g p r o d u c t s i n t o the o t h e r phase (12-13)· The advantage here i s t h a t not o n l y l i q u i d / s o l i d s e p a r a t i o n but a l s o c o n c e n t r a t i o n and p a r t i a l p u r i f i c a t i o n are a c c o m p l i s h e d at the same time. Compared w i t h o t h e r methods, a f a c t o r t h a t needs to be c o n s i d e r e d w i t h aqueous two-phase e x t r a c t i o n

2. HO Strategies for Large-Scale Protein Purification

23

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch002

i s the a d d i t i o n o f r e a g e n t s (polymers & s a l t s ) t o the system, which may c o m p l i c a t e downstream p r o c e s s i n g i f they have t o be removed a t the end. P a r t l y because o f t h i s v e r y i s s u e , l a r g e - s c a l e a p p l i c a t i o n s o f aqueous two-phase systems have been l i m i t e d t o p u r i f i c a t i o n o f i n d u s t r i a l enzymes where a c t i v i t y r a t h e r than a b s o l u t e p u r i t y i s needed . F o r s e c r e t e d p r o d u c t s o r i n t r a c e l l u l a r p r o d u c t s r e l e a s e d by p e r m e a b i l i z a t i o n , c e l l / l i q u i d s e p a r a t i o n f o l l o w e d by p r o d u c t c o n c e n t r a t i o n / d i a f i l t r a t i o n can be c o n v e n i e n t l y c a r r i e d out by cross-flow m i c r o f i l t r a t i o n or u l t r a f i l t r a t i o n . REFOLD. T h i s s t e p i s n e c e s s a r y i f the p r o t e i n i s i n an i n a c t i v e form ( d e n a t u r e d , r e d u c e d , e t c . ) . I t has been found t h a t o v e r e x p r e s s i o n o f f o r e i g n p r o t e i n s i n b a c t e r i a l systems o f t e n r e s u l t s i n formation o f i n c l u s i o n bodies. The p r o t e i n e x i s t s i n a r e d u c e d , p o l y m e r i c s t a t e which n e c e s s i t a t e s d i s s o l u t i o n and renaturation. T h i s s t e p i s q u i t e c r i t i c a l s i n c e i t not o n l y a f f e c t s the o v e r a l l y i e l d but through the n a t u r e o f the i m p u r i t i e s g e n e r a t e d w i l l a l s o d i c t a t e the subsequent p u r i f i c a t i o n t r a i n . For secreted p r o d u c t s , which tend t o be i n t h e i r a c t i v e form, r e f o l d i s not normally necessary. In the case o f i n c l u s i o n b o d i e s , t h e r e f o l d s t e p t y p i c a l l y c o n s i s t s o f f i r s t d i s s o l v i n g the s o l i d s i n a s t r o n g c h a o t r o p e such as g u a d i n i n e h y d r o c h l o r i d e , sodium t h i o c y a n a t e o r u r e a , f o l l o w e d by renaturing/oxidative r e f o l d i n g process. T h i s i s a complex p r o c e s s w i t h major y i e l d l o s s due t o a g g r e g a t i o n (_16). Key parameters f o r m i n i m i z i n g a g g r e g a t i o n o r m a x i m i z i n g the r e f o l d y i e l d a r e pH, temperature, time, and c o n c e n t r a t i o n o f dénaturants. A d d i t i v e s such as d e t e r g e n t s have a l s o been used ( 1 7 ) . The p r i n c i p l e a t work here seems t o be the d r i v e towards an o p t i m a l h y d r o p h o b i c / h y d r o p h i l i c b a l a n c e e x e r t e d on the p r o t e i n by i t s environment: s u f f i c i e n t h y d r o p h o b i c f o r c e i s needed t o cause the p r o t e i n t o r e f o l d but the same f o r c e a l s o l e a d s t o a g g r e g a t i o n . A great deal o f p r o p r i e t a r y information r e l a t e d to p r a c t i c a l r e f o l d i n g o f p r o t e i n s has been g e n e r a t e d i n the p r i v a t e s e c t o r s . C o n t r a r y t o t h e o r e t i c a l b e l i e f s , a number o f p r o t e i n s have been found t o r e f o l d q u i t e e f f i c i e n t l y (RF e f f i c i e n c y > 7 0 % ) a t h i g h c o n c e n t r a t i o n s ( g / L l e v e l ) u s i n g f a i r l y simple p r o c e d u r e s . It appears t h a t s i n c e p r o t e i n r e f o l d i n g i s not w e l l u n d e r s t o o d and s i n c e each p r o t e i n may be u n i q u e , g e n e r a l i z a t i o n t o the p o i n t o f e x c l u d i n g e x p e r i m e n t a l i n v e s t i g a t i o n may not be w a r r a n t e d a t t h i s time. GROSS PURIFICATION. The main purpose here i s t o remove as many i m p u r i t i e s as p o s s i b l e i n a simple s t e p o r s t e p ( s ) . F o r t h i s , b a t c h a d s o r p t i o n and p r e c i p i t a t i o n seem t o be most e f f e c t i v e . Batch adsorption i s p a r t i c u l a r l y e f f e c t i v e f o r dealing with d i l u t e s o l u t i o n s by s e l e c t i n g an a d s o r b e n t t h a t w i l l b i n d the p r o d u c t . S i n c e h i g h s e l e c t i v i t y i s not c r i t i c a l a t t h i s s t a g e , an a d s o r b e n t w i t h h i g h c a p a c i t y and some s p e c i f i c i t y f o r a p a r t i c u l a r p r o d u c t i s not t o o d i f f i c u l t t o f i n d . Common a d s o r b e n t s a v a i l a b l e c o m m e r c i a l l y are e i t h e r charged o r h y d r o p h o b i c . A f f i n i t y adsorbents using metal c h e l a t e o r dye l i g a n d s a r e v e r y e f f e c t i v e f o r group s p e c i f i c i n t e r a c t i o n s and s h o u l d be e x p l o i t e d when a p p r o p r i a t e . With

24

PROTEIN PURIFICATION

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch002

w e l l - t h o u g h t out a p p l i c a t i o n s , t h i s approach c o u l d a c c o m p l i s h c l a r i f i c a t i o n , c o n c e n t r a t i o n , and p a r t i a l p u r i f i c a t i o n i n p r a c t i c a l l y one i n t e g r a t e d s t e p , as i l l u s t r a t e d i n one o f the case studies discussed l a t e r . I f the s o l u t i o n i s f r e e o f p a r t i c u l a t e s , a d s o r p t i o n i n column mode can be done, which a l l o w s some l e v e r a g e f o r improved p u r i f i c a t i o n d u r i n g e l u t i o n . P r e c i p i t a t i o n can be a v e r y p o w e r f u l method f o r g r o s s purification. P r o t e i n p r e c i p i t a t i o n can be brought about by v a r i o u s means such as changes i n pH o r temperature o f the s o l u t i o n , by a d d i t i o n o f s a l t s ( e . g . ammonium s u l f a t e ) , w a t e r - s o l u b l e polymers (e.g. p o l y e t h y l e n e g l y c o l , p o l y e t h y l e n e imine, p o l y a c r y l i c a c i d ) , i n o r g a n i c f l o c c u l a n t s ( s i l i c a o r a l u m i n a , b i o p r o c e s s i n g a i d s from Rohm and Haas, c e l l d e b r i s remover from Whatman), o r o r g a n i c s o l v e n t s (e.g. a l c o h o l s ) . An e x c e l l e n t r e v i e w o f these p r e c i p i t a t i o n methods i s g i v e n by B e l l e t a l (_18). S a l t i n g out i n which molar c o n c e n t r a t i o n s o f s a l t s such as ammonium s u l f a t e are used i s a v e r y common method f o r p r o t e i n p r e c i p i t a t i o n . This approach, however, has many drawbacks: low s e l e c t i v i t y , h i g h s e n s i t i v i t y to o p e r a t i n g c o n d i t i o n s , and down stream c o m p l i c a t i o n s ( s a l t removal & d i s p o s a l ) . S i m p l e r and more e f f e c t i v e methods such as change i n pH o r temperature, use o f p o l y e l e c t r o l y t e s alone o r i n c o m b i n a t i o n w i t h n e u t r a l polymers ( 1 9 , 2 0 ) s h o u l d not be o v e r l o o k e d . There are two ways to use p r e c i p i t a t i o n . One convenient approach i s to p r e c i p i t a t e most o f the i m p u r i t i e s l e a v i n g the product i n s o l u t i o n f o r f u r t h e r p r o c e s s i n g . For t h i s , f l o c c u l a t i o n i s v e r y e f f e c t i v e . The p r e c i p i t a t e i s a network o f c e l l d e b r i s , e x t r a n e o u s p r o t e i n s , c o l o r e d c o n t a m i n a n t s , and, w i t h a n i o n exchange f l o c c u l a n t s such as p o l y e t h y l e n e i m i n e (2Λ) , n u c l e i c a c i d s . Instead o f p r e c i p i t a t i n g c o n t a m i n a n t s , i n some c a s e s i t may be e a s i e r to p r e c i p i t a t e the p r o d u c t . T h i s , however, would n e c e s s i t a t e s o l i d r e c o v e r y and r e d i s s o l u t i o n , which means a d d i t i o n a l p r o c e s s i n g s t e p s and y i e l d r e d u c t i o n . While the above methods appear common and tend not to be h i g h l y s e l e c t i v e , they can be v e r y e f f e c t i v e i f used a p p r o p r i a t e l y , e s p e c i a l l y f o r rDNA p r o d u c t s , because o f the f o l l o w i n g two r e a s o n s . F i r s t , most p r a c t i c a l rDNA p r o c e s s e s a c h i e v e f a i r l y h i g h e x p r e s s i o n ( 1 0 - 3 0 % o f t o t a l c e l l u l a r p r o t e i n s ) , so h i g h l y s o p h i s t i c a t e d t e c h n i q u e s are not u s u a l l y r e q u i r e d to i n c r e a s e the p u r i t y to 70-90%. Second, the p r o t e i n s o f i n t e r e s t b e i n g f o r e i g n to the b a c t e r i a l systems are l i k e l y to have v e r y d i f f e r e n t p r o p e r t i e s ( p i , h y d r o p h o b i c i t y , s i z e , heat s t a b i l i t y , e t c . ) t h a t s h o u l d be e x p l o i t e d f o r simple y e t e f f e c t i v e p u r i f i c a t i o n . T h i s w i l l be i l l u s t r a t e d i n one o f the case s t u d i e s . A n o t h e r e f f e c t i v e method f o r g r o s s p u r i f i c a t i o n i s l i q u i d - l i q u i d e x t r a c t i o n , e s p e c i a l l y aqueous two-phase systems (12-15)· These, however, have l i m i t e d a p p l i c a t i o n s so f a r because o f a number o f r e a s o n s : r e l a t i v e l y new method, h i g h polymer c o s t ( f o r PEG-dextran s y s t e m s ) , and the need to remove p h a s e - f o r m i n g r e a g e n t s ( p o l y m e r s , s a l t s ) from the p r o d u c t s . HIGH RESOLUTION PURIFICATION. V a r i o u s forms o f chromatography ( T a b l e IV) are p r e d o m i n a n t l y used f o r the f i n a l p u r i f i c a t i o n o f p r o t e i n s to homogeneity ( 2 2 ) . Ion-exchange chromatography i s w i d e l y used due to i t s v e r s a t i l e a p p l i c a b i l i t y f o r p r o t e i n s , h i g h c a p a c i t y

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch002

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and r e s o l u t i o n . Based on l i t e r a t u r e i n f o r m a t i o n as w e l l as our e x p e r i e n c e , ion-exchange or r e v e r s e p h a s e / h y d r o p h o b i c i n t e r a c t i o n s chromatography can have remarkable r e s o l u t i o n d e s p i t e i t s s u p p o s e d l y n o n s p e c i f i c mode o f i n t e r a c t i o n s (charge o r h y d r o p h o b i c i t y ) . The n a t u r e o f the i n t e r a c t i o n between macromolecules ( w i t h t h e i r t h r e e - d i m e n s i o n a l s t r u c t u r e s ) and s u r f a c e s i s such t h a t unexpected s p e c i f i c i t y may r e s u l t from the dynamics o f m u l t i s i t e i n t e r a c t i o n s (23). With the l a r g e number o f parameters a v a i l a b l e f o r manipulation i n chromatographic s e p a r a t i o n — type o f r e s i n , degree o f l o a d i n g , washing c o n d i t i o n s , e l u t i o n s t r a t e g y ( s t e p , g r a d i e n t , d i s p l a c e m e n t o r c o m b i n a t i o n ; type o f b u f f e r o r s o l v e n t ) — r e s o l u t i o n o f m o l e c u l e s w i t h minor d i f f e r e n c e s i n amino a c i d r e s i d u e s o r between monomer and o l i g o m e r s c o u l d be a c h i e v a b l e . Very good r e v i e w a r t i c l e s and books can be found on the use o f chromatography f o r p u r i f i c a t i o n (24-26). Naveh, i n an e x c e l l e n t r e v i e w on s c a l e - u p s t r a t e g i e s f o r p r o t e i n p u r i f i c a t i o n ( 4 ) , o f f e r e d some p r a c t i c a l c o n s i d e r a t i o n s f o r u t i l i z i n g chromatography i n a p u r i f i c a t i o n scheme. He d i s c u s s e d the importance o f m a i n t a i n i n g c o n s i s t e n t f e e d s o l u t i o n ( e . g . p r o d u c t c o n c e n t r a t i o n and i m p u r i t y p r o f i l e ) due to o v e r l o a d i n g c o n d i t i o n s ; the s i g n i f i c a n c e o f dynamic c a p a c i t y as r e l a t e d to l i n e a r f l o w r a t e , s o l u t i o n pH and i o n i c s t r e n g t h ; and the r o l e o f m a t r i x p a c k i n g w i t h r e s p e c t to column p r e s s u r e drop through the c h r o m a t o g r a p h i c c y c l e . Chromatography u s i n g a f f i n i t y i n t e r a c t i o n s i s an important method but one has to be v e r y c a r e f u l i n the c h o i c e o f l i g a n d s . A n t i b o d i e s are g e n e r a l l y not a p r a c t i c a l c h o i c e due to h i g h c o s t , low c a p a c i t y , and f a i r l y poor s e l e c t i v i t y f o r c l o s e l y - r e l a t e d m o l e c u l e s (monomer/dimer, d e n a t u r e d , w r o n g l y - f o l d e d ) . Receptors appear to a b e t t e r c h o i c e w i t h r e g a r d s to these a s p e c t s , as found by Hoffmann La Roche i n the p u r i f i c a t i o n o f i n t e r f e r o n . M e t a l i o n s and dyes c o u l d be v e r y s e l e c t i v e f o r some p r o t e i n s , e s p e c i a l l y i f they are m o d i f i e d f o r enhanced i n t e r a c t i o n s . A unique a s p e c t o f a f f i n i t y methods i s t h a t , due to the s t r o n g i n t e r a c t i o n i n v o l v e d , they are a powerful t o o l f o r c o n c e n t r a t i o n / p u r i f i c a t i o n of d i l u t e s o l u t i o n s . As such, they c o u l d be viewed as c o n c e n t r a t i o n & g r o s s - p u r i f i c a t i o n methods, which need to be f o l l o w e d w i t h , f o r example, chromatography f o r the f i n a l p u r i f i c a t i o n . Even though t h i s o r d e r o f usage may seem s t r a n g e , i t makes sense i f one r e c o g n i z e s t h a t , due to l o c a l i z e d i n t e r a c t i o n s and the n a t u r e o f s i n g l e - s t a g e b i n d / r e l e a s e , most a f f i n i t y methods can not u s u a l l y s e p a r a t e s i m i l a r m o l e c u l e s . FINAL CONCENTRATION. At t h i s p o i n t i n the p r o c e s s , the u s u a l r e q u i r e m e n t i s p r o d u c t c o n c e n t r a t i o n w i t h o r w i t h o u t b u f f e r exchange ( e i t h e r f o r s t a b i l i t y and/or i n p r e p a r a t i o n f o r the f o r m u l a t i o n s t e p ) p r i o r to the d r y i n g s t e p . Both c o n c e n t r a t i o n and b u f f e r exchange can be c o n v e n i e n t l y c a r r i e d out i n one s t e p u s i n g ultrafiltration. F o r p r o d u c t s t h a t are not s t a b l e i n a c r o s s - f l o w f i l t r a t i o n environment ( h i g h c i r c u l a t i n g r a t e , l o n g p r o c e s s i n g time) o r t h a t have a s t r o n g tendency to f o u l membranes, the a l t e r n a t i v e i s g e l filtration. T h i s i s a m i l d e r method f o r b u f f e r exchange and removal o f s a l t s or low m o l e c u l a r weight c o n t a m i n a n t s ; i t i s , however, not s u i t a b l e f o r h a n d l i n g l a r g e volumes.

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Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch002

SOME PRACTICAL CONSIDERATIONS

RECOMMENDED INITIAL ACTIVITIES. Our e x p e r i e n c e shows t h a t the f i r s t c r i t i c a l s t e p i n the p r o c e s s development i s to e s t a b l i s h a r e l i a b l e assay f o r the p r o d u c t . Without t h i s , i t would be v e r y d i f f i c u l t t o a s s e s s the performance o f v a r i o u s methods and s e p a r a t i o n schemes. Next, one s h o u l d a l s o c h a r a c t e r i z e the s t a r t i n g s o l u t i o n i t s e l f — i n a d d i t i o n to the p u r i f i e d p r o d u c t , which i s commonly done. The purpose here i s t o look f o r main d i f f e r e n c e s between the p r o d u c t and c o n t a m i n a n t s , which w i l l serve as the b a s i s f o r d e v i s i n g a sound p u r i f i c a t i o n process. G e l e l e c t r o p h o r e s i s i s a q u i c k way to a s s e s s the s i z e (MW) and p u r i t y d i s t r i b u t i o n o f p r o t e i n s i n the m i x t u r e . P r o t e i n n e t charge and h y d r o p h o b i c i t y can be s t u d i e d w i t h a d s o r b e n t s ( a n i o n / c a t i o n , h y d r o p h o b i c ) o r aqueous two-phase systems. The i s o e l e c t r i c p o i n t s ( p i ) o f p r o t e i n s i n s o l u t i o n can be d e t e r m i n e d by i s o e l e c t r i c focusing. In a d d i t i o n , s o l u t i o n c h a r a c t e r i s t i c s such as s t a b i l i t y as a f u n c t i o n o f temperature and time, h a n d l i n g , e t c . , s h o u l d be c a r e f u l l y noted and taken i n t o account i n the p r o c e s s development. T h i s not o n l y w i l l minimize p r o c e s s i n g c o m p l i c a t i o n s a t the l a r g e - v o l u m e s t a g e but sometimes may a l s o o f f e r c l u e s f o r unique p u r i f i c a t i o n approaches.

SOME GUIDELINES FOR PURIFICATION PROCESS DEVELOPMENT Approaches f o r s e l e c t i o n o f e a r l y s t e p s i n the p u r i f i c a t i o n t r a i n : . reduce p r o c e s s volume e a r l y on . e l i m i n a t e components o f extreme n a t u r e : p a r t i c u l a t e s , small s o l u t e s , large aggregates, n u c l e i c a c i d s , e t c . A p p r o p r i a t e methods here a r e a d s o r p t i o n ( h y d r o p h o b i c o r i o n -exchange), p r e c i p i t a t i o n / f l o c c u l a t i o n , u l t r a f i l t r a t i o n , and a f f i n i t y adsorption. I n t e g r a t i o n o f P u r i f i c a t i o n Steps: . s t e p s s h o u l d be complementary t o one a n o t h e r both i n degree o f p u r i f i c a t i o n as w e l l as i n p r o c e s s flow t o a c h i e v e the f i n a l g o a l s . I t s h o u l d always be kept i n mind t h a t o p t i m i z a t i o n ( y i e l d , p u r i t y & c o s t ) i s done f o r the whole p r o c e s s not f o r any s i n g l e s t e p . T h i s i s to a v o i d b e i n g t r a p p e d i n t o u n d u l y m a x i m i z i n g a s t e p which may n o t make much d i f f e r e n c e i n the whole scheme. . minimize the number o f s o l v e n t s and b u f f e r s used. While t h i s may sound t r i v i a l and i s n o t t h a t c r i t i c a l a t the bench s c a l e , u n n e c e s s a r y s o l v e n t o r b u f f e r exchanges w i l l be c o s t l y and time consuming on a l a r g e - s c a l e . A l s o , avoid, i f p o s s i b l e , b u f f e r s that are expensive, c o m p l i c a t e d t o p r e p a r e , o r d i f f i c u l t to pH ( T r i s b u f f e r s ) Ease o f O p e r a t i o n : The key p o i n t here i s the s i m p l e r the p r o c e s s and the more s t r a i g h t f o r w a r d the c o n d i t i o n s , the l e s s l i k e l y f o r i t to f a i l on a l a r g e - s c a l e .

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CASE STUDIES

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch002

T a b l e V l i s t s the p o s s i b l e s o u r c e s o f p r o t e i n / p e p t i d e p r o d u c t s . Only a few r e p r e s e n t a t i v e r e a l examples a r e d i s c u s s e d here t o demonstrate the a p p l i c a b i l i t y o f the o u t l i n e d s t r a t e g i e s . MICROBIAL, INTRACELLULAR, INSOLUBLE (E.G., INCLUSION BODIES). An i n t e r e s t i n g case i n t h i s c a t e g o r y i s the p u r i f i c a t i o n p r o c e s s f o r f i b r o b l a s t b e t a - i n t e r f e r o n p r e s e n t e d by Hershenson o f Cetus Corp (9)· The i n t e r f e r o n was made i n the form o f i n c l u s i o n b o d i e s . The c e l l s were f i r s t homogenized to r e l e a s e the p r o d u c t , f o l l o w e d by c e n t r i f u g a t i o n t o b r i n g down the i n c l u s i o n b o d i e s l e a v i n g c e l l d e b r i s i n the s u p e r n a t a n t . The r e c o v e r e d i n c l u s i o n b o d i e s were then d i s s o l v e d and the p r o t e i n r e f o l d e d . I n the c l o n i n g o f i n t e r f e r o n , the odd c y s t e i n e r e s i d u e t h a t does not p a r t i c i p a t e i n the d i s u l f i d e f o r m a t i o n was d e l i b e r a t e l y r e p l a c e d w i t h s e r i n e t o m i n i m i z e m i s p a r i n g i n the r e f o l d i n g p r o c e s s . T h i s was done t o improve the refold efficiency. A f t e r the c o m p l e t i o n o f the r e f o l d s t e p , t h e r e are two t y p e s o f c o n t a m i n a n t s p r e s e n t : E. c o l i - d e r i v e d ( p r o t e i n s , e n d o t o x i n s , n u c l e i c a c i d s ) , and p r o d u c t - l i k e ( d i m e r / a g g r e g a t e s o f i n t e r f e r o n and i t s m o d i f i e d f o r m s ) . T h i s type o f m i x t u r e i s f a i r l y t y p i c a l f o r rDNA p r o d u c t s made by E. c o l i i n the form o f i n c l u s i o n bodies. An u n d i s c l o s e d " p r e t r e a t m e n t " s t e p ( g r o s s p u r i f i c a t i o n ?) was c a r r i e d o u t next to p r e p a r e a c l e a n e d - u p l o a d s o l u t i o n f o r the f i n a l p u r i f i c a t i o n s t e p i n which r e v e r s e phase chromatography was used t o s e p a r a t e i n t e r f e r o n from i t s o t h e r monomeric forms as w e l l as from i t s polymers. For the p u r i f i c a t i o n o f animal growth hormones, which f o l l o w s more o r l e s s the same scheme d i s c u s s e d above f o r b e t a - i n t e r f e r o n , we d i s c o v e r e d a v e r y p o w e r f u l p r e c i p i t a t i o n method f o r the g r o s s p u r i f i c a t i o n step ( 1 9 - 2 0 ) . B a s i c a l l y a n e u t r a l polymer and a charged polymer, both w a t e r - s o l u b l e , were used s i m u l t a n e o u s l y t o b r i n g about p r e c i p i t a t i o n o f almost a l l o f the c o n t a m i n a n t s ( F i g u r e s 4 & 5 ) . The r o l e o f the charged polymer i s t o form charged complex w i t h the c o n t a m i n a n t s . The n e u t r a l polymer enhances the p r e c i p i t a t i o n o f the complex as w e l l as improves the charged polymer specificity. Both p u r i t y and y i e l d i n e x c e s s o f 9 0 % c o u l d be achieved i n a s i n g l e p r e c i p i t a t i o n step. Chromatography c o u l d then be used f o r the f i n a l p u r i f i c a t i o n . MICROBIAL, EXTRACELLULAR, SOLUBLE. An example here i s the p u r i f i c a t i o n o f a p e p t i d e ( m o l e c u l a r weight about 3 0 0 ) made by a fungal fermentation. The b r o t h c o n t a i n e d a l o t o f f i n e p a r t i c l e s as w e l l as s t r i n g y , s l i m y s u b s t a n c e s . The p e p t i d e c o n c e n t r a t i o n and i t s p u r i t y i n the b r o t h were v e r y low ( F i g u r e 6 ) . Thus the s o l u t i o n needs t o be c l a r i f i e d and, as the p r o p o s e d s t r a t e g y i n d i c a t e s , s i g n i f i c a n t l y concentrated, p r e f e r a b l y with p a r t i a l p u r i f i c a t i o n , b e f o r e a h i g h - r e s o l u t i o n p u r i f i c a t i o n method i s used. By s c r e e n i n g the recommended i s o l a t i o n and g r o s s p u r i f i c a t i o n methods ( T a b l e I &, I I I ) we were a b l e to q u i c k l y d e v e l o p a v e r y e f f i c i e n t , e a s i l y s c a l a b l e p r o c e s s i n which a simple a d s o r p t i o n / e x t r a c t i o n s t e p was used t o a c h i e v e s u b s t a n t i a l c o n c e n t r a t i o n and g r o s s p u r i f i c a t i o n . Shown i n F i g u r e 7 , the b r o t h was f i r s t s u b j e c t e d to a c o a r s e f i l t r a t i o n s t e p m a i n l y t o remove l a r g e p a r t i c l e s and s t r i n g y

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TABLE V. 1)

MICROBIAL, INTRACELLULAR, INSOLUBLE e.g.

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch002

SOURCES OF PROTEINS AND PEPTIDES

i n s u l i n , a n i m a l growth hormones, b e t a - i n t e r f e r o n AP3-Rec A f u s i o n p r o t e i n , i n t e r l e u k i n - 2

C h a r a c t e r i s t i c s : i n c l u s i o n or r e f r a c t i l e bodies p r o d u c t i n r e d u c e d , a g g r e g a t e d forms high product c o n c e n t r a t i o n 2)

MICROBIAL, INTRACELLULAR, SOLUBLE e.g.

L-aspartase, factor

human growth hormone, tumor

necrosis

C h a r a c t e r i s t i c s : products s u s c e p t i b l e to p r o t e o l y t i c d e g r a d a t i o n and h i g h l y c o n t a m i n a t e d w i t h s o l u b l e c e l l u l a r components upon cell lysis. 3)

MICROBIAL, EXTRACELLULAR (SOLUBLE) e.g.

4)

IGF-1,

TISSUE CULTURE e.g.

detergent

enzymes, r e n n i n

(EXTRACELLULAR & SOLUBLE)

t-PA, monoclonal a n t i b o d i e s ,

interleukin-4

C h a r a c t e r i s t i c s : v e r y low p r o d u c t c o n c e n t r a t i o n major c o n t a m i n a n t s : serum p r o t e i n s o r p r o t e i n a d d i t i v e s (BSA, e t c . ) 5)

PEPTIDE SYNTHESIS e.g.

AP3

C h a r a c t e r i s t i c s : no m i c r o b i a l p r o t e i n s / e n d o t o x i n s high product concentration c l o s e a n a l o g s as major i m p u r i t i e s

HO

Strategies for Large-Scale Protein Purification

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch002

100

DEAE-DEXTRAN LEVEL (g / g proteins) F i g u r e 4. The use o f PEG and DEAE-dextran f o r p r o t e i n p r e c i p i t a t i o n from a f e r m e n t a t i o n b r o t h c o n t a i n i n g an animal growth hormone.

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PROTEIN PURIFICATION

Figure 5 . SDS-PAGE o f E. c o l i crude e x t r a c t (Lane 1 ) and a f t e r p r e c i p i t a t i o n w i t h v a r i o u s c o m b i n a t i o n s o f n e u t r a l and charged polymers (Lane 2 - 8 ) . Dark bands at bottom are b o v i n e growth hormone.

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch002

2. HO Strategies for Large-Scale Protein Purification 31

32

PROTEIN PURIFICATION

Fermentation Broth ι Coarse Filtration

Batch Adsorption

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch002

ι Filtration / Wash

Extraction

clear solution 10x

concentration

70-80% yield significant purification

Chromatography 99%

purity

τ Figure

7.

P u r i f i c a t i o n process developed

f o r the

fungal

peptide.

materials. A h y d r o p h o b i c r e s i n (XAD s e r i e s from Rohm & Hass) was then added d i r e c t l y to the c l o u d y f i l t r a t e to a d s o r b the p e p t i d e as w e l l as some o t h e r components. The subsequent s i m p l e f i l t r a t i o n / w a s h s t e p removed a l l the f i n e p a r t i c u l a t e s i n s o l u t i o n by e x p l o i t i n g the l a r g e s i z e d i f f e r e n c e between the r e s i n and the particulates. E x t r a c t i o n o f the r e s i n r e s u l t e d i n a c l e a r s o l u t i o n t h a t was 1 0 - f o l d c o n c e n t r a t e d w i t h 7 0 - 8 0 % y i e l d and s u b s t a n t i a l peptide p u r i f i c a t i o n (Figure 8 ) . The f i n a l p u r i f i c a t i o n u s i n g chromatography to get to 9 9 % p u r i t y was g r e a t l y s i m p l i f i e d owing to the r e l a t i v e l y pure and c o n c e n t r a t e d l o a d s o l u t i o n . TISSUE CULTURE (EXTRACELLULAR, SOLUBLE). For p u r i f y i n g p r o t e i n s made by t i s s u e c u l t u r e ( e . g . , m o n o c l o n a l a n t i b o d i e s , t-PA) S c o t t and coworkers at I n v i t r o n ( 2 7 ) s u g g e s t e d s e v e r a l schemes i l l u s t r a t e d i n F i g u r e 9 f o r c o n d i t i o n e d media. T h e i r recommended scheme ( # 2 ) i s more o r l e s s c o n s i s t e n t w i t h our p r o p o s e d s t r a t e g y . A key c h a r a c t e r i s t i c o f t i s s u e c u l t u r e i s t h a t p r o d u c t l e v e l s tend to be q u i t e low. C o n c e n t r a t i o n i s thus a n e c e s s a r y f i r s t s t e p , f o r which u l t r a f i l t r a t i o n comes i n handy. For v e r y d i l u t e s o l u t i o n s , however, a f f i n i t y a d s o r p t i o n may have to be used a l s o f o r the concentration s t e p to m i n i m i z e p r o d u c t l o s s due to n o n s p e c i f i c b i n d i n g and l o n g p r o c e s s i n g time a s s o c i a t e d w i t h u l t r a f i l t r a t i o n . For media c o n t a i n i n g serum p r o t e i n s , p r e c i p i t a t i o n c o u l d be an e f f e c t i v e and simple g r o s s p u r i f i c a t i o n method f o r removing t h e s e p r o t e i n s b e f o r e moving on to chromatography.

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch002

HO

33

Strategiesfor Large-Scale Protein Purification

ο CO CO
90% c o u p l i n g e f f i c i e n c y was a c h i e v e d . The r e s i d u a l i m m u n o r e a c t i v i t i e s a t v a r i o u s a n t i b o d y l o a d i n g s on the m a t r i x a r e summarized i n T a b l e I I I . In accordance w i t h the o b s e r v a t i o n o f o t h e r s , (26) h i g h a n t i b o d y l o a d i n g s r e s u l t e d i n lower r e s i d u a l i m m u n o r e a c t i v i t i e s , p o s s i b l y due t o s t e r i c h i n d r a n c e . Residual

Immunoreactivities

of Various

Immunosorbents

The i m m u n o r e a c t i v i t i e s r e t a i n e d by the immunosorbents depend n o t o n l y upon the f a c t o r s d i s c u s s e d p r e v i o u s l y b u t a l s o upon the c h e m i c a l n a t u r e o f the a c t i v a t e d m a t r i c e s used. T a b l e IV l i s t s t h e v a r i o u s immunosorbents w i t h r e s p e c t t o t h e i r r e s i d u a l immunoreacti­ v i t i e s . F o r p r a c t i c a l reasons, the a n t i b o d y l o a d i n g was chosen a t 8-15 mg/ml g e l . A t t h i s a n t i b o d y d e n s i t y a p p r o x i m a t e l y 1 mg recombinant p r o t e i n ( i n t e r f e r o n - a l p h a 2a) c a n be p u r i f i e d p e r ml g e l . N e u t r a l pH was chosen f o r a n t i b o d y c o u p l i n g . Among the immunosorbents s t u d i e d , the a d i p i c d i h y d r a z i d e d e r i v a ­ t i v e had the b e s t a n t i g e n b i n d i n g c a p a c i t y due t o the o r i e n t e d c o u p l i n g through the c a r b o h y d r a t e m o i e t i e s o f the Fc domain o f t h e monoclonal a n t i b o d y . S i n c e the N H S - d e r i v a t i v e s o f agarose and NuGel were r e a d i l y a v a i l a b l e , we used them f o r a l l o f our s t u d i e s . The NuGel immunosorbent has the added advantages o f b e i n g a d u r a b l e b e d s u p p o r t and a l l o w i n g a f o u r - f o l d i n c r e a s e i n f l u x (23) as compared to the c r o s s - l i n k e d agarose. These a r e i m p o r t a n t f a c t o r s t o c o n s i ­ der when s c a l i n g up the p r o d u c t i o n o f recombinant p r o t e i n s . D e t e c t i o n and P r e v e n t i o n o f A n t i b o d y

Leaching

from

Immunosorbents

The c o v a l e n t bond formed between the a n t i b o d y and the m a t r i x d u r i n g i m m o b i l i z a t i o n may n o t be c o m p l e t e l y s t a b l e . T r a c e amounts o f i m m o b i l i z e d a n t i b o d y may l e a c h from the column d u r i n g the immuno­ a f f i n i t y p u r i f i c a t i o n p r o c e s s , thereby c o n t a m i n a t i n g the f i n a l b u l k product. D e t e c t i o n Method. A s e n s i t i v e , n o n - c o m p e t i t i v e , sandwich ELISA i s used t o d e t e c t a n t i b o d y l e a c h i n g from the immunosorbent d u r i n g column o p e r a t i o n s ( d a t a n o t shown). The lower l i m i t o f the a s s a y ' s s e n s i t i v i t y i s 0.1 ng/ml. S t a b i l i z a t i o n o f Immobilized Monoclonal A n t i b o d i e s . The use o f g l u t a r a l d e h y d e c r o s s l i n k i n g to prevent immobilized p r o t e i n leakage has been r e p o r t e d p r e v i o u s l y ( 2 7 ) . We have s u c c e s s f u l l y used t h i s

11.

BAILON AND ROY

157

Recovery of Recombinant Proteins

100

80

c Φ υ

60

»_ Φ

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch011

û.

40

20

ο % Recovery of Immunoreactivity Following NHS Coupling -

• % Recovery of Immunoreactivity Following CMO Coupling • NHS Coupling Efficiency • CHO Coupling Efficiency I I I 10 Coupling pH

F i g u r e 2. E f f e c t o f pH on C o u p l i n g E f f i c i e n c y and I m m u n o r e a c t i v i t y .

Table I I I .

Ab C o u p l e d (mg/ml) 0.6 3.1 6.2 11.7 17.7 24.0

E f f e c t o f Antibody

Binding Capacity (mg/ml) 0.13 0.45 0.76 1.43 1.86 1.76

Concentration

R e s i d u a l Immunoreactivity Expected Observed Recovery (nmoles/ml) (%) 8 39 78 148 224 304

7 23 40 74 97 92

89 60 51 50 43 30

Monoclonal antibody to i n t e r f e r o n - a l p h a A a t v a r i o u s concentra­ t i o n s (1-25 mg/ml) was i m m o b i l i z e d on NuGel-NHS e s t e r d e r i v a ­ t i v e a c c o r d i n g t o the p r o c e d u r e d e s c r i b e d i n t h e t e x t . I m m u n o r e a c t i v i t i e s a r e c a l c u l a t e d t a k i n g i n t o a c c o u n t t h e two b i n d i n g s i t e s o f IgG p e r m o l e c u l e and t h e m o l e c u l a r w e i g h t o f 158 Kd. M o l e c u l a r weight o f r I F N - a l p h a 2a i s t a k e n as 19.2 Kd f o r the c a l c u l a t i o n o f observed i m m u n o r e a c t i v i t i e s .

158

PROTEIN PURIFICATION

t e c h n i q u e to s t a b i l i z e the c o v a l e n t l y bonded a n t i b o d y and the r e s u l t s are summarized i n T a b l e V. At low c o n c e n t r a t i o n s o f g l u t a r a l d e h y d e and under c o n t r o l l e d c o n t a c t time the a n t i b o d y l e a c h i n g from immunosorbents was r e d u c e d to n o n - d e t e c t a b l e l e v e l s , w i t h o u t s i g n i f i c a n t l o s s i n immunoreactivities .

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch011

F a s t Assays

f o r M o n i t o r i n g Downstream P u r i f i c a t i o n

Steps

The s u c c e s s o f l a r g e - s c a l e p u r i f i c a t i o n p r o c e s s e s depends upon the a v a i l a b i l i t y o f r a p i d and r e l i a b l e a s s a y s f o r m o n i t o r i n g the downstream p u r i f i c a t i o n s t e p s . B i o a s s a y s and ELISAs a r e time consuming and o f t e n g i v e ambiguous r e s u l t s , e s p e c i a l l y i n the e a r l y s t a g e s o f the p u r i f i c a t i o n scheme. Roy e t a l (28) r e p o r t e d the use o f an automated h i g h - p e r f o r m a n c e immunosorbent a s s a y f o r m o n i t o r i n g recombinant l e u k o c y t e A i n t e r f e r o n p u r i f i c a t i o n s t e p s . S i m i l a r immunoa d s o r b e n t a s s a y s are found to be a f a s t and s i m p l e way o f m o n i t o r i n g the downstream p u r i f i c a t i o n o f o t h e r recombinant p r o t e i n s . Immunoaffinity

Purification

Procedures

In t h i s s e c t i o n , we f o c u s our a t t e n t i o n on the p r o c e d u r e s i n v o l v e d i n the i m m u n o a f f i n i t y p u r i f i c a t i o n o f c l i n i c a l grade recombinant p r o t e i n s from c o l i and c u l t u r e d c e l l s u p e r n a t a n t s . T h e o r e t i c a l a s p e c t s o f the f a c t o r s i n v o l v e d i n the l a r g e - s c a l e i m m u n o a f f i n i t y p u r i f i c a t i o n p r o c e s s have been r e v i e w e d by Chase ( 2 9 ) . In h i s review a r t i c l e , Sharma (30) has p r e s e n t e d an e x c e l l e n t overview o f the r e c o v e r y o f recombinant p r o t e i n s from E . c o l i . Use o f immunoa f f i n i t y chromatography i n the p u r i f i c a t i o n o f b i o m o l e c u l e s from c u l t u r e d c e l l s i s d e s c r i b e d by B o s c h e t t i e t a l ( 3 1 ) . G e n e r a l p u r i f i c a t i o n schemes f o r the p r o d u c t i o n o f p h a r m a c e u t i c a l grade recombinant p r o t e i n s from m i c r o b i a l and mammalian s o u r c e s are g i v e n below i n F i g u r e 3. S o l u b i l i z a t i o n and R e n a t u r a t i o n o f Recombinant P r o t e i n s E x t r a c t i o n o f the d e s i r e d p r o t e i n from JL_ c o l i i n i t s n a t i v e form poses unique problems, e s p e c i a l l y when the p r o t e i n i s e x p r e s s e d i n h i g h c o n c e n t r a t i o n s i n an i n s o l u b l e form w i t h i n the i n c l u s i o n b o d i e s . The c e l l s a r e d i s r u p t e d by m e c h a n i c a l , enzymatic or chemic a l means. For example, i n t e r f e r o n i s e x t r a c t e d by s i m p l y s t i r r i n g the f r o z e n and thawed c e l l s i n b u f f e r s c o n t a i n i n g dénaturants l i k e g u a n i d i n e h y d r o c h l o r i d e (GuHCl) and n o n - i o n i c d e t e r g e n t s such as T r i t o n X-100, Tween-20, e t c . Recombinant i n t e r l e u k i n - 1 i s s o l u b i l i z e d by s i m p l e e x t r a c t i o n a f t e r h o m o g e n i z a t i o n . S i n c e recombinant i n t e r l e u k i n - 2 ( r I L - 2 ) i s e x p r e s s e d i n JL_ c o l i i n h i g h c o n c e n t r a t i o n s w i t h i n the i n c l u s i o n b o d i e s , s p e c i a l t r e a t m e n t s a r e required. These i n c l u d e h o m o g e n i z a t i o n , i s o l a t i o n o f i n c l u s i o n b o d i e s , washing the i n c l u s i o n b o d i e s to remove unwanted c e l l u l a r p r o t e i n s , s o l u b i l i z i n g the r I L - 2 w i t h s t r o n g dénaturants such as 7M GuHCl and f i n a l l y , d i l u t i n g the e x t r a c t and g i v i n g the p r o t e i n enough time to r e f o l d . S t u d i e s c o n d u c t e d by L i g h t (32) have shown t h a t o p t i m a l r e f o l d i n g o c c u r s when the p r o t e i n c o n c e n t r a t i o n i s a t o r below the micromolar range. Consequently, r e l a t i v e l y large d i l u t i o n s o f the d e n a t u r e d e x t r a c t s , f o l l o w e d by a g i n g f o r v a r i o u s

11.

BAILON AND ROY

Recovery ofRecombinant Proteins

T a b l e IV. R e s i d u a l I m m u n o r e a c t i v i t i e s

Immunosorbents Ab Coupled (mg/ml)

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch011

Agarose-NHS NuGel-NHS Agarose-CI NuGel-CI Agarose-CHO NuGel-CHO NuGel-Hydrazide

Immunoreactivities

o f V a r i o u s Immunosorbents

B i n d i n g Capac. (mg/ml)

Residual Immunoreactivity Recovery Observed Expected (nmoles/ml) (%)

a r e c a l c u l a t e d as i n T a b l e I I I .

T a b l e V. D e t e c t i o n and P r e v e n t i o n o f A n t i b o d y

Glutaraldehyde % (v/v) 0 0 0 1

* **

50 50 34 25 41 48 73

74 70 54 40 77 76 77

148 140 157 158 186 157 106

1.43 1.34 1.03 0.77 1.48 1.45 1.47

11.7 11.1 12.4 12.5 14.7 12.4 8.4

159

0 1 5 0

Leaching

Agarose-NHS ng/ml* %**

Agarose-CHO ng/ml %**

8 2 0 0

10 2 0 0

100 97 96 96

2 2 5 1

1 7 0 0

100 99 96 95

Antibody l e a c h i n g Residual Immunoreactivities Immunoadsorbents were t r e a t e d w i t h b i f u n c t i o n a l f o l l o w e d by N a B H ^ r e d u c t i o n .

glutaraldehyde

I E.coli Cells I Cell Membrane Preparation Extraction, Dilution and Concentration/Diafiltration

Cell Culture Supernatant

Immunoadsorbent Column 1

Gel Filtration

j

Concentration/Diafiltration

ι

~~

Bulking F i g u r e 3. I m m u n o a f f i n i t y

P u r i f i c a t i o n Schemes f o r Recombinant P r o t e i n s

160

PROTEIN PURIFICATION

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch011

p e r i o d s o f time are r e q u i r e d . The r e s u l t s o f such an a g i n g o r r e f o l d i n g experiment c o n d u c t e d w i t h 7M GuHCl e x t r a c t e d r I L - 2 a f t e r 4 0 - f o l d d i l u t i o n w i t h PBS i s g i v e n i n T a b l e V I . In some i n s t a n c e s , the i n c l u s i o n o f r e d u c i n g agents l i k e DTT i n the e x t r a c t i o n b u f f e r i s h e l p f u l i n s o l u b i l i z i n g the recombinant p r o t e i n s . When r e d u c i n g a g e n t s a r e used i n t h e e x t r a c t i o n b u f f e r , care s h o u l d be taken t o lower the c o n c e n t r a t i o n o f the r e d u c i n g agents so t h a t they w i l l n o t a d v e r s e l y a f f e c t the a f f i n i t y adsorbents which c o n t a i n d i s u l f i d e bonds; f o r example a n t i b o d y o r r e c e p t o r adsorbent columns. I n g e n e r a l , each recombinant p r o t e i n may r e q u i r e customized e x t r a c t i o n p r o c e d u r e s . U s u a l l y no s p e c i a l treatments a r e needed f o r the c e l l c u l t u r e s u p e r n a t a n t s and they can be a p p l i e d to the a d s o r b e n t column a f t e r a s i m p l e f i l t r a t i o n step. Adsorption A d s o r p t i o n i s one o f the most c r i t i c a l a s p e c t s o f i m m u n o a f f i n i t y chromatography. D u r i n g a d s o r p t i o n , the crude m a t e r i a l i s k e p t i n a b u f f e r which a l l o w s maximum a d s o r p t i o n . I n o r d e r t o e n s u r e t h a t no p r o d u c t i s wasted d u r i n g the a d s o r p t i o n phase, s u f f i c i e n t c o n t a c t time between the s o l u b l e a n t i g e n and the immunosorbent i s m a i n t a i n e d by c a r e f u l l y c h o o s i n g the f l o w r a t e . Washing The purpose o f washing t h e immunosorbent immediately after adsorption i s two-fold: (1) t o remove the c r u d e m a t e r i a l s from w i t h i n o r s u r r o u n d i n g the immunosorbent beads and (2) t o remove m a t e r i a l s n o n - s p e c i f i c a l l y bound e i t h e r t o the s u p p o r t o r t o the i m m o b i l i z e d a n t i b o d y . N o n - s p e c i f i c b i n d i n g t o the s u p p o r t c a n be m i n i m i z e d , b u t i s r a r e l y e l i m i n a t e d c o m p l e t e l y . E l e c t r o s t a t i c as w e l l as hydrophobic i n t e r a c t i o n s between the IgG m o l e c u l e and extraneous m a t e r i a l s i n the crude e x t r a c t a r e another s o u r c e o f n o n - s p e c i f i c b i n d i n g . These n o n - s p e c i f i c a l l y bound c o n t a m i n a n t s c a n u s u a l l y be reduced t o low l e v e l s by washing e x t e n s i v e l y w i t h b u f f e r s c o n t a i n i n g s a l t s a t n e u t r a l o r s l i g h t l y a l k a l i n e pH o r by i n c l u s i o n o f low c o n c e n t r a t i o n s o f n o n - i o n i c d e t e r g e n t s i n the s t a r t i n g m a t e r i a l s and i n a l l b u f f e r s used f o r washing. Elution The e l u t i o n o f adsorbed a n t i g e n from the immunosorbent i s a c h i e v e d by c a u s i n g the d i s s o c i a t i o n o f the a n t i g e n - a n t i b o d y complex. N o n - s p e c i f i c e l u t i o n methods a r e commonly used f o r the d e s o r p t i o n o f a n t i g e n s . These e l u e n t s i n v o l v e low o r h i g h pH b u f f e r s , p r o t e i n dénaturants such as u r e a o r GuHCl and c h a o t r o p i c agents l i k e potassium t h i o c y a n a t e . I f the a n t i g e n i n v o l v e d i s s t a b l e a t a c i d i c pH and i s r e a d i l y e l u t e d from the immunosorbent under t h e s e c o n d i t i o n s , an e l u e n t o f c h o i c e i s a low pH (kî 42,47 Formic acid DÎP 48,49 Acetic acid DÎP 50 Cyanogen bromide MÏ 51,52 BNPS-skatole W 53 o-iodosobenzoic acid Wi 54 N-chlorosuccinimide Wi d Enzyme Commercial Sources ? Chymotrypsin W i and YI and Fi 45 BoCbCz Si, Collagenase P-XiG-P' 15,37-40 BoCbSi Endoproteinase Lys-C KÎ 8 46 BoCb Enterokinase D-D-D-D-Ki Table VI Si Factor X I-E-G-Ri V Table Π Bo Si Kallikrein P-F-RiM 18 BoCbSi Renin Y-I.H-P-F-H-LW 8 CbSi H64A subtilisin BPN' A-A-H-Yi Table IV r Thrombin R-G-P-Rii' Table III Bo Si Trypsin Rj, and K i ° 11,41-44,77 BoCbCzPiSi Ubiquitin protein peptidase Ubiquitini Ρ Table V N/A Chemical methods for fragmenting polypeptides are reviewed in ±. Many additional examples of using cyanogen bromide to cleave fusion proteins are given in Table 2 from 1. Several different methods for cleaving fusion proteins on the C-terminal side of Trp are compared in 55- Nilsson, B.; Forsberg, G.; Hartmanis, M. Methods Enzvmol.. in press. Primary and secondary sub-site specificities of α-chymotrypsin are examined in 56- Consensus site in collagen which is cleaved by collagenase (57). # Cleavage sites for Lys-C with melittin as a substrate (Boehringer Mannheim Biochemicals: manufacturer's.literature). Enterokinase cleavage site in its natural substrate, trypsinogen (5$)· Factor X cleavage sites in its natural substrate, thrombin (59). J Kinetic parameters with a large number of chromogenic and fluorogenic substrates are summarized in 25 and 26· Cleavage site for plasma kallikrein in kininogen (60). 'Minimal fragment of natural substrate, angiotensinogen, required for cleavage by renin (36). Most favorable sequence identified from analysis of tetrapeptide p-nitroanilide substrates (2). Thrombin cleavage site in its natural substrate fibrinogen (61). ° Primary and sub-site specificities are reviewed in £2.. Ρ Available evidence suggests that ubiquitin as the N-terminal part of a fusion protein is necessary but not sufficient for cleavage to occur (22). ? Commercial sources of enzyme are provided only as a guide to availability - other unnamed sources may be superior: Bo, Boehringer Mannheim Biochemicals; Cb, Calbiochem; Cz, Calzyme; Pi, Pierce; Si, Sigma Chemical Company; N/A not commercially available. H64A subtilisin BPN' variants may be obtained for non-commercial research use upon request to the author or Dr James A. Wells at the Department of Biomolecular Chemistry, Genentech Inc, 460 Point San Bruno Boulevard, South San Francisco, CA 94080. BNPS-skatole, 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine. b

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of the target site may be compromised by neighboring sequences on either side of the scissile bond. One strategy for enhancing the accessibility to proteases is to flank the target sequence on both sides with short stretches of glycine residues Q2) or on one side if the correct terminus of the protein of interest is required after cleavage (13). An alternative strategy to enhance substrate accessibility is to perform the digests under denaturing or reducing conditions (provided that this is compatible with the protease used) or by denaturation of the substrate prior to digestion (14). It may be possible to design a C-termi­ nal fusion protein such that the target linker is an inherently accessible site in the affinity handle, e.g. by overlapping a known protease sensitive site CD-

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Problems Encountered in Site-Specific Proteolysis A major problem encountered in attempting site-specific proteolysis is that of non-specific cleavage at additional sites to the target site. This may be due to the "site-specific protease" itself or contaminating proteolytic activity in preparations of either the protease or fusion protein. Contaminating proteases may be overcome by the judicious addition of protease inhibitors to the digest, or by further purification of the substrate or enzyme (15). In the case where the additional cleavage events are due to the "site-specific protease", optimizing the digestion conditions to give partial proteolysis may give an acceptable yield of intact target protein (16). Failing this, one may have to adopt an alternative enzymatic (1Q) or chemical cleavage (12) method. A second significant difficulty in achieving site-specific proteolysis is inefficient (or no) cleavage at the target site. This may reflect inaccessibility of the target site (see above) or the substrate specificity of the protease on the C-terminal side of the scissile bond. Many of the proteases in Table I will not cleave on the N-terminal side of proline. One strategy to circumvent this problem is to add amino acids to the N-terminus of the protein of interest and subsequently remove them using an aminopeptidase (18) or diaminopeptidase. If other methods do not enhance cleavage at the target site it may be necessary to try a different protease and fusion protein (19). An infrequent problem is that of insolubility of the fusion protein or cleavage products, which may usually be overcome by including low concentrations of dénaturant (or detergent) in the digest, provided that this is compatible with the cleavage method used (20). Proteolysis in vivo is sometimes a major problem in the use of fusion proteins. It is highly desirable to obtain fusion proteins as free of proteolysis products as possible: if the fusion protein is heterogeneous (or contaminated with proteases) it may difficult to interpret the outcome of digestion experiments or to separate the protein of interest away from degradation products (P.C., unpublished data). A variety of strategies have been used to curb proteolysis in vivo, including the use of protease deficient host strains and optimization of growth media and temperature (reviewed in 1-2. Uhlén, M . ; Moks, T. Methods Enzymol.. in press). Intracellular expression of fusion proteins in E. coli often results in the accumulation of the fusion protein in insoluble aggregates known as "inclusion" or "retractile bodies". This has the benefit of protecting the fusion protein from proteolysis as well as allowing the fusion protein to be readily separated from most E. coli proteins and also nucleic acids. A disadvantage is that it necessitates refolding the protein of interest into the native conformation, although in many cases this is possible (see Kelley, R. F.; Winkler, M . E. In Genetic Engineering: Principle and Methods: Setlow, J., Ed.; Plenum: London; Vol. 12, in press). Insulin-like growth factor II (IGF-II) is susceptible to proteolysis in E. coli but has been successfully expressed in a soluble form using a dual affinity fusion protein strategy (21). A tripartite fusion protein was constructed in which IGF-II was flanked on the N-terminal side by the IgG-binding domains of staphylococcal protein A and on the C-terminal side by the albumin-binding domains of streptococcal protein G. Recovery of full length IGF-II was ensured by sequential affinity purifications on IgG and albumin matrices. Furthermore this was facilitated by the observation that IGF-II in the tripartite fusion protein is less prone to proteolytic degradation in E. coli than when expressed as the N-terminal fusion protein lacking the albumin-binding domains. IGF-II was then excised from the tripartite fusion by the use of CNBr which also leaves a homoserine or homoserine lactone at the C-terminus. Thus one limitation of this dual affinity fusion protein strategy is that existing cleavage methods include specificity determinants of one or more residues on the N-terminal side of the scissile bond, which may preclude generation of the correct C-terminus. A potential solution of this difficulty is the use of H64A subtilisin BPN' variants (see below). A potential limitation (not a problem for IGF-II) of dual affinity fusions is that of compromising the correct folding of the protein of interest, which may necessitate refolding in vitro (see Kelley, R. F.; Winkler, M . E. In Genetic Engineering: Principle and Methods: Setlow, J., Ed.; Plenum: London; Vol. 12, in press).

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Survey of Enzvmes Used for Site-Specific Proteolysis Factor X . To date the most widely used protease for site-specific proteolysis of fusion proteins has been the blood coagulation factor X . This is a consequence of the pioneering work of Nagai and Th0gersen, who developed this cleavage method in conjunction with a versatile E. coli intracellular expression system (see 22 for a practical review). Factor X has been used successfully with a variety of fusion protein systems (Table II) including those in which the N-terminal region is protein A, β-galactosidase, maltose binding protein or glutathione S transferase, where efficient affinity purification methods are available (reviewed by Uhlén, M.; Moks, T. Methods EnzymoL. in press). a

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Table II. Fusion Proteins Cleaved by Factor X

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Substrate Residues* P3 P2 PI PI' Ε R V G Ε G R S Ε G R V Ε G R G Ε G R S Ε G R D Ε G R A Ε G R M Ε G R G Ε G R F Ε G R S Ε G R M ? Ε G R Ε G R D Ε G R Κ Ε G R Κ Ε G R S Ε G R H Ε G R Τ Ε G R G Ε G R Ν Ε G R Τ Ε G R Τ Ε G R G Ε G R M Ε G R Y Ε G R S

P2' H Ρ L L Y D Ρ A Ε Τ S Ν ? À Ε G D G V D S F ?

i Ε A Ρ

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Unique cleavage Fusion protein site? Ref. Yes* 63 cII/B-globin cil / p-globin (mutant) Yes 22 cil / oc-globin Yes 22 cil / myoglobin Yes 22 cII/TMV coat protein No 17 cil / actin Yes 22 cII/MLC No 22 cil / troponin C Yes 64 ? cII/ΤΠΙΙΑ 22 cII/MATal No 16 cil / ferredoxin Yes 65 cII/PDE γ subunit Yes 66 cII/MLC/IGF-II receptor tail Yes 67 cII/MOPC315V Yes 68 β-gal / ribonucleaseA No* 9 P-gah-375/pl5orp24 NoAla. Specificity determinants for subtilisin extendfromP4 to P2' (80). This enzyme is also known as Genenase I. Analysis of tetrapeptide p-nitroanilide substrates suggests that A is preferable to F at the P4 position Q). X represents the 20 common amino acids except proline and isoleucine. /N-succinylated. S Very slow cleavage within the Ζ domain was detected with a His residue at the ΡΓ position (see text). Nilsson, B.; Forsberg, G.; Hartmanis, M.; Methods Enzvmol.. in press. ND, not determined; AP, alkaline phosphatase; ACTHi_io* residues 1 to 10 of adrenocorticotropic hormone. a

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PROTEIN PURIFICATION Table V. Fusion Proteins Cleaved by Ubiquitin Protein Peptidase Unique Substrate residues* cleavage P4 P3 P2 PI P i ' P2' Fusion protein site ? Ref. L R 32 G ubiquitin / relaxin A chain Yes G L Q L R G G D ubiquitin / relaxin B chain Yes 32 s ubiquitin /Xis L R G G M Y Yes 32 ubiquitin / X* L R G G G Yes 32 s L R G G E F ubiquitin / rlx Yes c L R G G N T ubiquitin / GAP Yes c Nomenclature of Schechter and Berger (£) - see Table II. X corresponds to: G S-P-G-E-L-E-F-T-G-R-R-F-T-T-S. Liu, C.-C; Miller, H. I.; Kohr, W. J.; Silber, J. I. J. Biol. Chem.. in press. Xis, product of Xis gene from bacteriophage λ; rlx, 38 residue peptide including 32 residues from human prorelaxin; GAP, rat gonadotropin associated protein. a

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Cleavage of target linkers has always occurred with the histidine residue at the expected P2 position of the substrate (F-A-H-YÎ and A - A - H - Y l - see Table IV). In a few cases cleavage has also been detected at an additional site with a histidine residue at the ΡΓ position (P. C , J. Wells, R. Vandlen, K. Miller and S. Braxton, unpublished data). Further studies are underway to investigate the substrate requirements for proteolysis assisted by histidine at ΡΓ. It may be possible to exploit this phenomenon for site-specific proteolysis of tripartite (21) or C-terminal fusion proteins, which both include extensions C-terminal to the protein of interest. Ubiquitin Protein Peptidase. Several different enzymes are involved in the removal of ubiquitin from proteins in eukaryotes including ubiquitin protein peptidase, which has been cloned from yeast (32). Ubiquitin protein peptidase has been used for site-specific proteolysis of a number of fusion proteins (Table V). However its utility appears to be limited by a requirement for ubiquitin at the N-terminus of a fusion protein substrate (32). Furthermore this enzyme requires the presence of a reducing agent for activity in vitro and it is most efficacious against low molecular weight substrates. The C-terminal glycine (G76) in ubiquitin is apparently important since replacement by either valine or cysteine (or deletion) abolishes detectable cleavage (32). The specificity for the residue on the C-terminal side of the scissile bond (ΡΓ) is evidently broad, with at least E, D, C, G, Τ and M representing favorable residues. Only proline at Ρ Γ seems to be incompatible with cleavage. A unique feature of ubiquitin protein peptidase compared with other proteases shown in Table I, is that suitable fusion proteins may be specifically cleaved in vivo in E. coli over-expressing ubiquitin protein peptidase (32) or by the endogenous ubiquitinhydrolyzing activity in yeast (22). This provides a means for the heterologous expression of eukaryotic proteins and generating the correct N-termini, which may not be possible by direct expression, because a methionine residue derived from the initiation codon is often left at the N-terminus. Enterokinase. The serine protease, enterokinase, activates trypsinogen to trypsin in vivo and in vitro (34) by specific cleavage of the peptide bond between K6 and 17. Enterokinase has been used for site-specific proteolysis of fusion proteins in which the N-terminal 8 residues includes the required cleavage sequence but also represents the epitope for a Ca -dependent monoclonal antibody which enables affinity purification of the fusion protein (flag™ system - see Table VI). Enterokinase is capable of generating products with at least E, A, T, L and I at the N-terminus of the protein of interest. Enterokinase is commercially available (see Table I) but may require further purification to remove contaminating proteases such as chymotrypsin, trypsin and elastase (35). 2+

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Table VI. Fusion Proteins Cleaved by Enterokinase Unique Substrate residues* cleavage P4 P3 P2 PI ΡΓ Ρ2' Fusion protein site? Ref. D D D Κ Α Ρ Yes 81 fW7IL-3 D D D Κ Α Ρ flag*7IL-4 Yes 81 D D D Κ Α Ρ flag*/IL-2 No 35 D D D Κ Α Ρ flag*/GM-CSF No 35 D D D Κ Α Ρ flag*/CSF-I No 35 D D D Κ Α Ρ flag*7G-CSF No 35 Nomenclature of Schechter and Berger (© - see Table II. Flag corresponds to the octapeptide sequence: D-Y-K-D-D-D-D-K QS. A small amount of internal cleavage was detected representing < 10% of the total fusion protein. IL-3, IL-4 and IL-2 are interleukins 3, 4 and 2 respectively; GM-CSF, granulocyte-macro­ phage colony stimulating factor; CSF-I, colony stimulating factor; G-CSF, granulo­ cyte colony stimulating factor. c c c

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Renin. The aspartyl protease, renin, specifically cleaves angiotensinogen to release angiotensin I in vivo (Table I). Murine submaxillary gland renin has been used for sitespecific proteolysis of a fusion protein (190 K) between Epstein-Barr virus membrane antigen and β-galactosidase (&). Quantitative cleavage at the target linker (H-P-F-H-LiLV) was obtained with little (if any) cleavage at additional sites. The specificity determinants for renin appear to extend for one or more residues on the C-terminal side of the scissile bond, which may limit its utility (26)· Collaeenase. Although microbial collagenase was one of the first proteases used for site-specific proteolysis of fusion proteins (37). it has not yet enjoyed widespread use. A major limitation is that collagenase linkers Q£, 37-39) are long (-60 residues) and contain multiple cut sites for collagenase, which often gives rise to product heterogeneity. Furthermore, even if cleavage of the linker proceeds to completion, the protein of interest is left with an extension of at least a few residues from collagen. Recently however these problems with collagenase have been largely overcome: efficient cleavage of a fusion protein between alkaline phosphatase and adrenocorticotropic hormone (ACTH) has been demonstrated at short target linkers, G-(P-X-G) -P, where η > 2 (4Q). Furthermore it was possible to generate ACTH with the correct N-terminus by subsequent cleavage with dipeptidyl aminopeptidase-IV (Table VII). A variety of collagenases are commercially available (see Table I) but further purification is generally Q£, 37-39) but not always (40) required to remove contaminating proteases. n

Trypsin and Chymotrypsin. If the protein of interest is highly resistant to proteolysis, it may be possible to achieve site-specific proteolysis using proteases such as trypsin (11,4L· 44., 22) or chymotrypsin (45). which are cheap, widely available, and very well characterized. For example, intact human myoglobin was released from a fusion protein with the N-terminus of bacteriophage λ cil protein by trypsin cleavage (11). This had the side-benefit of degradation of contaminating proteins thus decreasing the need for further purification enabling myoglobin to be economically and efficiently prepared on a gram scale. The action of trypsin is readily restricted to arginine residues by citraconylation (reversible) of lysine residues (41). Endoproteinase Lvs-C. The serine endoproteinase Lys-C has primary substrate specificity similar to trypsin but narrower in that it cleaves only on the C-terminal side of lysine residues. Lys-C has been used to specifically cleave a fusion protein between trpE (residues 1 to 320) and epidermal growth factor, which has no lysine residues (46). This cleavage was difficult to reproduce because of variation between different batches of enzyme. Today however, reliable albeit expensive commercial sources of Lys-C from Lysobacter enzymogenes are available (see Table I).

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PROTEIN PURIFICATION Table VII. Enzymes for Modification of Amino Termini Enzyme Substrate Specificity* Example Substrate Ref. H N-MiA-P... Met-IL2 82 Aminopeptidase M H N-M>lX-P H N-AiP-P... Ala-IL6 18 Aminopeptidase P° H N-XiP DAP-I** (cathepsin C) H N-X!-X iX3 H N-M-ViF-P... Met-Asp-bGH 83 H N-M-DlF-P... Met-Val-bGH 83 X ,X3*P; H N-M-ViF-P... Ala-Glu-hGH 84 Xl*K DAP-IV H N-Xi-X iX3 H N-G-PlS-Y... Gly-Pro-ACTH 40 X = Ρ (or A) For details see 85. Commercially available from Bo, Cb, Si, and Pi - see Table I. Not commercially available (?). Commercially availablefromBo and Si. bGH and hGH are bovine and human growth hormones respectively; DAP, dipeptidylaminopeptidase. b

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Kallikrein. Plasma kallikrein is a serine protease that specifically cleaves kininogen to release bradykinin in vivo. Human plasma kallikrein has been used successfully to cleave a fusion protein containing human interleukin-2 at the N-terminus and interleukin-6 (IL-6) at the C-terminus (18). An extra alanine residue was included before the N-terminal proline of IL-6 to permit cleavage by kallikrein, which will not cleave on the N-terminal side of proline. It was then possible to generate the correct N-terminus of IL-6 by digestion with E. coli aminopeptidase Ρ (Table VII). Aminopeptidases and Dipeptidvlaminopeptidases. Several enzymes have been used to cleave one or more residues from the N-terminus of proteins (Table VII) present after cleavage with some chosen endoprotease Q£, 4Q). The use of these exoproteases also provides an alternative strategy to the use of ubiquitin protein peptidase (see above) for generating the correct N-terminus of heterologously expressed eukaryotic proteins. Conclusion The availability of suitable proteases for site-specific proteolysis has in the past been a major limitation to the use of fusion proteins. This problem has been extensively eroded by more extensive characterization and wider availability of known proteases, by the discovery of new highly specific proteases, and by tailoring the substrate specificity of existing proteases by protein engineering. Acknowledgments The author thanks Drs Bjôm Nilsson, Mathias Uhlén, Steven Weame and Chung-Cheng Liu for generously communicating manuscripts prior to publication and helpful discussions, and Dr Jim Wells for continued support Legend of Symbols The single letter amino acid code has been used: A, Ala; C, Cys; D, Asp; E, Glu; G, Gly; H, His; I, De; K, Lys; L, Leu; M , Met; N , Asn; P, Pro; Q, Gin; R, Arg; S, Ser, T, Thr; V, Val; W,Trp. Literature Cited 1. 2. 3.

Harris, T. J. R. In Genetic Engineering; Williamson, R., Ed.; Academic: London, 1983; Vol. 4, p 127. Marston, F. Α. Ο. Biochem. J. 1986, 240, 1. Kleid, D. G.; Yansura, D.; Small, B.; Dowbenko, D.; Moore, D. M.; Grubman, M. J.; McKercher, P. D.; Morgan, D. O.; Robertson, B. H.; Bachrach, H. L. Science 1981, 214, 1125.

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48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73.

13. CARTER 74. 75. 76. 77. 78. 79.

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RECEIVED January 30,1990

Chapter 14

Purification Alternatives for IgM (Human) Monoclonal Antibodies G. B. Dove, G. Mitra, G. Roldan, M. A. Shearer, and M.-S. Cho

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch014

Cutter Biological Laboratory, Miles, Inc., Berkeley,CA94704

Methods suitable for purification of IgM monoclonal antibodies from serum-free tissue culture supernatants are described in this case study. We review techniques found to be useful in purifying proteins and the techniques applied to establish u t i l i t y . Partitioning techniques include polyethylene glycol (PEG) precipitation, size exclusion chromatography, anion and cation exchange chromatography, hydroxylapatite chromatography, and immunoaffinity. Modification techniques include the use of enzymes (e.g. DNAse). Protein purity is achieved primarily with precipitation, size exclusion chromatography, and immunoaffinity. DNA removal is greatest with anion exchange, immunoaffinity,and a combination of DNAse and size exclusion chromatography. Virus is partitioned most effectively through hydroxylapatite and immunoaffinity. A cascade of several appropriate steps provides contaminant protein clearance of >100x (purity greater than 99%), DNA clearance of >l,000,000x, and virus clearance of >100,000x. This paper presents a case study of the definition of purification processes for monoclonal IgMs produced by tissue culture fermentation. We review techniques that we have found to be useful and the approaches taken to establish their utility. The monoclonal antibodies are specific to various bacterial antigens for use as a therapeutic product. Serum-containing and serum-free (supplemented with proteins) broths are purified, although the purification methods are optimized for serum-free media. Purification is necessary to remove contaminants introduced by the media and the cells. Contaminants include media components (albumin, transferrin, insulin, and many serum components), nucleic acids, viruses, and other cellular products. 0097-^156790/D427-O194$06.00A) © 1990 American Chemical Society

14. DOVE ET AL.

Purification Alternatives for IgM Monoclonal Antibodies

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch014

Polymers, high molecular weight aggregates and fragments of IgM and albumin must be removed as well. There a r e s e v e r a l approaches t o p u r i f i c a t i o n i n terms o f p r o p e r t i e s (Table 1). The common p r o p e r t i e s are s i z e , charge and i o n i c i n t e r a c t i o n , and a f f i n i t y . Separation i s optimized by ex­ p l o i t i n g (e.g. size between IgM and albumin) or creating differences (e.g. DNAse) between the product IgM and contaminants. Techniques discussed i n t h i s study include f i l t r a t i o n s , pre­ c i p i t a t i o n with PEG and s a l t s (1^. 2^ 3), size exclusion chromatogra­ phy (41. 5. 6), anion (4^. 5)and c a t i o n exchange (7), contact with hydroxylapatite (8_«_ 9) , immunoaff i n i t y , a d d i t i o n of reagents (e.g. DNAse (10)) and combinations of these techniques (11).

Materials and Methods. Monoclonal (human) antibodies of c l a s s M (m-IgM) were derived from human Β lymphocyte c e l l l i n e s , designated A, B, C, D, and E. A n t i ­ body from each l i n e was directed toward a d i f f e r e n t , s p e c i f i c bacte­ r i a l antigen. C e l l s were grown i n suspension c u l t u r e o r hollow f i b e r . P o l y c l o n a l plasma-derived IgM was obtained from Cohn f r a c ­ t i o n I I I (ethanol f r a c t i o n a t i o n of human plasma (12). Chemicals were reagent-grade. S a l t s were obtained from Mallinckrodt, Paris, Ky. Enzymes were obtained from Sigma, St. Louis, Mo. Chromatography r e s i n s and equipment, unless noted otherwise, were obtained from Pharmacia, Uppsala, Sweden. Hydroxylapatite (DNA-Grade Bio-Gel HTP) was obtained from Bio-Rad L a b o r a t o r i e s , Richmond, Ca. Proteins were characterized by SDS-polyacrylamide gels using a 2-10% agarose gradient, stained with either Coomasie Blue or s i l v e r . General product and contaminant analysis were quantitated by Pharma­ c i a FPLC (Fast Protein L i q u i d Chromatography) system with a column matrix o f Superose 6, u t i l i z i n g s i z e e x c l u s i o n chromatography. Buffers defined the state as native, reduced, or denatured. Absorbance at 280 nm. was used f o r approximate measurements. Various ELISA were developed to provide a consistent basis f o r loss of IgM as w e l l as denaturation during processing. An antigen ELISA using anti-u chain IgG detected the Fc region and i s u s e f u l i n monitoring y i e l d . A f u n c t i o n a l ELISA i n d i c a t e d antibody binding e f f i c i e n c y to antigen. Epstein-Barr v i r u s (EBV) was derived from B95-8 c e l l s (13). EBV-specific nuclear antigen (EBNA) was demonstrated by an a n t i ­ complementary immunofluorescence (ACIF) assay with m o d i f i c a t i o n s (14). Residual native DNA was assayed by dot b l o t h y b r i d i z a t i o n analysis (15. 16). P32-labeled DNA was prepared by n i c k - t r a n s l a t i o n of host c e l l DNA isolated from culture harvests of c e l l l i n e C. The DNA was s p i k e d i n t o v a r i o u s p r o c e s s steps and r e c o v e r e d ( 1 7 ) . Samples were spotted to f i l t e r paper, p r e c i p i t a t e d and washed with 10% t r i c h l o r o a c e t i c acid (TCA) and measured by s c i n t i l l a t i o n coun­ ter (LKB, model #1217 Rack Beta).

195

PROTEIN PURIFICATION

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Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch014

Table 1.

Component

IgM

I s o l a t i o n Parameters f o r P u r i f i c a t i o n

Size (xlOOO MW)

800

I s o e l e c t r i c Point (Neutral Charge)

Other Characteristics

pH 6-6.5

Contaminants : Albumin DNA

69 variable

pH 5 pH 5

Viruses

>1000

pH 4-6

HAPT DNAse

Basis of Separation: Albumin DNA

SEC

Viruses All

SEC

IEC

SEC: size exclusion chromatography IEC: ion exchange chromatography HAPT: hydroxylapatite DNAse: degradation by DNAse Inactivation: elimination of b i o l o g i c a l a c t i v i t y

HAPT DNAse Inactivation Immunoaffinity

14. DOVE ET A L

Purification Alternatives for IgM Monoclonal Antibodies

Clearance values were calculated by a r a t i o of concentrations: (Contaminants) p u r i f i c a t i o n step (IgM) = Clearance b

e

f

o

r

e

(Contaminants) i (IgM) For example, 99% removal of contaminants with 100% y i e l d generates a factor of lOOx. Contaminants included proteins, DNA, and viruses. a

f

t

e

r

p

u

r

i

f

i

c

a

t

o

n

s

t

e

p

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch014

Results and Discussion. P r e c i p i t a t i o n . C l a r i f i e d harvests were concentrated on 100,000 MW membrane, adjusted to s p e c i f i c pH and p r e c i p i t a t e d with PEG (Table 2, c e l l l i n e B). Low pH and high PEG concentration resulted i n the highest y i e l d o f IgM and the lowest p u r i t y . I t should be noted that, as the p u r i t y of the i n i t i a l m a t e r i a l increased, the y i e l d from p r e c i p i t a t i o n with PEG increased. S t a b i l i t y data substantiate t h i s observation; IgM Is s t a b i l i z e d i n s o l u t i o n by proteins (e.g. albumin). Further, IgM at a concentration of l e s s than 50 ug/ml p r e c i p i t a t e d poorly. P r e c i p i t a t i o n of high molecular weight aggregates with low concentrations of PEG were studied. 1% and 2% PEG produced only s l i g h t reductions of aggregates, as measured by FPLC-Superose 6. P r e c i p i t a t i o n with other agents and r e p r e c i p i t a t i o n o f an i n i t i a l p r e c i p i t a t i o n were examined, u t i l i z i n g ammmonium s u l f a t e , dextran sulfate, ethanol, and boric acid (Table 2). With the excep­ t i o n of ammonium s u l f a t e , these agents d i d not p r e c i p i t a t e IgM at the c o n c e n t r a t i o n s t e s t e d . R e p r e c i p i t a t i o n gave low y i e l d s and unremarkable p u r i t y with a l l agents. Size Exclusion Chromatography (Gel F i l t r a t i o n ) . I n i t i a l experiments with plasma-derived IgM demonstrated good separation of IgM from contaminants on Sephacryl S-300, an a c r y l i c based g e l . However, separation and y i e l d were poor with monoclonal IgMs. Sepharose CL6B, an agarose based g e l , produced e x c e l l e n t r e s o l u t i o n from albu­ min. A t y p i c a l a n a l y t i c a l chromatogram o f a PEG p r e c i p i t a t e on FPLC-Superose 6 (30 χ 1 cm. dia.) i s shown i n Figure 1, compared to a t y p i c a l l a r g e - s c a l e run (80 χ 37 cm. d i a . ) . The leading ( l e f t side) peak consisted of aggregates of IgM and albumin, the second ( l a r g e s t ) peak was IgM, and the t h i r d peak was p r i m a r i l y albumin with minor low molecular weight fragments. Clearance of DNA was approximately lOx. Yields were improved by high s a l t concentration, which reduced non-specific binding and increased s t a b i l i t y . Howev­ er, several workers have demonstrated improved separation with the use of both low and high ionic strength buffers (4j_ 18) . Degradation of DNA by DNAse. Endogenous or exogenous DNAses degrade the contaminant DNA by enzymatic cleavage, changing the s i z e and charge of the DNA and thereby a l t e r i n g the e f f i c i e n c y of the separa­ t i o n cascade between the product (IgM) and contaminant (DNA).

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PROTEIN PURIFICATION

198

Table 2.

Agent

P r e c i p i t a t i o n by Various Agents

Concentration of agent (%)

pH

Yield (%)

Purity (% IgM)

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch014

Samples and agent were mixed f o r 1 hr. at 4 C. Line B, 200 ug/ml. PEG 10 PEG 8 PEG 5

7.4 7.4 7.4

84 75 20

80-95 95+ 95+

PEG PEG PEG PEG

5 5 10 10

5.5 7.4 5.5 7.4

66 10 95 86

95+ 98+ 80-95 80-95

E, 100 ug/ml, Sulfate 18 Sulfate 18 Sulfate 24 Sulfate 31

6.5 7.2 7.2 7.2

7 0 49 57

45

Line C, 100 ug/ml, Amm. Sulfate 24 PEG 12

7.2 5.5

79 96

80 60

39 80 0 71 2 0 0

85 80

Line Amm. Amm. Amm. Amm.

The PEG p r e c i p i t a t e of l i n e C was reprecipitated by each of the following: PEG 10 PEG 12 Amm. Sulfate 11 Amm. Sulfate 24 Dextran Sulfate 10 Ethanol 25 Boric Acid 5 5.0

Amm:

Ammonium

90 38

90

Purification Alternatives for IgM Monoclonal Antibodies

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch014

14. DOVE ET AL.

Fraction Figure 1. A. B.

Chromatograms of size exclusion (SEC). FPLC-Superose 6, 30 χ 1.0 cm. d i a . Sepharose CL-6B, 80 χ 37 cm. d i a . Load: p r e c i p i t a t e by PEG.

199

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200

PROTEIN PURIFICATION

Exogenous DNAses were added to the p u r i f i c a t i o n processes to accelerate the e f f e c t of the endogenous enzymes, and degraded v i r t u a l l y a l l of the DNA q u i c k l y to allow separation. S p e c i f i c a l l y , i n p r e c i p i t a t i o n s or s i z e exclusion chromatography, reduction of the molecular weight of the contaminanting DNA improved separation from a high molecular weight product (e.g. IgM). Bovine pancreas DNAse was immobilized on an agarose matrix to characterize and demonstrate controlled degradation of a p u r i f i e d DNA preparation passed through a column of the matrix. The p u r i f i e d DNA p r e p a r a t i o n c o n s i s t e d i n i t i a l l y of molecular weight 1,000,000. Two column passes resulted in an approximate b e l l d i s t r i b u t i o n with a broad range of 1,000,000 to 10,000 daltons and median at molecular weight 100,000. Many passes r e s u l t e d i n a narrow range of approximately 10,000 daltons. DNA degradation was monitored by s i z e exclusion chromatography and SDS-PAGE (Figure 2). In a separate experiment, a harvest of IgM was passed through the DNAse column and then fractionated on FPLC-Superose 6. DNA clearance was 10,000x, compared to lOx without DNAse digestion (10). Anion Exchange. Anion exchange on DEAE-Sepharose was optimized f o r IgM p u r i f i c a t i o n from DNA. The f o l l o w i n g b u f f e r conditions were studied: T r i s , pH 8.0; phosphate, pH 6.5; sodium acetate, pH 6.5; and no buffer. E l u t i o n was achieved by a l i n e a r gradient of sodium chloride. Separation from other proteins was marginal, but removal of albumin was s l i g h t l y superior i n the phosphate buffer. The s a l t buffers gave comparable reduction i n DNA (10,000x), with the unbuffered system giving l,000x. To demonstrate removal of DNA more c l e a r l y , several preparations were bound to DEAE-Sepharose: a) a p a r t i a l l y p u r i f i e d IgM preparation, b) a p u r i f i e d preparation of DNA, and c) a combination of both. Preparations were bound i n 0.05 M T r i s , 0.05 M NaCl, pH 8.0 and eluted by l i n e a r gradient of sodium chloride. Referring to Figure 3A, IgM was recovered i n the f i r s t e l u t i o n peak at 0.15 M NaCl. The second and t h i r d e l u t i o n peaks at 0.2 and 0.34 M NaCl contained native DNA. Figure 3B shows e l u t i o n of the DNA preparat i o n at 0.3 and 0.35 M NaCl. Mixing the p u r i f i e d IgM and DNA preparations and repeating the elution yielded a precise superimposition of the two chromatograms (Figure 3C), i n d i c a t i n g the two e n t i t i e s eluted independently. T y p i c a l chromatograms are shown i n Figure 4, with e l u t i o n by l i n e a r gradient and step elution. A small amount of protein did not bind, i n d i c a t e d by the peak on the l e f t . A high s a l t s t r i p a f t e r e l u t i o n of the IgM produced a peak of s i m i l a r magnitude, which contained IgM, DNA, and albumin. A step e l u t i o n at 0.15 M NaCl gave DNA clearances l i s t e d i n Table 3. Native DNA was assayed by dot blot hybridization. Clearance studies with P32 labeled DNA gave s u b s t a n t i a l l y lower clearance factors. P32 labeled DNA derived from harvests of c e l l l i n e C gave approximately twice the clearance of v i r a l DNA. Other anion exchange resins gave comparable or poorer r e s u l t s for IgM. Use of Q-Sepharose resulted i n s l i g h t l y t i g h t e r binding of both IgM and DNA. Repetitive chromatography on DEAE did not r e s u l t in increased removal of DNA from IgM.

Purification Alternativesfor IgM Monoclonal Antibodies

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch014

DOVE ET AL.

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Figure 2. DNA degradation by immobilized DNAse. Chromatograms (FPLC-Superose 6) of: A. 0 passes across DNAse column(initial DNA prep.). B. 2 passes. C. 14 passes.

202

Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch014

PROTEIN PURIFICATION

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