Purification of Fermentation Products. Applications to Large-Scale Processes 9780841208902, 9780841211018, 0-8412-0890-5

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Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.fw001

Purification of Fermentation Products

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.fw001

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

ACS SYMPOSIUM SERIES 271

Purification of Fermentation Products Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.fw001

Applications to Large-Scale Processes Derek LeRoith,

EDITOR

National Institutes of Health

Joseph Shiloach,

EDITOR

National Institutes of Health

Timothy J. Leahy,

EDITOR

Millipore Corporation

Based o n a symposium sponsored by the Division of Microbial and Biochemical Technology

American Chemical Society, Washington, D.C. 1985

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.fw001

Library of Congress Cataloging in Publication Data Purification of fermentation products. (ACS symposium series, ISSN 0097-6156; 271) Includes bibliographies and indexes. 1. Fermentation—Congresses. 2. Separation (Technology)—Congresses. I. LeRoith, Derek, 1945. II. Shiloach, Joseph. III. Leahy, Timothy J., 1949. IV. American Chemical Society. Division of Microbial and Biochemical Technology. V. Series. TP156.F4P87 1985 660.2'8449 84-24316 ISBN 0-8412-0890-5

Copyright © 1985 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of thefirstpage 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., 21 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 thefirstpage 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 UNITED STATES OF AMERICA

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

ACS Symposium Series M . Joan Comstock, Series Editor

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.fw001

Advisory Board Robert Baker

Robert Ory

U.S. Geological Survey

USDA, Southern Regional Research Center

Martin L. Gorbaty Exxon Research and Engineering Co.

Geoffrey D. Parfitt

Roland F. Hirsch

James C. Randall

U.S. Department of Energy

Carnegie-Mellon University

Phillips Petroleum Company

Herbert D. Kaesz

Charles N. Satterfield

University of California—Los Angeles

Massachusetts Institute of Technology

Rudolph J. Marcus Office of Naval Research

Vincent D. McGinniss Battelle Columbus Laboratories

Donald E. Moreland

W. D. Schultz Oak Ridge National Laboratory

Charles S. Tuesday General Motors Research Laboratory

Douglas B. Walters

USDA, Agricultural Research Service

National Institute of Environmental Health

W. H. Norton

C. Grant Willson

J. T. Baker Chemical Company

IBM Research Department

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.fw001

FOREWORD The ACS S Y M P O S I U M SERIES 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 SERIES 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 since symposia may embrace both types of presentation.

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

PREFACE

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.pr001

THE

ADVENT OF GENETIC ENGINEERING through the use of recombinant DNA techniques has stimulated wide interest in creating useful biologically derived products. There exists today a significant family of products that has been traditionally synthesized through the action of microorganisms. Typically, the manufacture of a biologically derived product begins with mass cultivation of the desired microorganism, a process known as fermentation. Fermentation, however, represents only one step in the overall scheme of product formation. It is equally important to recover and purify the desired product from the fermentation after the organism has been cultivated. Often, that product is present in relatively low concentrations compared to the other components of the fermentation, and the challenge lies in separating what is wanted from what is not in an efficient, economical, and timely fashion. Although great strides have been made in the genetic manipulation of microorganisms, a concomitant increase in new product-recovery methods has lagged behind. Recently, though, many groups of researchers in both academia and industry have initiated active development programs in the area of fermentation product recovery and purification. This book presents some of these newer approaches to product recovery. The emphasis is on large-scale processes where several approaches to product isolation are required to obtain pure material. Each author discusses state-of-the-art approaches to product purification that uniquely fit the material to be isolated. Two separation technologies are highlighted here:filtrationand chromatography. Both technologies have existed for some time, but only recently have they found wider use in fermentation processes. The chapters in this book describe some of the newest applications of these technologies to product purification. The authors provide a cross section of viewpoints from academia and industry and consider both the practical and theoretical aspects of biological processing. We wish to thank all of the authors for their contributions to this book. We also thank the Division of Microbial and Biochemical Technology, and specifically, Dr. Larry Robertson, for sponsorship of this work. Finally, we

ix In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

wish to express our appreciation to the editorial staff of the ACS Books Department with special thanks to Robin Giroux. DEREK LEROITH

National Institutes of Health J O S E P H SHILOACH

National Institutes of Health

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.pr001

T I M O T H Y J. L E A H Y

Millipore Corporation October 5, 1984

χ In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

1 Processing Cell Lysate with Tangential Flow Filtration

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch001

R A Y M O N D G A B L E R and M A R Y RYAN Millipore Corporation, Bedford, M A 01730

Operating conditions and the corresponding performance of a membrane filter system have been examined for recovering proteins from a bacterial lysate. Specifically, the dependence of membrane flux on average trans membrane pressure and recirculation rate has been investigated for both whole cells and lysate. The recovery of an intracellular protein was simulated by adding a marker protein, IgG, to the lysate after cell lysis. Protein concentrations were measured at each step of the processing scheme to determine the distribution of IgG and how much could be recovered. Operating parameters that influence the flow of IgG through a microporous membrane have also been studied. Since the introduction of genetic engineering on a practical scale in the 1970's, there has been increased interest in the production of biological products using large scale fermentation. Both new products that had not been commercially available previously in sufficient quantities, and old products whose production costs are now potentially cheaper have been in the spotlight. One of the difficulties that is encountered with some types of recombinantly derived proteins, however, is the purification steps. Because E. coli does not secrete recombinant proteins into the growth media, the cells must be lysed which increases the difficulty of separating the desired protein from all other biological constituents. Purification of a protein from a lysate requires a number of steps to first eliminate cell fragments and debris from soluble material and to then separate the desired protein from other soluble components. Techniques that have been classically used in lysate processing and protein isolation include: centrifugation, open column chromatography and precipitation. Tangential flow filtration can now be added to the list. (JO Tangential flow filtration has been most extensively used for concentrating cells(2f3)9 concentrating and washing proteins (A) and removing pyrogens from solutions(5»6). By passing the process solution tangential or parallel to the filter, and recirculating this fluid back to the original container, it is possible to filter solu0097-6156/85/0271-0001 $06.00/0 © 1985 American Chemical Society In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch001

2

PURIFICATION O F FERMENTATION PRODUCTS

t i o n s t h a t would n o r m a l l y c l o g a dead ended f i l t r a t i o n system. A l s o , w i t h f i l t r a t i o n , i t i s p o s s i b l e to perform gross f r a c t i o n a t i o n s based on s i z e . These two f a c t o r s make i t f e a s i b l e t o p r o c e s s c e l l l y s a t e s w i t h membrane f i l t e r s i n o r d e r t o s e p a r a t e c e l l d e b r i s from p r o t e i n s , and t o a l s o c o n c e n t r a t e the crude f r a c t i o n p r i o r t o t h e n e x t p u r i f i c a t i o n s t e p . F i l t r a t i o n o f f e r s advantages over o t h e r t e c h n i q u e s i n t h a t r e l a t i v e l y l i t t l e energy i s needed compared t o c e n t r i f u g a t i o n , and t h e f i l t r a t i o n system i s c l o s e d and w i l l n o t produce a e r o s o l s . A l s o , t h e equipment can be s c a l e d up t o h a n d l e large quantities. The purpose o f t h i s work i s s e v e r a l f o l d . F i r s t , we wanted t o determine t h e magnitude o f f i l t r a t i o n r a t e s t h a t a r e p o s s i b l e w i t h l y s a t e s compared t o whole c e l l s , and t o i d e n t i f y t h o s e o p e r a t i o n a l parameters t h a t i n f l u e n c e membrane f l u x . Second, an e f f o r t was made to i n v e s t i g a t e how s e v e r a l d i f f e r e n t methods o f g e n e r a t i n g a l y s a t e s u s p e n s i o n would a f f e c t membrane p r o c e s s i n g , i f a t a l l . T h i r d , we wanted t o i n v e s t i g a t e t h e q u a n t i t a t i v e l y r e c o v e r y o f a s p e c i f i c p r o t e i n from a l y s a t e w i t h f i l t r a t i o n , and l a s t , an e f f o r t was made t o d e t e r m i n e what o p e r a t i o n a l parameters a r e most i m p o r t a n t i n m a x i m i z i n g t h e f l o w o f p r o t e i n through t h e f i l t e r ? I n s h o r t , t h i s work was d e s i g n e d as t h e f i r s t s t e p i n documenting t h e performance o f membrane systems f o r p r o c e s s i n g l y s a t e s . To answer t h e above i s s u e s , a model system was d e v i s e d i n w h i c h a s p e c i f i c p r o t e i n was added t o an IS. c o l i l y s a t e . The l y s a t e was then p r o c e s s e d through t h e f i l t r a t i o n s t e p s and t h e p r o t e i n r e c o v e r e d . The p r o t e i n chosen was human IgG which has a r e l a t i v e l y l a r g e m o l e c u l a r w e i g h t (160,000 D a l t o n s ) . I t was reasoned t h a t i f a l a r g e p r o t e i n c o u l d be s e p a r a t e d from c e l l d e b r i s and passed through a membrane and r e c o v e r e d , then a s m a l l e r p r o t e i n t h a t i s t y p i c a l o f a recombinant p r o c e s s , s h o u l d prove t o be much e a s i e r . M a t e r i a l s and Methods F e r m e n t a t i o n s . IS. c o l i was grown i n a d e f i n e d media w i t h t h e f o l l o w i n g composition: -2 PO.

0.05M -3 10 M

4

MgS0 CaCl

4

4

10" M

2

5

FeSO, 4 (NH ) 4

10" M 2

S0

4

Glucose

0.2% 0.5%

The a n t i f o a m used was 2% o c t a d e c a n o l i n m i n e r a l o i l ( 0 . 5 m l / l i t e r of f e r m e n t a t i o n b r o t h ) . The f e r m e n t o r s were batched i n 11 l i t e r q u a n t i t i e s , and growth l a s t e d about 24 hours a t 37°C. R o t o r speed was 400· RPM and t h e a e r a t i o n r a t e was 5 l i t e r s / m i n u t e . A M i c r o g e n (New Brunswick S c i e n t i f i c ) o r a Chemap 14 l i t e r f e r m e n t o r was u s e d . F i n a l v i a b l e counts were t y p i c a l l y g r e a t e r than l O ^ / m l . Membrane S e p a r a t i o n s .

The membrane f i l t e r s e p a r a t i o n

system

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch001

1.

G A B L E R A N D RYAN

Lysate Processing with Tangential Flow Filtration

3

( M i l l i p o r e C o r p o r a t i o n , XX8140000) c o n s i s t e d of a membrane h o l d e r ( P e l l i c o n ) , a 4 g a l l o n per minute r o t a r y vane pump (Procon) and the membranes t h e m s e l v e s . The f i l t e r s were e i t h e r 0.45 m i c r o n m i c r o porous (Durapore) o r 100,000 NMWL u l t r a f i l t r a t i o n membranes. A l l t u b i n g and c o n n e c t i o n s were 1/2 i n c h . A m a n i f o l d f l o w bypass was a t t a c h e d t o the pump so f l u i d c o u l d be i n t r o d u c e d i n t o the f i l t r a t i o n system w i t h o u t a sudden surge o f p r e s s u r e b u i l d u p ( B u l l e t i n AB822, M i l l i p o r e C o r p o r a t i o n ) . I n a l l c a s e s , 5 square f e e t of membrane were used. F i g u r e 1 shows a s c h e m a t i c of the equipment used and how i t was plumbed t o g e t h e r . A l l t o g e t h e r t h e r e were t h r e e modes o f o p e r a t i o n used f o r v a r i o u s a p p l i c a t i o n s r e p o r t e d h e r e . For c o n c e n t r a t i n g c e l l s o r l y s a t e , the system was o p e r a t e d e x a c t l y as shown i n F i g u r e 1. F i l t r a t e was c o l l e c t e d s e p a r a t e l y w h i l e the r e t e n t a t e was c i r c u l a t e d back t o the o r i g i n a l h o l d i n g tank. I n the t o t a l r e c y c l e mode of o p e r a t i o n , the f i l t r a t e l i n e was p l a c e d i n the c e l l o r l y s a t e h o l d i n g r e s e r v o i r so the c e l l s or l y s a t e c o n c e n t r a t i o n remained cons t a n t as a f u n c t i o n o f t i m e . The r e a s o n f o r p e r f o r m i n g t o t a l r e c y c l e i s to i s o l a t e p r o c e s s v a r i a b l e s i n d e t e r m i n i n g the independent i n f l u e n c e of t r a n s membrane p r e s s u r e o r r e c i r c u l a t i o n r a t e on f l o w through the membrane. A c o n s t a n t volume wash mode of o p e r a t i o n i s a c c o m p l i s h e d by c o n t i n u o u s l y a d d i n g wash b u f f e r to the c e l l o r l y s a t e s u s p e n s i o n a t the same r a t e t h a t f i l t r a t e i s c o l l e c t e d . F o r a l l modes of o p e r a t i o n , t h e r e was no f i l t r a t e back p r e s s u r e . The i n l e t p r e s s u r e f o r the f i l t e r h o l d e r i s measured on the upstream s i d e o f the membrane a s the f l u i d e n t e r s . The o u t l e t p r e s sure i s a l s o m o n i t o r e d on the upstream s i d e of the membrane as the r e t e n t a t e e x i t s the f i l t e r h o l d e r d e v i c e . The d i f f e r e n c e between the i n l e t and o u t l e t p r e s s u r e i s p r o p o r t i o n a l to the c i r c u l a t i o n r a t e w h i l e the sum of the i n l e t and o u t l e t p r e s s u r e s i s p r o p o r t i o n a l t o the average t r a n s membrane p r e s s u r e . C e l l L y s i s . C e l l l y s i s was a c c o m p l i s h e d e i t h e r e n z y m a t i c a l l y w i t h lysozyme or with- s o n i c a t i o n . S o n i c a t i o n . Fermentor b r o t h s were reduced i n volume v i a f i l t r a t i o n t o s e v e r a l l i t e r s o r l e s s . T h i s reduced volume was then c o n t i n u o u s l y c i r c u l a t e d through a s o n i f e r (Branson C e l l D i s r u p t e r 185)· Water from an i c e b a t h was passed through the j a c k e t t o d i s s i p a t e heat generated from the s o n i f e r . L y s i s was checked v i s u a l l y by m o n i t o r i n g the number of whole c e l l s seen i n a wet mount under a n o p t i c a l m i c r o s c o p e a t 1000X m a g n i f i c a t i o n . Lysozyme. A f t e r f e r m e n t a t i o n , the c e l l b r o t h was reduced i n volume v i a f i l t r a t i o n t o one l i t e r o r l e s s . The c o n c e n t r a t e d c e l l s were then p e l l e t e d and washed w i t h 0.85% s a l i n e . The washed c e l l s were resuspended i n 0.1M EDTA, pH 8.0 and a l l o w e d to s i t a t room temperat u r e f o r about 45 minutes w i t h g e n t l e s t i r r i n g . Lysozyme was t h e n added t o enough 0.5M N a C l , pH 8.0, to b r i n g the t o t a l volume t o l / 2 0 t h o f the o r i g i n a l c u l t u r e volume. The lysozyme c o n c e n t r a t i o n was 2 mg/ml i n t h i s s u s p e n s i o n . The m i x t u r e was suspended i n 0.1% sodium d e s o x y c h o l a t e t o f i n a l i z e the l y s i s . T h i s p r o c e d u r e ( 7 ) p r o duced a v e r y v i s c o u s s u s p e n s i o n . The v i s c o s i t y was reduced by addi n g e i t h e r s t r e p t o m y c i n s u l f a t e o r DNAse. I n e i t h e r c a s e , the r e s u l t i n g s u s p e n s i o n was a l l o w e d t o s i t o v e r n i g h t w i t h g e n t l e s t i r r i n g .

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

4

PURIFICATION OF FERMENTATION PRODUCTS

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch001

IgG, DNAse. Human, f r e e z e d r i e d IgG was purchased from Sigma Chemic a l ( c a t . no. HG-11) as Cohn f r a c t i o n IT. The IgG was resuspended i n p h y s i o l o g i c a l s a l i n e o r c e l l c o n c e n t r a t i o n f i l t r a t e and the IgG was added to t h e l y s e d c e l l s t o a f i n a l c o n c e n t r a t i o n of around 0.1%0.2%. DNAse was a l s o purchased from Sigma C h e m i c a l . IgG A s s a y . Samples and c o n t r o l s t o be assayed f o r IgG were c o l l e c t e d i n 5 ml q u a n t i t i e s . From these samples, 750 m i c r o l i t e r s were c e n t r i fuged f o r 10 minutes a t 1250 RPM. The supernant f l u i d was c o l l e c t e d , and 250 m i c r o l i t e r s were i n j e c t e d i n t o a Beckman ICS A n a l y z e r I I . The a n a l y z e r would mix a p p r o p r i a t e amounts o f a n t i IgG a n t i b o d y w i t h the sample a u t o m a t i c a l l y and measure the change i n l i g h t s c a t t e r i n g i n t e n s i t y as the a n t i g e n - a n t i b o d y r e a c t i o n p r o g r e s s e d . The f o r m a t i o n of the i m m u n o p r e c i p i t i n complex proceeds g r a d u a l l y a t f i r s t , then r a p i d l y and f i n a l l y p r o g r e s s e s through a peak v a l u e . I f the sample IgG i s w i t h i n the c o r r e c t c o n c e n t r a t i o n range, the f i n a l peak r a t e f o r the change i n l i g h t s c a t t e r i n g i n t e n s i t y i s p r o p o r t i o n a l t o the sample IgG c o n c e n t r a t i o n . Normal human s e r a of known IgG c o n c e n t r a t i o n was used f o r c a l i b r a t i o n and c o n t r o l s . W i t h l y s a t e samples, t h i s assay t e c h n i q u e was r e p r o d u c i b l e t o about 10%. Results A schematic i l l u s t r a t i n g how membrane f i l t e r s a r e used t o p r o c e s s l y s a t e s i s shown i n F i g u r e 2. The o v e r a l l purpose of the p r o c e s s i n g i s to s e p a r a t e an i n t r a c e l l u l a r p r o d u c t , t y p i c a l l y a p r o t e i n , from the b u l k of l a r g e c e l l fragments or d e b r i s , and to c o n c e n t r a t e the p r o t e i n t o a s m a l l w o r k a b l e volume. B a s i c a l l y , t h e r e a r e f o u r f i l t r a t i o n s t e p s i n t h i s p r o c e s s . A f t e r the c e l l s a r e grown, the c e l l s a r e c o n c e n t r a t e d t o a r e l a t i v e l y s m a l l volume. T h i s i s accomp l i s h e d i n the f i r s t f i l t r a t i o n s t e p . A f t e r l y s i s , the l y s a t e i s c o n c e n t r a t e d and washed i n the second and t h i r d s t e p s r e s p e c t i v e l y . Washing i s performed i n o r d e r t o enhance the passage of p r o t e i n through the membrane i f the p r o t e i n remains i n t h e r e t e n t a t e d u r i n g the c o n c e n t r a t i o n of the l y s a t e . Both c o n c e n t r a t i o n and washing can use the exact same microporous membrane. I n the l a s t s t e p , an u l t r a f i l t r a t i o n membrane i s used t o c o n c e n t r a t e the c r u d e l y f r a c t i o n a t e d p r o t e i n s o l u t i o n i n p r e p a r a t i o n f o r the n e x t p u r i f i c a t i o n s t e p w h i c h i s u s u a l l y a chromatographic p r o c e s s o f one s o r t o r a n o t h e r . The i n f o r m a t i o n p r e s e n t e d i n the f o l l o w i n g f i g u r e s r e p r e s e n t s two types of r e s u l t s . F i r s t , t h e r e i s the e v i d e n c e f o r IgG r e c o v e r y from membrane f i l t r a t i o n as a f u n c t i o n of d i f f e r e n t b i o c h e m i c a l p r o c e s s i n g schemes. Second, t h e r e i s the d a t a w h i c h d e s c r i b e s the f l o w r a t e s o f f l u i d or t h e f l u x through the membrane as a f u n c t i o n of different operating conditions. F i g u r e 3 i s a schematic r e p r e s e n t i n g a f i l t r a t i o n p r o c e s s of c e l l s , l y s a t e and p r o t e i n s o l u t i o n i n w h i c h the l y s a t e was formed by s o n i c a t i o n . I n i t i a l l y , 22.3 l i t e r s or c e l l s were c o n c e n t r a t e d down to 4.8 l i t e r s w i t h 0.45 m i c r o n microporous membrane. The c e l l s were l y s e d and the IgG added i n t h e amount of 11.1 grams (6.8 l i t e r s t o t a l l y s a t e ) . The l y s a t e was then c o n c e n t r a t e d t o 1.3 l i t e r s . D u r i n g t h e l y s a t e c o n c e n t r a t i o n , 7.6 grams of IgG passed through the 0.45 m i c r o n pore s i z e membrane and 2.5 grams remained i n the r e t e n t a t e . R e s p e c t i v e f l u i d volumes were 5.5 l i t e r s and 1.3 l i t e r s . A c o n s t a n t volume wash was performed on the 1.3 l i t e r s o f r e t e n t a t e i n

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

1.

G A B L E R A N D RYAN

Lysate Processing with Tangential Flow Filtration

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch001

RETENTATE

F i g u r e 1. Schematic o f equipment f o r p r o c e s s i n g l y s a t e s . The t a n g e n t i a l f l o w f i l t e r i s i n the s t a c k e d sheet c o n f i g u r a t i o n w h i c h allows high i n l e t pressure.

GROW CELLS

CONCENTRATE CELLS (MF) •

CONCENTRATE PROTEIN (UF)

•

LYSE CELLS

RETENTATEXV

J

S

^?\ S*™

hi-ι «Aie ^ • • Y T ^ SEPARATE . / • • Λ : CELL i DEBRIS (MF) FILTRATE

RETENTATE

F i g u r e 2. F i l t r a t i o n steps f o r p r o c e s s i n g l y s a t e . A l l s t e p s , except the p r o t e i n c o n c e n t r a t i o n , use a m i c r o p o r o u s membrane. P r o t e i n c o n c e n t r a t i o n i s a c c o m p l i s h e d by a UF membrane.

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

5

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch001

6

PURIFICATION O F FERMENTATION PRODUCTS

an e f f o r t to s e p a r a t e the r e m a i n i n g 2.5 grams o f IgG from the c e l l d e b r i s . A f t e r 5 l i t e r s o f wash was c o l l e c t e d i n the f i l t r a t e , 2.4 grams o f IgG had a l s o passed the membrane showing t h a t the IgG can be r e c o v e r e d and e f f e c t i v e l y s e p a r a t e d from the l y s a t e . The 5.5 l i t e r s from t h e o r i g i n a l l y s a t e c o n c e n t r a t e was then c o n c e n t r a t e d w i t h a 100,000 MWC0 UF membrane. I n the c o n c e n t r a t i o n , o n l y 5.2 grams o f IgG was r e c o v e r e d , however, none of the IgG passed the UF membrane. I n an e f f o r t t o d e t e r m i n e i f the l o s s o f IgG i n the UF concent r a t i o n o f the crude p r o t e i n f r a c t i o n shown i n F i g u r e 3 r e p r e s e n t e d a r e a l i s s u e o r j u s t a m a t t e r of c o l l e c t i n g a l l the r e t e n t a t e from the dead volume, the c o n c e n t r a t i o n s t e p was r e p e a t e d . This time, p a r t i c u l a r a t t e n t i o n was p a i d t o f l u s h i n g out the r e t e n t a t e hoses and dead s p o t s i n the system. F i g u r e 4 shows the r e s u l t s o f t h i s work. The l y s a t e f i l t r a t e (10.5 l i t e r s , 7.2 grams o f IgG) was s p l i t i n t o two f r a c t i o n s o f 5.9 l i t e r s and 4.8 l i t e r s r e s p e c t i v e l y and concent r a t e d under two s e p a r a t e o p e r a t i n g c o n d i t i o n s . O p e r a t i n g a t an i n l e t p r e s s u r e o f 80 p s i and c o n c e n t r a t i n g the 5.9 l i t e r s t o 1.4 l i t e r s , 3.7 grams o f IgG was r e c o v e r e d immediately which r e p r e s e n t s 95% o f t h a t expected. S u b s e q u e n t l y , 1.5 l i t e r s o f p h y s i o l o g i c a l s a l i n e was r e c i r c u l a t e d through the f i l t r a t i o n system f o r 5 minutes and t h e n assayed f o r the r e m a i n i n g IgG. Recovery of another 0.2 grams o f IgG ( r e m a i n i n g 5%) was found, so t o g e t h e r w i t h the i n i t i a l 3.7 grams o f IgG, the t o t a l amount expected was r e c o v e r e d . For o p e r a t i n g c o n d i t i o n s o f 40 p s i i n l e t p r e s s u r e and 0 o u t l e t p r e s s u r e , 88% o f the IgG was r e c o v e r e d . R e s u l t s from F i g u r e 4 i n d i c a t e t h a t under b o t h h i g h and moderate i n l e t p r e s s u r e s , the IgG c o u l d be r e c o v e r e d from the concentration step. F i g u r e 5 shows the f l o w decay f o r the c e l l s u s p e n s i o n c o n c e n t r a t i o n s t e p o f F i g u r e 3. The f l o w r a t e decays g r a d u a l l y w i t h time w i t h an average f i n a l r a t e around 700 ml/min. I n l e t p r e s s u r e was 90 p s i , and t h e r e was a 4.6X r e d u c t i o n i n volume i n about 15 m i n u t e s . The f l o w decay c u r v e i n F i g u r e 5 i s t y p i c a l f o r an 15. c o l i c o n c e n t r a t i o n w i t h a new 0.45 m i c r o n pore s i z e m i c r o p o r o u s membrane. F i g u r e 6 i s the c o r r e s p o n d i n g f l o w decay curve f o r the s o n i f i e d l y s a t e suspension. A g a i n , t h i s i s a t y p i c a l f l o w decay w i t h t i m e . The f l u x f o r the l y s a t e i s l e s s than t h a t seen f o r the whole c e l l c o n c e n t r a t i o n . The f l o w r a t e , however, i s s t i l l s i g n i f i c a n t and the e q u i l i b r i u m r a t e c o r r e s p o n d s t o 23 g a l l o n s per square f o o t per day (GFD). I n o t h e r e x p e r i m e n t s , f l o w r a t e s f o r l y s a t e c o n c e n t r a t i o n a r e a l s o seen t o be r e l a t i v e l y c o n s t a n t showing l i t t l e decay. F i g u r e 7 i s a p l o t o f the f l o w r a t e over time f o r the c o n s t a n t volume wash s t e p used t o pass the IgG r e m a i n i n g i n the r e t e n t a t e a f t e r the i n i t i a l c o n c e n t r a t i o n of the l y s a t e . As expected, w i t h the l y s a t e volume r e m a i n i n g c o n s t a n t a t about 1.3 l i t e r s , t h e r e i s l i t t l e f l o w decay w i t h t i m e . The arrows i n F i g u r e 7 i n d i c a t e the commencement o f the a d d i t i o n of one l i t e r a l i q u o t s o f s a l i n e s o l u t i o n a t a r a t e matching t h a t o f the f i l t r a t e . F i v e square f e e t o f an 0.45 m i c r o n pore s i z e f i l t e r was used f o r t h i s p r o c e d u r e . F i g u r e 8 shows the r e s p e c t i v e f l o w decays f o r the crude p r o t e i n f r a c t i o n c o n c e n t r a t i o n w i t h the 100,000 MWCO UF membrane. I n l e t p r e s s u r e s were 80 p s i and 40 p s i r e s p e c t i v e l y . The h i g h e r i n l e t p r e s s u r e gave a h i g h e r f l u x compared to the 40 p s i i n l e t p r e s s u r e a s would be expected. When the i n l e t p r e s s u r e i s i n c r e a s e d , b o t h the average TMP and a l s o the r e c i r c u l a t i o n r a t e i n c r e a s e . Based on the d a t a i n F i g u r e 8 a l o n e , i t i s not p o s s i b l e t o determine i f the h i g h e r

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

1.

G A B L E R A N D RYAN

Lysate Processing with Tangential Flow Filtration

1

E. coli (22.3L) C O N C E N T R A T E C E L L S (4.8L)

f L Y S E C E L L S (4.8L) (SONICATION)

f M E M B R A N E P R O C E S S I N G O F L Y S A T E (6.8L;11.1g) (0.45μ MF)

\

RETENTATE (1.3L; 2.5g)

FILTRATE (5.5L; 7.6g)

Ψ

Ψ

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch001

W A S H (5L SALINE)

/ RETENTATE (1.3L;0.3g)

™

PROTEIN C O N C E N T R A T I O N (100,000 MWCO)

\

/

FILTRATE (5L;2.4g)

\

RETENTATE (0.8L; 5.2g)

FILTRATE (4.6L; Og)

F i g u r e 3 . D i s t r i b u t i o n of IgC f o r the d i f f e r e n t process s t e p s . Both the f l u i d volumes and the t o t a l IgC content a r e g i v e n f o r a l l r e t e n t a t e s and f i l t r a t e s throughout the whole sequence.

E. coli (10.8L) C O N C E N T R A T E C E L L S (1.2L)

f LYSE CELLS(1.2L) (SONICATION)

f P R O C E S S L Y S A T E (12L, 9.1g)

/

\

RETENTATE (1.4L;1.5g)

FILTRATE (10.5L; 7.2g)

f C O N C E N T R A T E PROTEIN (SPLIT FILTRATE) 40/0 RETENTATE (.95L; 2.1g) WASH STEP

(1.75L; 0.65g)

Ψ

FILTRATE (3.5L; 0g)

%80/0 RETENTATE (1.4L; 3.7g)

FILTRATE (4.45L; Og)

(1.3L;0.2g)

F i g u r e 4. C o n c e n t r a t i o n o f p r o t e i n under d i f f e r e n t o p e r a t i n g c o n d i t i o n s . IgC can be r e c o v e r e d q u a n t i t a t i v e l y under two s e t s o f o p e r a t i n g p r e s s u r e s by a UF membrane.

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

8

PURIFICATION O F FERMENTATION PRODUCTS

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch001

1000|

2

4

6

8

10

12

14

TIME (minutes)

F i g u r e 5. Flow decay curve f o r c o n c e n t r a t i n g E . c o l i whole c e l l s w i t h a 0.45 μπι microporous (Durapore) membrane. I n l e t p r e s s u r e was 90 p s i . The i n i t i a l volume was 22.3 l i t e r s ; the f i n a l volume, 4.8 l i t e r s .

2 200 c 100

2

4

6

8

10

12

14

16

TIME (minutes)

F i g u r e 6. Flow decay curve f o r c o n c e n t r a t i n g E . c o l i l y s a t e w i t h a 0.45 μπι microporous (Durapore) membrane. I n l e t p r e s s u r e was 90 p s i ; o u t l e t p r e s s u r e , 30 p s i . The i n i t i a l volume was 6.8 l i t e r s ; the f i n a l volume, 1.3 l i t e r s .

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

G A B L E R A N D RYAN

~ 400 ADD |1 liter 1

Ε

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch001

g

Lysate Processing with Tangential Flow Filtration

ADD 1 liter

ADD 1 liter

\

ADD 1 liter

ADD 1 liter

\

\

300

200

< AC t 100

6 8 10 TIME (minutes)

12

14

F i g u r e 7. Flow versus time f o r the l y s a t e wash w i t h 5 square f e e t of a 0.45 μπι microporous (Durapore) membrane. L y s a t e volume (1.3 l i t e r s ) remains c o n s t a n t d u r i n g the wash. I n l e t p r e s s u r e was 90 p s i ; o u t l e t p r e s s u r e , 30 p s i .

F i g u r e 8. P r o t e i n c o n c e n t r a t i o n f l o w decay curves f o r two d i f f e r ­ ent o p e r a t i n g c o n d i t i o n s w i t h a 100,000 MWCO UF membrane. Key: •, P. = 80 p s i ; ·, P. = 40 p s i . m

r

in

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

10

PURIFICATION O F FERMENTATION PRODUCTS

c i r c u l a t i o n r a t e o r h i g h e r TMP i s r e s p o n s i b l e f o r the h i g h e r f l u x . The a b s o l u t e f l u x f o r the p r o t e i n s o l u t i o n w i t h the UF membrane i s more t h a n h a l f t h a t seen f o r c o n c e n t r a t i n g c e l l s w i t h an 0.45 m i c r o n microporous membrane. The p r o t e i n f l u x i s h i g h e r than might be ex­ p e c t e d , however, the p r o t e i n f r a c t i o n has a l r e a d y been c l a r i f i e d through the 0.45 m i c r o n pore s i z e membrane and, hence, i s a r e l a t i v e ­ ly clean solution. I n an e f f o r t t o d e t e r m i n e whether the t r a n s membrane p r e s s u r e (TMP) o r r e c i r c u l a t i o n r a t e a r e more i m p o r t a n t i n d e t e r m i n i n g the membrane f l u x f o r c o n c e n t r a t i n g c e l l s and l y s a t e o r p r o t e i n , t o t a l r e c y c l e experiments were performed. F i g u r e 9 i l l u s t r a t e s a t y p i c a l response of f l u x t o average TMP f o r whole c e l l s as c i r c u l a t i o n r a t e i s held constant. I t i s seen t h a t as TMP i s i n c r e a s e d , the f l u x a l s o i n c r e a s e s . F i g u r e 10 shows the response of f i l t r a t i o n r a t e t o c i r c u l a t i o n r a t e as the average TMP i s h e l d c o n s t a n t , a g a i n f o r whole c e l l s . Beyond a minimum r e c i r c u l a t i o n r a t e where Ρ-^ - Pout equals about 20 or 30 p s i , not much g r e a t e r f l u x i s generated by i n c r e a s i n g the c i r c u l a t i o n r a t e . C e l l l y s a t e s o l u t i o n s generated by s o n i c a t i o n e x h i b i t a c o m p l e t e l y analagous b e h a v i o r except the abso­ l u t e f l o w r a t e s a r e l e s s than f o r whole c e l l s . So, f o r c o n c e n t r a t i o n of e i t h e r c e l l s or l y s a t e w i t h a m i c r o p o r o u s membrane, the average TMP i s most dominant i n i n c r e a s i n g the f i l t r a t e r a t e ; the g r e a t e r the average TMP, the h i g h e r the f l u x . T h i s s h o u l d not i m p l y , how­ e v e r , t h a t c i r c u l a t i o n i s n o t an i m p o r t a n t v a r i a b l e t o manage. F i g u r e 11 i s a f l o w schematic where the l y s a t e has been p r o ­ duced by a lysozyme d i g e s t . The f i n a l volume of the l y s a t e was 10.6 l i t e r s and a t o t a l o f 12.7 grams o f IgG was assayed i n the l y s a t e suspension. When lysozyme was used to l y s e the c e l l s , a v e r y v i s c o u s s o l u t i o n was c r e a t e d due t o the l a r g e s t r a n d s o f DNA t h a t are gener­ a t e d compared t o the s o n i c a t i o n method. W i t h s o n i c a t i o n , the DNA tends t o be b r o k e n up i n t o s m a l l e r fragments because of the shear f o r c e s generated. To reduce the v i s c o s i t y of the l y s a t e s u s p e n s i o n , s t r e p t o m y c i n s u l f a t e was added w h i c h p r e c i p i t a t e s the DNA. The l y s a t e was t h e n c o n c e n t r a t e d to 700 ml w i t h an 0.45 m i c r o n pore s i z e m i c r o p o r o u s membrane. A f t e r c o n c e n t r a t i o n , 6 grams of the IgG r e ­ mained i n the r e t e n t a t e , w h i l e o n l y 3.1 grams passed through the membrane i n t o the f i l t r a t e , C o n c e n t r a t i o n of the p r o t e i n s o l u t i o n from 9.65 l i t e r s t o 900 ml w i t h the UF membrane d i d r e c o v e r a l l of the 3.1 grams of IgG i n i t i a l l y i n the f i l t r a t e from the microporous membrane. I n an e f f o r t t o develop a method w h i c h would r e c o v e r more o f the IgG from the l y s a t e c o n c e n t r a t i o n r e t e n t a t e , s e v e r a l p r o c e d u r a l changes were made. F i r s t , DNAse 1 was added to the lysozyme p r o ­ duced l y s a t e i n o r d e r t o reduce the s i z e of the DNA. Second, a more e x t e n s i v e washing procedure was i n s t i t u t e d . F i g u r e 12 shows the r e s u l t s o b t a i n e d w i t h these changes. I n i t i a l l y , t h e r e was 3.5 grams of IgG i n the l y s a t e r e t e n t a t e . A f t e r a 5 l i t e r c o n s t a n t volume wash w i t h s a l i n e , 2.2 grams o f IgG were found i n the f i l t r a t e , o r 61% of the IgG had been s e p a r a t e d from the r e t a i n e d c e l l d e b r i s . Next, 5 l i t e r s of s a l i n e was added t o the 400 m l . D u r i n g t h i s c o n c e n t r a t i o n s t e p , an a d d i t i o n a l 0.69 grams (20%) o f IgG was r e c o v e r e d i n the f i l ­ t r a t e . F i n a l l y , a n o t h e r 5 volume c o n s t a n t volume wash w i t h 1.5 l i ­ t e r s o f s a l i n e was performed, and 0.25 grams of IgG was f l u s h e d through the membrane. A l l t o g e t h e r , w i t h the DNAse and the extended washing s t e p s , 88% o f the IgG i n i t i a l l y i n the r e t e n t a t e was r e -

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch001

η

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

1.

G A B L E R A N D RYAN

Lysate Processing with Tangential Flow Filtration

1-H 1.0 .9 H .8 FILTRATE -7 RATE (L/M) .5

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Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch001

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TMP (psi) F i g u r e 9. I n f l u e n c e of average transmembrane p r e s s u r e on membrane f l u x (0.45 μπι m i c r o p o r o u s ) . R e c i r c u l a t i o n r a t e (40 p s i ) i s kept c o n s t a n t as TMP i s v a r i e d . O p e r a t i o n i s i n the t o t a l r e c y c l e mode.

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In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

F i g u r e 17. R e l a t i o n s h i p between volume c o n c e n t r a t i o n ( f o r m a t i o n of v i r u s - r i c h r e t e n t a t e ) and a c t u a l v i r u s t i t e r d u r i n g 100,000 MWCO membrane u l t r a f i l t r a t i o n ( U F ) . Supernatant was c o n c e n t r a t e d 5.6 times by t h i s method. V i r u s was c o n c e n t r a t e d 3.8 times. The permeate c o n t a i n e d no v i r u s a c t i v i t y .

Process Time (min)

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch002

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RICKETTS ET AL.

45

Ultrafiltration Systems

these p r o c e d u r e s i s o f s u f f i c i e n t l y h i g h r e l i a b i l i t y t o be employed routinely. R o u t i n e d e c o n t a m i n a t i o n and c l e a n i n g f o l l o w i n g manufacturer's suggestions, r e q u i r e forethought t o insure a p p l i c a b i l i t y , s a f e t y , and thoroughness. The system d e c o n t a m i n a t i o n f o l l o w i n g c o n c e n t r a t i o n o f t h e HTLV i s a case i n p o i n t . T h i s agent i s n o t o n l y i m p l i c a t e d i n human m a l i g n a n c y , b u t i s now under i n v e s t i g a t i o n as a p o s s i b l e agent i n a c q u i r e d immune d e f i c i e n c y syndrome (AIDS). F o l l o w i n g c o n c e n t r a t i o n o f HTLV over 100K membrane, t h e system i s p r o c e s s e d i n s i t u by t h e p r o c e d u r e d e t a i l e d i n T a b l e V.

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch002

Table V. Membrane System D e c o n t a m i n a t i o n Procedure In s i t u , a l l waste t o k i l l tank: 1. NaOH, 1M, 1 l i t e r / s q . f t . membrane, f i l t r a t e v a l v e s c l o s e d . 2. Repeat s t e p 1 w i t h f i l t r a t e v a l v e s open. 3. A l l o w i n g s t a n d i n g (30 min.) w i t h s o l u t i o n . 4. NaOCL, 525 ppm ( 1 % C h l o r o x ) , 1 l i t e r / s q . f t . membrane. 5. A l l o w s t a n d i n g , o v e r n i g h t , w i t h s o l u t i o n . 6. F l u s h w i t h R/0 w a t e r , 205 l i t e r s / s q . f t . membrane. Containment Room 1. Remove f i l t e r s t a c k . 2. P l a c e i n sodium a z i d e (0.1%) o v e r n i g h t . 3. S t o r e f i l t e r s a t 4°C. In

situ 1. R e s e a l u n i t . 2. Steam s t e r i l i z e system, 30 min. 121°C. 3. S t e r i l i z e k i l l tank.

Low h a z a r d p r o c e s s i n g systems, such as those d i s c u s s e d f o r use w i t h m o n o c l o n a l a n t i b o d y and lymphokines a r e a l s o c l e a n e d i n s i t u , but under l e s s r i g o r o u s containment c o n d i t i o n s , d e t a i l e d i n T a b l e V L Membrane c l e a n i n g i s e s s e n t i a l l y t h a t suggested by t h e v e n d o r , m o d i f i e d i n c e r t a i n a p p l i c a t i o n s . Durapore i s l e s s c h e m i c a l l y r e s i s t a n t than p o l y s u l f o n e , and sodium h y d r o x i d e i s no l o n g e r recommended, b u t i s used as noted i n s t e p 3, a p p r o x i m a t e l y 1%, w i t h o u t s i g n i f i c a n t damage. T h i s i s needed as t h e C h l o r o x s t e p i s o m i t t e d f o r Largomycin I I p r o c e s s e s due t o t h e extreme s e n s i t i v i t y of t h a t agent t o c h l o r i n e . F l u x r a t e s s h o u l d r e t u r n t o no l e s s than 80% o f pre-use v a l u e s . Table V I . Low Hazard Membrane C l e a n i n g

3.

Polysulfone PBS, 0.5-2.0 l i t e r / s q . f t . NaOCl 1%, 1.0 l i t e r / s q . f t . , Stand o v e r n i g h t . NaOH, 1M, 30 m i n .

4. 5. 6.

R/0 w a t e r , 2-5 l i t e r / s q . f t . Redetermine f l u x r a t e . S t o r e wet 4©C

1. 2.

1. 2. 3. 4. 5. 6.

Durapore Same. NaOCl 0.1%, 0.5 l i t e r / s q . f t . 30 min. t o 1 h r . Not recommended. NaOH, 0.025M, 30 min. Same. Same. Same.

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

46

PURIFICATION OF FERMENTATION PRODUCTS

Steam S t e r i l i z a t i o n . C a r t r i d g e f i l t e r systems d i s c u s s e d f o r c e l l h a r v e s t a p p l i c a t i o n s p r e s e n t no d i f f i c u l t i e s i n steam s t e r i l i z a t i o n . They may be s t e r i l i z e d by a u t o c l a v i n g , w i t h v e n t s open (and f i l t e r e d ) f o r 1 h r . a t 121QC. The same u n i t s c o u p l e d t o f e r m e n t a t i o n e q u i p ment may be s t e r i l i z e d i n s i t u f o r the same time. The t a n g e n t i a l and s e r p e n t i n e f l o w membrane f i l t e r u n i t s t e s t e d are a v a i l a b l e i n non-autoclavable a c r y l i c o r s t e r i l i z a b l e s t a i n l e s s s t e e l . As mentioned e a r l i e r , the f i l t e r p a c k e t s f o r the s e r p e n t i n e f l o w system w i l l not w i t h s t a n d steam s t e r i l i z a t i o n . The Durapore and p o l y s u l f o n e f i l t e r s h e e t s f o r the l a r g e r u n i t s w i t h s t a n d 1 h r . at 121°C w e l l , but must be t h o r o u g h l y w e t t e d p r i o r t o s t e r i l i z a t i o n . O r i g i n a l i n s t r u c t i o n s f o r s e p a r a t e l y a u t o c l a v i n g the s t e e l u n i t pre-assembled and w e t t e d , suggest " f i n g e r - t i g h t e n i n g o n l y o f t h e r e t a i n i n g n u t s p r i o r t o s t e r i l i z a t i o n . When t h i s was done i n o u r l a b o r a t o r i e s , c o n c e n t r a t i o n o f e u k a r y o t i c c e l l c u l t u r e s was much s l o w e r and c l o g g i n g a much g r e a t e r problem than had been a n t i c i p a t e d . E x a m i n a t i o n o f the f i l t e r s themselves showed t h a t s i g n i f i c a n t s h r i n k a g e had o c c u r r e d , r e s u l t i n g i n r e d u c t i o n o f r e t e n t a t e and f i l t r a t e manifolds to a f r a c t i o n of t h e i r o r i g i n a l s i z e . Polye t h y l e n e end gaskets were used i n t h i s assembly, and w h i l e these a r e not recommended f o r a u t o c l a v i n g , they are a p p a r e n t l y s u f f i c i e n t l y r e s t r a i n e d by the s t a i n l e s s s t e e l b l o c k s t h a t they do n o t change dimensions s i g n i f i c a n t l y , except f o r c u r l i n g beyond t h e margins o f the f i l t e r s t a c k , n o r do the f i l t e r s themselves change d i m e n s i o n s i g n i f i c a n t l y . The s c r e e n s , however, do s h r i n k s i g n i f i c a n t l y c a r r y i n g the f i l t e r s h e e t s , t o w h i c h they adhere, w i t h them. T h i s r e s u l t s i n some w r i n k l i n g o f the f i l t e r s u r f a c e , b u t t h i s does not seem t o a d v e r s e l y a f f e c t f i l t r a t i o n . The adverse e f f e c t comes from the s h r i n k a g e o f the s t a c k away from the openings i n the end g a s k e t s . To c o u n t e r a c t t h i s , u n i t s t o be a u t o c l a v e d s e p a r a t e l y s h o u l d be torqued t o the t e n s i o n a p p r o p r i a t e t o the membranes employed. T h i s procedure i s comparable t o the assembly f o r i n s i t u steam s t e r i l i z a t i o n ( F i g u r e s 18 & 19) where the pre-wetted u n i t must be f u l l y t i g h t e n e d t o r e t a i n steam. Steam l i n e s s h o u l d have s u f f i c i e n t upstream f i l t e r s t o p r o t e c t a g a i n s t l o a d i n g membrane s u r f a c e s w i t h pyrogenic m a t e r i a l s . For i n s i t u s t e r i l i z a t i o n , t e m p e r a t u r e - s e n s i t i v e p e n c i l s a r e a g a i n used t o i n s u r e s t e r i l i z a t i o n temperature i s a c h i e v e d f o r adequate t i m e . The a u t o c l a v e d u n i t i s b e s t c o o l e d t o ambient temperatures i n a p r o t e c t i v e environment such as a l a m i n a r - f l o w c a b i n e t , and brought back t o proper torque when c o o l e d . I n s i t u s t e r i l i z e d systems, i f not c o n t a i n e d , s h o u l d be r e - t o r q u e d s e v e r a l times d u r i n g c o o l i n g t o i n s u r e i n t e g r i t y .

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch002

n

Conclusions C a r t r i d g e and membrane f i l t e r systems t e s t e d i n p r e l i m i n a r y s t u d i e s i n our l a b o r a t o r i e s have proved t o be a p p l i c a b l e t o e u k a r y o t i c c e l l c u l t u r e p r o c e s s e s , i . e . c e l l removal, semi-continuous c u l t u r e growth, c e l l c u l t u r e c o n c e n t r a t i o n and r e c y c l i n g . P r e s e n t membrane systems are l i m i t e d i n p r o c e s s i n g volume; c a r t r i d g e s a r e s c a l a b l e , but have a n a r r o w e r range o f a p p l i c a t i o n . M a n i p u l a t i o n o f v i r u s , even more than r o u t i n e c e l l c u l t u r e s , i s l i k e l y t o r e q u i r e s t r i n g e n t containment t e c h n i q u e s w h i c h w i l l g r e a t l y c o m p l i c a t e the apparatus and f a c i l i t i e s n e c e s s a r y f o r p r o c e s s i n g . S u b s t a n t i a l f o r e t h o u g h t and e x t e n s i v e t e s t i n g a r e r e q u i r e d i n the d e s i g n and o p e r a t i o n of these p r o c e s s i n g systems.

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

R1CKETTS E T A L .

Ultrafiltration Systems

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch002

2.

American Chemical Society Library 1155 16th St. N. w. Washington. D. C. 20036 In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

47

PURIFICATION OF FERMENTATION PRODUCTS

48

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch002

Procedure: 1.

Install membranes and tighten UF unit to recommended levels. Open all valves on membrane UF unit.

2.

Turn on steam supplies 1 and 2 and open drain at supply 3. Do not open supply 3.

3.

Allow 60 min sterilization at 121 °C as measured by heat-sensitive pencil.

4.

Turn off steam and close drain. Open valve to 300-liter permeate vessel to charge lines with sterile air to maintain positive pressure while cooling.

5.

During cool-down, re-torque membrane UF unit to recommended levels.

6.

System can be operated when membrane UF unit and pump head are cooled to room temperature. F i g u r e 19. _In s i t u steam s t e r i l i z a t i o n o f the membrane u l t r a f i l t r a t i o n system.

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

2.

RICKETTS E T A L .

Ultrafiltration Systems

49

Most o f t h e t e c h n i c s mentioned h e r e a r e a p p l i c a b l e t o s t e r i l i ­ z a t i o n , a s e p t i c p r o c e s s i n g and p o s t - u s e d e c o n t a m i n a t i o n , w i t h s i m i l a r r e s t r i c t i o n s t o those mentioned f o r v i r u s and c e l l c u l t u r e handling. P r o t e i n p r o c e s s i n g systems f o r c o n c e n t r a t i o n and/or p u r i f i c a ­ t i o n o f f e r a v e r y b r o a d range o f a p p l i c a t i o n w h i c h may be t a i l o r e d t o a g i v e n p r o t e i n p r o d u c t p u r i f i c a t i o n scheme. The p a r t i c u l a r a p p a r a t u s and o p e r a t i n g parameters employed must be s e l e c t e d and r e f i n e d t o t h e unique r e q u i r e m e n t s o f any g i v e n p r o t e i n as w e l l as those o f t h e b r o t h i n which i t o c c u r s .

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2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

B e l l i n i , W.J., Trudgett, Α., and McFarlan, D.E. J . Gen. V i r o l . 1979, 43, 633-9. Berman, D., Rohr, Μ. Ε., and Safferman, R. S. Appl. Environ. Microbiol. 1980, 40, 426-8. Gangemi, J . D., Connell, Ε. V . , Mahlandt, B. G., and Eddy, G. A. Appl. Environ. Microbiol. 1977, 34, 330-2. Klein, F . , Ricketts, R. T., Rohrer, T. R., Jones, W. I., Clark, P. M., Flickinger, M. C. Appl. Environ. Microbiol. 1984, 47, 1023-6. Lee, J. C., Hapel, A. J., and Ihle, J . N. J . Immunol. 1982, 128, 2393-8. Mathes, L. E . , Yohn, D. S., and ulsen, R. G. J . Clin. Microbiol. 1977, 5, 372-4. Olsen, A. C. Proc. Biochem. 1977, 7, 333-43. Rosenberry, T. L., Chen, J . F . , Lee, Μ. Μ., Moulton, Τ. Α., and Onigman, P. J . Biochem. Biophys. Methods. 1981, 1, 39-48. Sekla, L . , Stackin, W., Kay, C., and VanBucken Hout, L. Can. J . Microbiol. 1980, 26, 518-23. Shant, J . L . , and Webster, D. W. Proc. Biochem. 1982, 17, 27-32. Shibley, G.P., Manousos, Μ., Munch, K., Zelljadt, I., Fisher, L . , Mayyasi, S., Harwood, Κ., Steven, R., and Jensen, Κ. E. Appl. Environ. Microbiol. 1980, 40, 1044-8. Van Reis, R., Stromberg, R. R., Friedman, L. I., Kern, J., and Franke, J . J . Interferon Res. 1982, 2, 533-41. Valeri, Α., Gazzei, G., Botti, R., P e l l e z r i n i , V . , Corradeschi, Α., and Goldateschi, D. Microbiology 1981, 4, 403-12. Zoon, K. C., Smith, M. E . , Bridgen, P. Α., Zurnedden, D., and Anfinsen, C. B. Proc. Natl. Acad. S c i . U.S.A. 1979, 26, 5601-5.

RECEIVED August 31, 1984

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

3 Practical Aspects of Tangential Flow Filtration in Cell Separations JOSEPH Z A H K A and T I M O T H Y J . L E A H Y

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch003

Millipore Corporation, Bedford, M A 01730

Tangential flow filtration is an effective method for performing the separation of cells from a suspending liquid. Cell separation is the unit process of concentrating biomass that has grown during a fermentation. It typically represents the first step in the extraction and purification of product. There are two advantages of using tangential flow filtration over other unit processes to separate cells and products from fermentors. These are the ability: (l) to work in a closed system without generating aerosols; and (2) to effect a more complete removal of the cells from the fermentor effluent. In addition, Tangential Flow Filtration allows for convenient cell washing after concentration in the same system. The theory of tangential flow Filtration as it applies to cell separations is discussed. Major emphasis, however, is placed on presenting the relationship of experimental results to theoretical performance. Topics highlighted are: flux decay with time, effects of operating pressures and flow, membrane fouling, prefiltration requirements and filter geometries . In most fermentation processes, the fermentation is only the first step in the long process train which includes product formation, recovery, and purification. Commonly the step following fermentation is the separation of the cells from the soluble components in the growth medium. This paper discusses an alternative technique for achieving such a solid/liquid separation. In the case of extracellular products, emphasis is on the treatment of the product-containing broth. Cells, in this instance, are by-products of the fermentation. The processing of the cells is only important in terms of the potential loss of product with the discarded cells. The primary concern is the clarification of soluble product to increase the efficiency of such operations as product isolation and modification. Intracellular products, on the 0097-6156/ 85/0271 -0051 $06.00/0 © 1985 American Chemical Society In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch003

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o t h e r hand, r e q u i r e t h e c o n c e n t r a t i o n o f p r o d u c t - c o n t a i n i n g c e l l s ; t h e suspending l i q u i d i s d i s c a r d e d . T y p i c a l l y , t h e c o n c e n t r a t e d c e l l mass i s t h e n p r o c e s s e d t o f a c i l i t a t e r e l e a s e o f t h e p r o d u c t . The d e s i r e d c o n c e n t r a t i o n o f c e l l s i s o f t e n a f u n c t i o n o f how t h e c e l l s w i l l be p r o c e s s e d . F o r example, c e l l r u p t u r e by s o n i c a t i o n i s most e f f e c t i v e i n a narrow c e l l c o n c e n t r a t i o n range. S e v e r a l t e c h n i q u e s a r e used t o s e p a r a t e c e l l s from t h e ferment a t i o n b r o t h . The most common ones used i n l a r g e s c a l e f e r m e n t a t i o n s a r e continuous f l o w c e n t r i f u g a t i o n , f i l t e r p r e s s e s and r o t a r y drum vacuum f i l t r a t i o n . T a n g e n t i a l ( o r c r o s s ) f l o w f i l t r a t i o n (TFF) has been proposed as a f o u r t h a l t e r n a t i v e {2). I t i s w i d e l y used t o p r o c e s s s m a l l f e r m e n t a t i o n b a t c h e s (N

HN

COOH R OC

O ^ N

2

1

OH

IV >N

I

0 = CHNCH . O .

wκ

ι H,NCH HÇR

3

Ν

OH

3

OH

ι

III

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch004

R

2

HO

H

OH

HOCH

I CHoOCNH, 2

II ο F i g u r e 2. S t r u c t u r e s of p o l y o x i n s L, B, A, and K.

OH

OMe

V

R = CH = CH

M

R = CH

3

F i g u r e 3. S t r u c t u r e s of g i l v o c a r c i n s V and M.

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

2

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch004

PURIFICATION OF FERMENTATION PRODUCTS

20

40

60

80

10of

120

140

160

180

200 Minutes

10% Acetonitrile F i g u r e 4. S e p a r a t i o n o f a p o l y o x i n m i x t u r e . Sample: p o l y o x i n e x t r a c t (100 mg). Column: L i C h r o p r e p RP-18, 25-40 μπι, 2.5 χ 50 cm. M o b i l e phase: 0.015 M HFBA. Flow: 14 ml/min, 80 p s i . D e t e c t i o n : 254 nm.

Gilvocarcin V

Gilvocarcin M

Inj.

1

2

/

\

3

Hours

F i g u r e 5. S e p a r a t i o n of a g i l v o c a r c i n m i x t u r e . Sample: gilvo­ c a r c i n (40 mg). Column: L i C h r o p r e p RP-18, 25-40 μπι, 2.5 χ 50 cm. M o b i l e phase: 3 5 % a c e t o n i t r i l e . Flow: 8 ml/min, 80 p s i . D e t e c t i o n : 254 nm.

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

4.

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Preparative Reversed Phase HPLC

79

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch004

take p l a c e b e f o r e e l u t i o n o f Peak I . F i g u r e 6b shows an a n a l y t i c a l chromatogram o f the same crude m a t e r i a l e l u t e d w i t h a gradient of a c e t o n i t r i l e . I n t h i s case t h e f r o n t m a t e r i a l i s c o m p l e t e l y r e s o l v e d from t h e d e s i r e d peaks. When 2 g o f crude complex was chromatographed on a g l a s s column u s i n g a step g r a d i e n t a s u b s t a n t i a l c l e a n - u p was o b s e r v e d , b u t r e s o l u t i o n and throughput were s t i l l l i m i t e d by column s i z e and f l o w r a t e l i m i t a t i o n s (See F i g u r e 7 ) . T h i s s e p a r a t i o n took o v e r 6 hours and y i e l d e d m i n i m a l amounts o f p r o d u c t s . S c a l i n g up t o l a r g e r i n s t r u m e n t s was n e c e s s a r y . Scale-up instrumentaiton. Two c o m m e r c i a l l y a v a i l a b l e i n s t r u m e n t s were used f o r t h e s c a l e - u p work, a J.Y. Chromatospac 100 and a Waters Prep-500A equipped w i t h a Whatman Magnum 40 column, as d e s c r i b e d i n M a t e r i a l s and Methods. The JY u n i t uses an a x i a l compression system t o o b t a i n t i g h t p a c k i n g . I n o u r hands,"both systems d i s p l a y e d e q u i v a l e n t r e s o l u t i o n . The Waters u n i t w i t h t h e Whatman column had s l i g h t l y lower c a p a c i t y because i t s column was s m a l l e r , but s o l v e n t changes and sample i n j e c t i o n s were e a s i e r t o c a r r y out w i t h i t s r e c i p r o c a t i n g pump. I n both c a s e s , a Gow Mac v a r i a b l e w a v e l e n g t h UV d e t e c t o r was used a t 210 o r 254 nm. T h i s d e t e c t o r i s very d e s i r a b l e as i t shows a l i n e a r response t o very c o n c e n t r a t e d s o l u t i o n s o f compounds a t t h e i r o p t i m a l absorbances. D e t e c t i o n o f the d e s i r e d m a t e r i a l i s enhanced s i n c e t h i s d e t e c t o r can be used a t the same wavelength as t h a t used i n c o r r e s p o n d i n g a n a l y t i c a l work. I n our hands, the use o f a normal v a r i a b l e w a v e l e n g t h d e t e c t o r r u n a t a w a v e l e n g t h somewhat removed from t h e maximum, as o f t e n recommended, has f r e q u e n t l y g i v e n m i s l e a d i n g r e s u l t s due t o the numerous UV-absorbing contaminants i n fermen­ t a t i o n products. The c e l l o f the Gow Mac d e t e c t o r has a 0.1 mm p a t h l e n g t h making i t 100 times l e s s s e n s i t i v e t h a n an a n a l y t i c a l UV d e t e c t o r w i t h a 10 mm p a t h l e n g t h . The c e l l d e s i g n can a l s o h a n d l e 500 ml/min, the maximum f l o w r a t e o f both l a r g e s c a l e chromatographs. Both l a r g e s c a l e systems d i s p l a y e d lower r e s o l u t i o n (200-300 p l a t e s ) than t h e g l a s s columns when e v a l u a t e d w i t h t h e parabens, but they s t i l l had s u f f i c i e n t r e s o l v i n g power t o s e p a r a t e t h e paraben m i x t u r e ( F i g u r e 8 ) . I n t h e o r y , e f f i c i e n c y i s s t i l l s u f f i c i e n t f o r r e s o l v i n g the t h r e e major components i n F i g u r e 4 ( a l p h a ' s o f 1.6 and 1.9). U s i n g a l p h a = 1.6, Ν = 300, and k'= 10 ( t y p i c a l v a l u e s from F i g u r e 4 and c o n d i t i o n s used f o r p r e p a r a t i v e work) and t h e well-known ( 1 ) r e s o l u t i o n e q u a t i o n : R

s

= 1/4 ( a - l ) V ^ T k ' / ( k ' + l )

where R ( r e s o l u t i o n ) i s t h e r a t i o o f the d i s t a n c e between two peaks d i v i d e d by t h e i r average band w i d t h , a c a l c u l a t e d r e s o l u t i o n of 2.3 i s a c h i e v e d . Such a v a l u e i m p l i e s b a s e l i n e r e s o l u t i o n (_1) and i s w e l l i n excess o f t h a t needed t o r e s o l v e peaks I and I I ( F i g u r e 6a). (The minor peaks have r e l a t i v e a l p h a ' s o f o n l y 1.25 i m p l y i n g an R = 0.96, which i s s t i l l s u f f i c i e n t i f c e n t e r c u t s a r e taken.) These c a l c u l a t i o n s use p l a t e counts determined f o r i d e a l substances under n o n - o v e r l o a d c o n d i t i o n s . F o r c o n d i t i o n s s

s

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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PURIFICATION OF FERMENTATION PRODUCTS

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch004

Peak I

Inj.

1 5

L. 9 Minutes

F i g u r e 6a. A n a l y t i c a l s e p a r a t i o n s of a crude g l y c o p e p t i d e complex (isocratic). Sample: crude a n t i b i o t i c e x t r a c t . Column: Beckman U l t r a s p h e r e ODS, 5 μπι, 4.6 χ 150 mm. M o b i l e phase: 35% a c e t o ­ n i t r i l e i n 0.1 M KH2PO4PH 3.2. Flow: 1.5 ml/min. D e t e c t i o n : 220 nm.

F i g u r e 6b. A n a l y t i c a l s e p a r a t i o n s o f a crude g l y c o p e p t i d e complex ( g r a d i e n t ) . Sample: crude a n t i b i o t i c e x t r a c t . Column: Beckman U l t r a s p h e r e ODS, 5 μπι, 4.6 χ 150 inm. M o b i l e phase: 27 t o 37% a c e t o n i t r i l e i n 0.1 M KH2PO4PH 3.2. Flow: 1.5 ml/min. D e t e c t i o n : 220 nm.

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

4.

81

Preparative Reversed Phase HPLC

SITRIN ET AL.

ι

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch004

(50 mg)

Inj.

1

2

3

4

5

6

Hours

F i g u r e 7. S m a l l - s c a l e p r e p a r a t i v e s e p a r a t i o n of g l y c o p e p t i d e complex. Sample: crude a n t i b i o t i c e x t r a c t (2 g ) . Column: Merck L i C h r o p r e p RP-18, 25-40 μπι, 2.5 χ 50 cm. M o b i l e phase: to 30% a c e t o n i t r i l e i n 0.1 M KH P04pH 3.2. Flow: 14 ml/min, 90 p s i . D e t e c t i o n : 210 nm (Gow Mac). 2

I

'

>-

Inj.

30

60

Minutes

F i g u r e 8. S e p a r a t i o n o f parabens on Magnum 40 column (4.8 χ 50 cm). Sample: parabens and t a r t r a z i n e (60-80 mg each). Column: Whatman P a r t i s i l Prep 40 0DS-3 (37-60 μπι). M o b i l e phase: 6 0 % methanol. Flow: 100 ml/min. D e t e c t i o n : 254 nm.

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

82

PURIFICATION OF FERMENTATION PRODUCTS run a t h i g h l o a d i n g (>10 mg/g) lower p l a t e counts a r e observed but o f t e n o t h e r b e n e f i c i a l e f f e c t s such as those observed i n d i s p l a c e ment chromatography become e v i d e n t ( 1 8 ) . An example o f a p r e p a r a t i v e s e p a r a t i o n o f a p u r i f i e d f e r m e n t a t i o n e x t r a c t i n order t o o b t a i n s u i t a b l e e l u t i o n c o n d i t i o n s using the Magnum column, i s shown i n F i g u r e 9a. The sample (2 g ) , was i n j e c t e d through t h e pump i n a s o l v e n t system lower i n a c e t o n i t r i l e content than needed t o e l u t e Peak I . E l u t i o n w i t h a s t e p g r a d i e n t y i e l d e d ( a f t e r d e s a l t i n g ) each o f the pure components w i t h b e t t e r than 90% r e c o v e r y and p u r i t y (see F i g u r e 9b). Loading i n t h i s case was a p p r o x i m a t e l y 1 mg o f Component I p e r gram o f adsorbant.

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch004

Factors P o t e n t i a l l y Limiting

Scale-up

W i t h s u i t a b l e s o l v e n t systems worked o u t , the q u e s t i o n arose as t o what e x t e n t t h i s p r o c e s s c o u l d be s c a l e d up t o produce gram quant i t i e s o f each component. Three p o t e n t i a l l i m i t a t i o n s e x i s t e d : 1) S u p p l i e s o f p u r i f i e d i n t e r m e d i a t e , 2) l o a d i n g c a p a c i t y o f t h e column, 3) s i z e o f column and c o s t o f p a c k i n g . B y p a s s i n g complex i s o l a t i o n scheme. The m u l t i s t e p procedure used to prepare t h e s t a r t i n g m a t e r i a l f o r t h i s chromatographic s e p a r a t i o n was found t o be the primary b o t t l e n e c k . T h e r e f o r e , i n t h e next s c a l e - u p experiment, 25 g o f a crude XAD i s o l a t e ( c o n t a i n i n g 2.2 g o f Component I ) i n 4 l i t e r s o f 17% a c e t o n i t r i l e i n b u f f e r was pumped onto t h e column a t 250 ml/min. S e q u e n t i a l step e l u t i o n s (20, 22, 24 and 26% a c e t o n i t r i l e a t 250 ml/min) r e s u l t e d i n n e a r l y b a s e l i n e r e s o l u t i o n o f t h e t h r e e components ( F i g u r e s 10a and 10b) a g a i n w i t h h i g h r e c o v e r y and p u r i t y (>85%), i n l e s s than 3 hours. S i n c e s e p a r a t e experiments had i n d i c a t e d t h a t a c c e p t a b l e r e s o l u t i o n c o u l d be o b t a i n e d a t h i g h e r f l o w r a t e s , a l l s c a l e - u p work was performed a t 250 ml/min. Thus, t h e p r e p a r a t i v e r e v e r s e d phase column not o n l y y i e l d e d c h r o m a t o g r a p h i c a l l y pure p r o d u c t s but c o u l d a c h i e v e t h e p r e l i m i n a r y p u r i f i c a t i o n w i t h h i g h r e c o v e r y i n hours, a process which o t h e r w i s e took s e v e r a l days t o do. When 50 g ( c o n t a i n i n g a p p r o x i m a t e l y 3 g o f each component) was i n j e c t e d i n 6 l i t e r s o f b u f f e r a l o n e , no breakthrough was observed, and on g r a d i e n t e l u t i o n , t h e chromatogram shown i n F i g u r e 11a was o b t a i n e d . A l t h o u g h r e s o l u t i o n between the t h r e e components had d e t e r i o r a t e d , the complex had been e f f i c i e n t l y separated from t h e contaminants i n l e s s than 2 hours. T h i s f i r s t chromatography step g i v e s a "window" c u t which i s r e l a t i v e l y f r e e o f contaminants o f h i g h e r and lower p o l a r i t y and thereby more amenable t o rechromatography. Indeed, on rechromatography, a s u p e r i o r s e p a r a t i o n o f t h e complex i n t o i t s components o c c u r r e d . The p o o r l y r e s o l v e d m i x t u r e of Components 1,11 and I I I from F i g u r e 11a was p o o l e d , d i l u t e d w i t h b u f f e r and rechromatographed t o y i e l d , a f t e r d e s a l t i n g , 3 g each o f the pure components ( F i g u r e l i b ) . U s i n g t h e above twos t e p procedure ( i . e . s e v e r a l s e p a r a t i o n s on 50 g o r more o f crude e x t r a c t f o l l o w e d by rechromatography o f t h e pooled p u r i f i e d complex) over 10 grams o f the major component was o b t a i n e d , w i t h the same h i g h r e c o v e r y and p u r i t y as shown i n F i g u r e 9b.

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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SITRIN ET AL.

83

Preparative Reversed Phase HPLC

I

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch004

(550 mg)

Inj.

1

2

3

4

Hours

F i g u r e 9a. S m a l l - s c a l e p r e p a r a t i v e s e p a r a t i o n of g l y c o p e p t i d e complex on Magnum 40 column (4.5 χ 50 cm). Sample: purified a n t i b i o t i c (2 g ) . Column: Whatman P a r t i s i l Prep 40 ODS-3 ( 3 7 60 μπι). M o b i l e phase: 20 t o 26% a c e t o n i t r i l e i n 0.1 M KH2PO4PH 6.0. Flow: 100 ml/min. D e t e c t i o n : 210 nm.

Antibiotic Component

Initial Content

Isolated Weight

HPLC Purity

I II III

600 my 350 mg 250 mg

550 mg 250 mg 200 m g

>95% >90% >95%

Inj. F i g u r e 9b. Recovery data f o r s m a l l - s c a l e s e p a r a t i o n o f g l y c o ­ p e p t i d e complex on Magnum 40 column. Sample: p u r i f i e d component I . Column: Beckman U l t r a s p h e r e ODS, 5 μπι, 4.6 χ 150 mm). M o b i l e phase: 27 t o 37% a c e t o n i t r i l e i n 0.1 M KH P04pH 3.2. Flow: 1.5 ml/min. D e t e c t i o n : 220 nm. 2

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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PURIFICATION OF FERMENTATION PRODUCTS

% Acetonitrile 30 26 - 24 - 22 20

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch004

- 17.5

30

60

90

120

150 Minutes

Preparative Separation

F i g u r e 10a. Scale-up p r e p a r a t i v e s e p a r a t i o n of g l y c o p e p t i d e com­ p l e x (25 g r u n ) . Sample: crude a n t i b i o t i c complex (25 g ) . Column: Whatman P a r t i s i l Prep 40 ODS-3 (37-60 μπι) , Whatman Magnum 40 (4.8 χ 50 cm). M o b i l e phase: 17.5 t o 26% a c e t o n i t r i l e i n 0.1 M KH2PO4PH 6.0. Flow: 250 ml/min. D e t e c t i o n : 210 nm. m 1.3g

I

2.ôg

π 17g

Minutes

F i g u r e 10b. A n a l y s i s o f f r a c t i o n s f o r s c a l e - u p s e p a r a t i o n of g l y c o p e p t i d e complex (25 g r u n ) . Sample: p o o l s from 25 g r u n . Column: Beckman U l t r a s p h e r e ODS (5 μπι), 4.6 χ 150 mm. Mobile phase: 27 t o 37% a c e t o n i t r i l e i n 0.1 M KH2PO4PH 3.2. Flow: 1.5 ml/min. D e t e c t i o n : 220 nm.

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

4.

SITRIN ET AL.

85

Preparative Reversed Phase HPLC

%

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch004

Acetonitrile π 30

F i g u r e l i a . Scale-up p r e p a r a t i v e s e p a r a t i o n of g l y c o p e p t i d e com­ p l e x (50 g r u n ) . Sample: crude a n t i b i o t i c complex (50 g) i n 6 L b u f f e r . Column: Whatman P a r t i s i l Prep 40 ODS-3 (37-60 μπι), What­ man Magnum 40 (4.8 χ 50 cm). M o b i l e phase: 0 t o 287 a c e t o n i t r i l e i n 0.1 M KH P04pH 6.0. Flow: 250 ml/min. D e t e c t i o n : 210 nm. 0

2

F i g u r e l i b . Rechromatography of pooled f r a c t i o n s f o r s c a l e - u p s e p a r a t i o n of g l y c o p e p t i d e complex (50 g r u n ) . Column: What­ man P a r t i s i l Prep 40 0DS-3 (37-60 μπι) , Whatman Magnum 40 (4.8 χ 50 cm). M o b i l e phase: 10 t o 28% a c e t o n i t r i l e i n 0.1 M Kl^PO^pH 6.0. Flow: 250 ml/min. D e t e c t i o n : 210 nm.

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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86

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch004

The use o f the column as a c o n c e n t r a t o r i s n o t a b l e . P o o l s c o n t a i n i n g r e l a t i v e l y l a r g e volumes o f f r a c t i o n s (10-20 l i t e r s o r l a r g e r ) c a n be mixed w i t h e q u a l volumes o f b u f f e r t o d i l u t e out the a c e t o n i t r i l e and pumped onto the column w i t h complete r e t e n t i o n . Normal e l u t i o n c a n g i v e the d e s i r e d chromatographic s e p a r a t i o n o r , u s i n g a very s t r o n g s o l v e n t , the e n t i r e product c a n be c o n c e n t r a t e d i n t o two t o t h r e e l i t e r s , a volume s u i t a b l e f o r d e s a l t i n g , l y o p h i l i z a t i o n , e t c . Such a s t e p takes l e s s time than l y o p h i l i z a t i o n o r e v a p o r a t i o n o f the e n t i r e volume. Loading c a p a c i t y . The second f a c t o r which l i m i t s throughput, i n d i c a t e d e a r l i e r , i s t h e l o a d i n g c a p a c i t y o f the column. Table I shows the r e s u l t s o f a l o a d i n g study w i t h homogeneous Compound I w h i c h i n d i c a t e s t h a t " o v e r l o a d " (as d e f i n e d (I) by a 10% l o s s i n k ) o c c u r s a t l e v e l s o f 1-2 mg o f pure m a t e r i a l per g a d s o r b e n t . The k v a l u e s i n Table I are apparent k's s i n c e t h e chromtographic procedure uses a step g r a d i e n t i n o r d e r t o i n j e c t l a r g e volumes. A c t u a l k's a r e somewhat lower. A l s o , l o s s o f r e s o l u t i o n ( d e f i n e d by N) o c c u r s as l o a d i n g i n c r e a s e s beyond t h a t l e v e l . On r e c a l c u l a t i o n o f r e s o l u t i o n observed a t the h i g h e s t l e v e l t e s t e d (13 mg/g) u s i n g Ν = 53, a l p h a = 1.6 and k'= 16, a m i n i m a l l y a c c e p t a b l e v a l u e o f 1.0 i s o b t a i n e d . I n t h e o r y , t h i s p r o b a b l y r e p r e s e n t s the maximum l o a d i n g f o r s e p a r a t i o n o f Component I from Component I I . A t p r e s e n t , q u a n t i t i e s are n o t a v a i l a b l e t o c o n f i r m t h i s . Although s u b s t a n t i a l l y h i g h e r l o a d i n g s are p o s s i b l e by u s i n g displacement chromatography, t h e o v e r a l l throughput would s t i l l depend on the c a p a c i t y o f the column t o p u r i f y the l a r g e r amounts o f crude e x t r a c t s needed t o prepare the p r e c u r s o r s f o r t h e d i s p l a c e m e n t chromatography s t e p . The maximum l o a d i n g of crude e x t r a c t i s y e t t o be determined because o f supply c o n s i d e r a t i o n s . 1

1

Cost l i m i t a t i o n s . The t h i r d l i m i t a t i o n t o s c a l e up i s the s i z e o f column used and c o s t o f p a c k i n g . I n c a r r y i n g out l a r g e s c a l e work on crude e x t r a c t s , the c o s t o f p a c k i n g i s dependent on column life. We have found t h a t the p a c k i n g t u r n s dark i n c o l o r and l o s e s r e s o l u t i o n a f t e r s e v e r a l 50 g r u n s . However, i t can be c o m p l e t e l y r e g e n e r a t e d by e x t r u s i o n , s o a k i n g i n DMSO and r e p a c k ­ ing. Because the p a c k i n g c a n be r e g e n e r a t e d and reused, i t s modest c o s t ($700/kg) i s not a s e r i o u s o b s t a c l e t o i t s use on crude m a t e r i a l . Should f u r t h e r s c a l e - u p be r e q u i r e d , l a r g e r columns w i t h 4 and 20 times t h e c a p a c i t y as the Prep-40 column are a v a i l a b l e from Whatman, as a r e the i n d u s t r i a l u n i t s from Whatman and Waters. Conclusions In summary, we have demonstrated the p o t e n t i a l u t i l i t y o f p r e p a r a t i v e r e v e r s e d phase HPLC as a t o o l t o p u r i f y f e r m e n t a t i o n p r o d u c t s i n m u l t i g r a m s c a l e on l a b o r a t o r y i n s t r u m e n t s . Secondly, due t o i t s i n t r i n s i c s e l e c t i v i t y and c o m p a t i b i l i t y w i t h the use o f h i g h f l o w r a t e s , RPHLC i s a u s e f u l t o o l f o r r a p i d l y p u r i f y i n g crude f e r m e n t a t i o n e x t r a c t s t o a l e v e l o f p u r i t y e q u i v a l e n t t o t h a t o b t a i n e d by m u l t i s t e p time consuming p r o c e d u r e s . We

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

4. SITRIN ET AL.

Preparative Reversed Phase

87

HPLC

TABLE I . L o a d i n g Study - Component

Injection 5

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch004

Weight (g)

Volume* ) (ml)

Loading (mg/g)

c

k' >

Ν

R d) s

0.2

80

0.3

19.2

147

1.7

0.2

2000

0.3

19.3

142

1.7

0.6

220

1.0

17.8

122

1.6

1.2

440

2.0

17.7

121

1.6

2.4

880

4.0

17.2

95

1.4

5.0

1900

8.3

16.2

61

1.1

7.7

1000

12.8

17.4

51

1.0

a) E l u a n t :

24% CH3CN i n 0.1 M phosphate b u f f e r pH 6.0 a t 200 ml/min. Whatman Prep 40 ODS-3 P a c k i n g (37-60 m i c r o n ) i n Magnum 40 Column 50 X 4.8 cm; UV d e t e c t i o n a t 210 urn.

b) Sample d i s s o l v e d i n b u f f e r , no a c e t o n i t r i l e . p r e - e q u i l i b r a t e d w i t h same b u f f e r . c ) Apparent k', not c o r r e c t e d

Column was

f o r e q u i l i b r a t i o n time.

d) C a l c u l a t e d u s i n g a l p h a = 1.6.

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

PURIFICATION OF FERMENTATION PRODUCTS

88

recommend a two-step procedure t o get maximum throughput o f m a t e r i a l : 1) S e v e r a l runs, loaded as h e a v i l y as p o s s i b l e w i t h crude e x t r a c t s t o get a "window" c o n t a i n i n g the d e s i r e d m a t e r i a l ( s ) , 2) rechromatography o f pooled c u t s t o produce t h e pure product. F i n a l l y , the o v e r a l l l o s s i n p l a t e count i n g o i n g from a 5 m i c r o n a n a l y t i c a l column run a t low l o a d i n g w i t h i d e a l s u b s t a n c e s t o a l a r g e r p a r t i c l e p r e p a r a t i v e column (37-60 m i c r o n s ) run a t h i g h l o a d i n g w i t h complex s u b s t r a t e s i s indeed s t r i k i n g . N e v e r t h e l e s s , f o r p r e p a r a t i v e purposes such r e s o l u t i o n i s s t i l l s u f f i c i e n t t o separate compounds c l e a n l y w i t h h i g h r e c o v e r y p r o v i d e d t h a t a l p h a - v a l u e s are l a r g e enough (>1.5). I t would appear t h a t e x c e s s i v e c o n c e r n w i t h p l a t e counts i n such systems i s unwarranted u n l e s s very c l o s e s e p a r a t i o n s are b e i n g r u n .

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch004

Acknowledgment We g r a t e f u l l y acknowledge the e x c e l l e n t t e c h n i c a l a s s i s t a n c e o f Mr. George Udowenko and the support o f o t h e r members o f the SK&F Department o f N a t u r a l P r o d u c t s Pharmacology f o r s u p p l i e s o f f e r m e n t a t i o n p r o d u c t s . We thank Dr. James Chan f o r d a t a on the s t r u c t u r e o f the p u r i f i e d p o l y o x i n s .

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Snyder, L.; Kirkland, J. "Introduction to Modern Liquid Chromatography", Second Edition, John Wiley, New York, 1979. Kabra, P.; Maron, L. "Liquid Chromatography in Clinical Analysis", Humana Press, Clifton, NJ, 1981. Poppe, H.; Kraak, J. J. Chromatog. , 1983, 295, 395. De Jong, Α.; Poppe, H.; Kraak, J. J. Chromatog. 1981, 209, 432. De Jong, Α.; Poppe, H.; Kraak, J. J. Chromatog. 1978, 148, 127. De Jong, Α.; Kraak, J.; Poppe, H.; Nooitgedacht, F. J. Chromatog. 1980, 193, 181. Hupe, K.; Lauer, H. J. Chromatog. 1981, 203, 41. Coq, B.; Cretier, G.; Rocca, J. Anal. Chem. 1982, 54, 2271. Sugnaux, F.; Djerassi, C. J. Chromatog. 1982, 248, 373. Rabel, F. Am. Lab. 1980, 12, 126. Hupe, K.; Lauer, H.; Zech, K. Chromatographia 1980, 13, 413. Verzele, M.; Dewaele, C.; Van Dijck, J.; Van Haver, D. J. Chromatog. 1982, 249, 231. Kagan, M.; Kraevskaya, M.; Vasilieva, V.; Zinkevich, Ε. J. Chromatog. 1981, 219, 183. Fiedler, H. J. Chromatog. 1981, 209, 103. Horvath, C.; Nahum, Α.; Frenz, J. J. Chromatog. 1981, 218, 365. Kalasz, H.; Horvath, C. J. Chromatog. 1981, 215, 295. Kalasz, H.; Horvath, C. J. Chromatog. 1982, 239, 423. Horvath, C.; Frenz, J.; Rassi, Z. J. Chromatog. 1983, 255, 273.

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch004

4.

SITRIN ET AL.

Preparative Reversed Phase HPLC

89

19. Snyder, L. and Kirkland, J., ob. cit., Chapter 15, "Preparative Liquid Chromatography". 20. Nettleton, D. J. Liq. Chromatog. 1981, 4 (Supp. 1), 141. 21. Haywood, P.; Munro, G. In "Developments in Chromatog­ raphy - 2"; C.F.H. Knapman, Ed.; Applied Science Publishers, London 1981, p.33. 22. Eisenbeiss, F.; Henke, H. J. of High Res. Chromatog. and Chromatog. Comm. 1980, 2, 733. 23. Gasparrini, F.; Cacchi, S.; Cagliotti, L.; Misiti, D.; Giovannoli, M. J. Chromatog. 1980, 194, 239. 24. Merck Index, 10th Edition, M. Windholz, Ed., Rayway, NJ. 1983, pl303. 25. Isono, K.; Asahi, K.; Suzuki, S. J. Am. Chem. Soc. 1969, 91, 7490. 26. a) Nakano, H. et al. J. Antibiotics 1981, 34 266; b) Takahashi, K. et al. Ibid 1981, 34, 271. 27. Chan, J.; Yeung, Ε.; Roberts, G.; Sitrin, R. unpublished data. 28. Sitrin, R.; Chan, G.; DeBrosse, C.; Dingerdissen, J.; Hoover, J.; Jeffs, P.; Roberts, G.; Rottschaeffer, S.; Valenta, J.; Snader, K.; 24th Interscience Conference on Antimicrobial Agents and Chemotherapy 1984, Abs. No. 1137. RECEIVED September 7, 1984

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

5 Process Scale Chromatography The New Frontier in High Performance Liquid Chromatography

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch005

A. H .

1

HECKENDORF ,

E. A S H A R E , and C. R A U S C H

Waters Associates, Inc., Milford, M A 01757

In a process scale chromatograph, the concern for continuous utilization of an expensive investment in capital equipment plays a key role in the trade-offs of operation. The scale-up from the laboratory to the industrial scale equipment requires a system engineered to solve a problem greater than the task of purifying larger amounts of compound. The problem is one of operating a system at minimum cost and obtaining high yields of compound at high purity in as short a period of time as possible, and at as high a concentration as possible to minimize cost and solvent removal problems in the recovery process. The use of multiple column segments allows the flexibility of tailoring the column length to the difficulty of varieties of separations and through column sequence and dynamic column length control, many different modes of operation can be performed. High performance liquid chromatography has developed from the analytical scale to the process scale. The evolution of column technology was enhanced by a technique of radial compression, first developed in 1975. This technology was the result of the realization that rather than go to smaller and smaller particle technology to gain resolution in order to enhance the ability of a packed bed structure to resolve compounds, the spaces between particles could be decreased, thus decreasing the amount of dilution that would occur from a given mass transfer rate. This significant step in the evolution of analytical HPLC technology resulted in the ability to change direction away from open column chromatography to the utilization of high-speed, high-resolution, column technology. Current address: NEST Group, Southboro, MA 01772.

1

0097-6156/ 85/0271 -0091 $06.00/0 © 1985 American Chemical Society In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

92

PURIFICATION OF FERMENTATION PRODUCTS

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch005

I n t r o d u c t i o n t o t h e Technology o f Chromatography I f one compares t h e e f f i c i e n c y o r r e s o l v i n g power o f s m a l l e r p a r t i c l e m a t e r i a l s i n a packed bed s t r u c t u r e t o t h e l o a d r e q u i r e m e n t s o f p r e p a r a t i v e chromatography ( F i g u r e 1 ) , one c a n see t h a t , i n f a c t , r e s o l u t i o n d e c r e a s e s w i t h l o a d . Hence, t h e d r i v e toward s m a l l e r p a r t i c l e s i s a g o a l f o r t h e a n a l y t i c a l chromatographer, b u t i t i s n o t a m u t u a l l y shared g o a l f o r t h e p r e p a r a t i v e chromatographer. Because prep i s t h e r e l a t i v e enrichment o f one compound v s . a n o t h e r , i t i s t h e a b i l i t y t o i s o l a t e compounds from a packed bed s t r u c t u r e i n h i g h y i e l d and h i g h mass i n a s h o r t p e r i o d o f t i m e . T h i s R a d i a l Compression t e c h n o l o g y has been packaged i n a v a r i e t y o f forms, t h e s m a l l e s t o f which i s t h e SEP-PAK c a r t r i d g e f o r sample p r e p a r a t i o n f o r t h e a n a l y t i c a l chromatographer. A SEP-PAK c a r t r i d g e i l l u s t r a t e s how column t e c h n o l o g y c a n f u n c t i o n much l i k e a l i q u i d - l i q u i d e x t r a c t o r , w h i l e u t i l i z i n g an i m m o b i l i z e d bed. The SEP-PAK C-18 c a r t r i d g e i s f i l l e d w i t h s i l i c a g e l w h i c h i s c o v a l e n t l y bonded w i t h an o c t a d e c y l s i l a n e e t h e r . T h i s C-18 ( o c t a d e c y l ) c o a t i n g f u n c t i o n s l i k e t h e non-polar s o l v e n t i n a l i q u i d / l i q u i d e x t r a c t o r , and i f one passes a p o l a r s o l v e n t a c r o s s t h i s column, a p a r t i t i o n phenomenon o c c u r s . I f one needs t o s e p a r a t e t h e components o f a m i x t u r e , one c a n s e l e c t s o l v e n t c o n d i t i o n s which w i l l o p t i m i z e t h e p a r t i t i o n i n g a b i l i t y o f t h e n o n - p o l a r i m m o b i l i z e d phase. I f one uses w a t e r , t h e f u r t h e s t i n p o l a r i t y away from t h e n o n - p o l a r C-18 group, n e u t r a l p o l a r and n o n - p o l a r sample components a r e p a r t i t i o n e d i n t o t h e s u r f a c e c o a t i n g . I f t h e amount o f m i s c i b l e n o n - p o l a r s o l v e n t i n t h e m o b i l e phase i s s e l e c t i v e l y i n c r e a s e d , one c a n s e l e c t i v e l y e l u t e o r p a r t i t i o n away from t h a t bonded phase t h e v a r i o u s components. I f one i n c r e a s e s even f u r t h e r t h e non-polar component o f t h e m o b i l e phase, t h e a d d i t i o n a l components b e g i n t o e l u t e . Thus, r a t h e r q u i c k l y we c a n f r a c t i o n a t e a complex m i x t u r e i n t o s e v e r a l components, o r f r a c t i o n a t e a s i m p l e m i x t u r e i n t o i t s i n d i v i d u a l components. Column Technology Design The t e c h n o l o g y o f f e r e d t o t h e p r o c e s s i n d u s t r y i s a c o m b i n a t i o n o f column t e c h n o l o g y , column c h e m i s t r y and system a r c h i t e c t u r e o p t i m i z e d around a p a r t i c u l a r t a s k . The k e y element t o most c h r o m a t o g r a p h i c systems i s based on column d e s i g n , and t h e parameters f o r column performance a r e o p t i m i z e d . I f one were concerned about t h e e f f e c t i v e n e s s o f t h e packed bed s t r u c t u r e , and i n k e e p i n g i t i n p l a c e f o r l o n g p e r i o d s o f t i m e , and one wanted t o m i n i m i z e t h e use o f p r e s s u r e because t h e t r e n d i s towards h i g h e r p r e s s u r e s t o g e t b e t t e r r e s o l u t i o n , one needs an a l t e r n a t e approach t o s c a l i n g up column c a p a c i t y . One c a n g e t t h a t b e t t e r r e s o l u t i o n f o r a p a r t i c u l a r p a r t i c l e s i z e w i t h o u t t h e i n c r e a s e d p r e s s u r e t h r o u g h r a d i a l compression technology. The need t o improve r e s o l u t i o n as i n t h e a n a l y t i c a l w o r l d i s t h e r e , b u t i n t h e p r e p a r a t i v e w o r l d t h e r e i s a l s o t h e need t o c o n t r o l t h e bed s t r u c t u r e over l o n g p e r i o d s o f t i m e . The use o f massive o v e r l o a d and massive overworking o f t h e column t o serve p r o d u c t i o n p u r p o s e s , demands a more r i g i d c o n t r o l over t h e

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

HECKENDORF ET AL.

Process Scale Chromatography

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch005

104

w

103h

10um 37jum 75um

102

1

10

_1_ 1 02

1 03

1 04

ug SAMPLE / gm PACKING F i g u r e 1. Load vs.

particle

size.

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

94

PURIFICATION OF FERMENTATION PRODUCTS p o t e n t i a l c h a n n e l i n g and v o i d i n g o f t h a t bed s t r u c t u r e through s o l v e n t changes, s o l v e n t s t r i p p i n g , and g e n e r a l abuse t h a t o c c u r s i n the p r o c e s s environment t o c o n t r o l c o s t s . I f one can keep p r e s s u r e s low, one a l s o c a n i n c r e a s e t h e speed w i t h which one c a n f l o w t h r o u g h t h a t column and, t h u s , the s e p a r a t i o n time i s g o i n g t o be a t a minimum. One wants t o get the b e s t e f f i c i e n c y out o f t h a t bed s t r u c t u r e t h a t one can, r e g a r d l e s s o f t h a t f a c t t h a t one w i l l l o s e a good p o r t i o n o f t h a t e f f i c i e n c y through l o a d a b i l i t y . And, one i s g o i n g t o want t o have the b e s t c a p a c i t y f o r s e p a r a t i n g compounds t h a t one can w i t h a f i x e d bed l e n g t h .

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch005

R = 1/4

vHï

a-1 α

f

k k» +1

The r e s o l u t i o n e q u a t i o n i s composed o f t h r e e p a r t s : the e f f i c i e n c y parameter, N, the c h e m i s t r y o r s e l e c t i v i t y parameter, a l p h a , and the c a p a c i t y o r l o a d a b i l i t y parameter, k'· I n the r e s o l u t i o n e q u a t i o n f o r the p r e p a r a t i v e environment, t h i s e q u a t i o n i s a l r e a d y f i x e d by the c h e m i s t r y p a r a m e t e r s . F o r example, t h e c a p a c i t y f a c t o r i s s e t f o r the optimum l o a d from the a n a l y t i c a l d a t a ; t h e c h e m i s t r y o r s e l e c t i v i t y parameter i s s e t by t h e a n a l y t i c a l work-up where one h a s t e s t e d a l l the p o s s i b l e c o m b i n a t i o n s o f c h e m i s t r y r e q u i r e d t o get the s e p a r a t i o n ; and the e f f i c i e n c y parameter i s f i x e d by the amount o f l o a d t h a t i s g o i n g t o be p u t on t h a t bed structure. The e f f i c i e n c y , N, i s a f u n c t i o n o f the l e n g t h and the equivalent to a t h e o r e t i c a l plate.

height

Ν = L/H

The h e i g h t e q u i v a l e n t t o a t h e o r e t i c a l p l a t e i s composed o f t h r e e components o f the e q u a t i o n :

H = A + B/u + Cu

The A term (Eddy d i f f u s i o n ) i s the term m i n i m i z e d by r a d i a l c o m p r e s s i o n t e c h n o l o g y ; the Β term i s p o s i t i v e l y i n f l u e n c e d by l i n e a r v e l o c i t y i n c r e a s e s . I f one wants t o go t o h i g h e r speeds, the l o n g i t u d i n a l d i f f u s i o n ( t h e Β term) g e t s s m a l l e r . T h i s i s good because one i s g o i n g t o want t o u s e the system a t a s h i g h a r a t e as p o s s i b l e t o get the maximum throughput o u t o f t h i s i n v e s t m e n t . However, the C term i s n e g a t i v e l y a f f e c t e d by t h i s i n c r e a s e i n l i n e a r v e l o c i t y , u , So one needs some f l u i d v e l o c i t y c o n t r o l t o be a b l e t o o p t i m a l l y get the s e p a r a t i o n over the w i d e s t p o s s i b l e o p e r a t i n g range. Thus, the g o a l s a r e t o d r i v e the Η term down ( t h e h e i g h t e q u i v a l e n t t o a t h e o r e t i c a l p l a t e down), get the optimum u s e of p r e s s u r e , and t o m a i n t a i n Η over a wide dynamic f l o w range, so t h a t one can do a number o f d i f f e r e n t s e p a r a t i o n s on the same system a t d i f f e r e n t l i n e a r v e l o c i t i e s .

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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Process Scale Chromatography

I f one c o n s i d e r s t h e e f f e c t s o f p a c k i n g m a t e r i a l p a r t i c l e s i z e on l i n e a r v e l o c i t y , i t has been determined t h a t i f one d e c r e a s e s p a r t i c l e s i z e by a f a c t o r o f two, the p r e s s u r e i n c r e a s e s as i t s square. Thus, the p r o d u c t i v i t y i n c r e a s e i s p o t e n t i a l l y j e o p a r d i z e d f o r a n e s t h e t i c i n c r e a s e i n r e s o l u t i o n . One needs t o t e s t the a c t u a l r e s o l u t i o n a c h i e v e d i n chromatograms of " u n r e s o l v e d peaks" by t a k i n g f r a c t i o n s t h r o u g h the r e g i o n o f i n t e r e s t and analyzing t h e i r composition. A n a l o g o u s l y , the c h o i c e o f pore s i z e on p r o d u c t i v i t y must be e v a l u a t e d t o o . The r e c e n t i n t e r e s t i n l a r g e pore ( g r e a t e r t h a n 300 angstrom) p a c k i n g m a t e r i a l s f o r chromatography o f macromolecules such a s p e p t i d e s and p r o t e i n s was i n i t i a t e d t o a l l o w h i g h e r r e c o v e r i e s from each c h r o m a t o g r a p h i c r u n , a s w e l l a s t o e l i m i n a t e "memory" e f f e c t s from entrapped m o l e c u l e s on subsequent r u n s . However, the l a r g e r the pore s i z e , the s m a l l e r the s u r f a c e area becomes. T h i s r e s u l t s i n lower c a p a c i t y f a c t o r s , k , a s w e l l a s lower t o t a l c a p a c i t y from a chromatographic system. Thus, t e s t i n g of r e l a t i v e l o a d a b i l i t y and c a p a c i t y from the p a c k i n g m a t e r i a l s i s n e c e s s a r y t o o p t i m i z e the p r o d u c t i v i t i e s o f any system. A s i g n i f i c a n t c o n s i d e r a t i o n i n the p r o d u c t i o n environment i s the c o n c e n t r a t i o n of the p r o d u c t i n the e l u e n t due t o the c o s t s a s s o c i a t e d w i t h sample r e c o v e r y systems and compound s t a b i l i t y during recovery. C o n c e n t r a t i o n i s a f u n c t i o n of the l e n g t h and d i a m e t e r o f t h e column. The w i d e r the column and the l o n g e r i t i s , the more d i l u t e the p r o d u c t stream i s going t o be. Thus, the more energy and t i m e r e q u i r e d t o r e c o v e r the components of i n t e r e s t , and the h i g h e r the p o t e n t i a l f o r p r o d u c t y i e l d l o s s e s due to degradat i o n of p u r i f i e d p r o d u c t . Thus, c o n s i d e r a t i o n of techniques t o prevent product d i l u t i o n i n a chromatographic system are i m p o r t a n t . The amount o f time i n v o l v e d w i t h t h e s e p a r a t i o n i s g o i n g t o be a f u n c t i o n of the l e n g t h of the column, the l i n e a r v e l o c i t y , and the c a p a c i t y term we d i s c u s s e d above. I f one wants to get the most f l e x i b l e column system f o r a v a r i e t y of s e p a r a t i o n problems, what one i s g o i n g t o want t o do i s o p t i m i z e each o f t h e s e terms ( f l u i d v e l o c i t y , c o n c e n t r a t i o n and s e p a r a t i o n t i m e ) . What one i s f o r c e d t o c o n s i d e r i s the a b i l i t y t o have a dynamic column l e n g t h c o n t r o l b u i l t i n t o the system a r c h i t e c t u r e , w h i c h w i l l m i n i m i z e the amount o f d i l u t i o n , s h o r t e n the amount o f time t o get components o u t , keep the p r e s s u r e a t i t s l o w e s t p o s s i b l e l e v e l f o r maximum o p e r a t i n g c a p a b i l i t y , and not s a c r i f i c e the a b i l i t y t o s e p a r a t e compounds. I f one i n c o r p o r a t e s r a d i a l c o m p r e s s i o n technology i n t o the c h r o m a t o g r a p h i c system, i t o f f e r s one the a b i l i t y t o segment the column i n t o s h o r t segments w i t h o u t s a c r i f i c i n g f l u i d d i s t r i b u t i o n o r i n c r e a s i n g p r o d u c t d i l u t i o n t h r o u g h t h i s s e g m e n t a t i o n o f the bed; w h i l e , a t the same t i m e , i t o f f e r s the a b i l i t y t o get the maximum e f f i c i e n c y of our sample f e e d r a t e w i t h r e s p e c t t o the column l e n g t h . I n F i g u r e 2, one can see t h a t the r a t i o of the column l e n g t h a v a i l a b l e over the minimum l e n g t h n e c e s s a r y t o get a s e p a r a t i o n , p l o t t e d a g a i n s t the e f f i c i e n c y of the sample feed r a t e , goes t h r o u g h a n optimum. And, depending on l o a d , t h i s optimum can s h i f t , so t h a t the need f o r h a v i n g a v a r i a b l e l e n g t h column w i t h i n a s i n g l e system i s a p p a r e n t , e s p e c i a l l y when one compares t h a t same L over L min. r a t i o a g a i n s t the e f f i c i e n c y of s o l v e n t u t i l i z a t i o n .

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1

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch005

PURIFICATION OF FERMENTATION PRODUCTS

EFFICIENCY OF SAMPLE FEED RATE

LOAD

COLUMN LENGTH / LENGTH (MIN)

SOLVENT UTILIZATION

LOAD

COLUMN LENGTH / LENGTH (MIN) F i g u r e 2.

E f f e c t s o f dynamic column l e n g t h c o n t r o l .

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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I f one c a n f i x t h e column l e n g t h a t a n optimum f o r t h e sample feed r a t e , t h e n one c a n f i x t h e amount o f s o l v e n t r e q u i r e d t o g e t t h a t separation.

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch005

F l u i d Velocity Control One must l o o k a t t h e f a c t o r s a f f e c t i n g sample d i s t r i b u t i o n t o see how one c a n c o n t r o l t h i s f l u i d v e l o c i t y parameter, t o a l l o w s e g m e n t a t i o n o f t h e column. S c a l i n g parameters f o r sample d i s t r i b u t i o n i n v o l v e f l u i d d i s t r i b u t i o n , c o n t r o l o v e r temperature and, a s has been d i s c u s s e d , a p r e s s u r e c o n s i d e r a t i o n a s w e l l as a m i x i n g volume c o n c e r n . I f one does not c o n t r o l m i x i n g volumes a d e q u a t e l y , t h e r e w i l l be an a u t o m a t i c i n c r e a s e i n t h e volume o f t h e p r o d u c t . I f one u t i l i z e s a l a r g e volume t a p e r a t t h e end o f t h e column t o c o n t r o l f l u i d v e l o c i t y , a s has been done i n t h e l a b o r a t o r y column t e c h n o l o g i e s , one c a n g e t a smooth a d d i t i o n o f sample onto t h e column a t l o w l i n e a r v e l o c i t i e s . However, i n a p r o d u c t i o n environment where one i s going t o t r y t o o p t i m a l l y pump t h a t bed s t r u c t u r e , one c a n see from t h e Van Deemeter p l o t comparison t o t h e f l u i d v e l o c i t y p r o f i l e s w i t h i n t h i s schematic o f a column ( F i g u r e 3) t h a t one w i l l be o p e r a t i n g a t d i f f e r e n t l i n e a r v e l o c i t i e s w i t h i n t h a t d i s t r i b u t i o n o r i f a c e . T h i s adds volume t o t h e p r o d u c t , and c o n s e q u e n t l y , t h e r e ' s a l o s s o f r e s o l v i n g power w i t h i n t h e system. C o n s i d e r i n g t h e e f f e c t o f momentum on t h e volume o f t h e peak t h r o u g h t h i s schematic h e r e , one had a p o i n t s o u r c e o f sample and s o l v e n t a t h i g h l i n e a r v e l o c i t i e s , t h e c e n t e r o f t h e peak would be d r i v e n downwards w i t h i n t h e column because o f momentum. I f one u s e s a d i s t r i b u t i o n p l a t e t o i n t e r r u p t t h a t momentum and t o a d e q u a t e l y d i s t r i b u t e t h e sample a c r o s s t h e bed s t r u c t u r e , l e s s o f a momentum parameter w i l l be i n t r o d u c e d t o t h a t s e p a r a t i o n . This l a t t e r s i t u a t i o n c a n be a c h i e v e d most e f f e c t i v e l y i f one has e l i m i n a t e d the w a l l e f f e c t through r a d i a l compression technology, and has o b t a i n e d a u n i f o r m d e n s i t y bed t h r o u g h t h e same technology. S c h e m a t i c a l l y , t h i s f l u i d d i s t r i b u t i o n system has been a c h i e v e d on o u r t e c h n o l o g y t h r o u g h t h e use o f a s e t o f t h i n , l o w volume d i s k s , supported by t h e f o r c e o f t h e a x i a l b r i d g i n g o f t h e p a c k i n g under r a d i a l c o m p r e s s i o n , so t h a t t h e sample i s p u t on i n a u n i f o r m manner, r e g a r d l e s s o f f l o w r a t e through t h e bed. S c h e m a t i c a l l y , one c a n g e t a v a r i e t y o f band p r o f i l e s from column d e s i g n and bed s t r u c t u r e parameters. On t h e t o p l e f t o f F i g u r e 4 i s one p r o f i l e from a h i g h l i n e a r v e l o c i t y f l o w o f t h e column. The c e n t e r p r o f i l e shows t h e e f f e c t o f w a l l e f f e c t and p o i n t source d i s t r i b u t i o n o f sample and s o l v e n t a t h i g h l i n e a r v e l o c i t i e s . I f one e l i m i n a t e s t h e w a l l e f f e c t by u t i l i z i n g v e r y wide d i a m e t e r columns and s m a l l e r p a r t i c l e p a c k i n g , b u t s t i l l has a p o i n t source d i s t r i b u t i o n o f sample and s o l v e n t , one w i l l g e t band d i s t o r t i o n i n t h e c e n t e r o f t h e band a t h i g h e r f l u i d v e l o c i t i e s . The n e t r e s u l t i n a p r o c e s s environment would be a broad d i l u t e p r o d u c t peak. I f one c a n combine w a l l e f f e c t , bed d e n s i t y and f l u i d d i s t r i b u t i o n c o n t r o l , one c a n narrow t h i s band and i n c r e a s e the c o n c e n t r a t i o n o f p r o d u c t , a s i l l u s t r a t e d on t h e bottom o f F i g u r e 4.

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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FAVORED FLOW PATTERNS

UNIFORM S T R U C T U R E ELIMINATION O F F A V O R E D F L O W

F i g u r e 4.

Band s p r e a d i n g .

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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System A r c h i t e c t u r e H a v i n g d e s i g n e d an optimum column f o r s e p a r a t i o n , we can now i n c o r p o r a t e t h i s t e c h n o l o g y i n t o t h e optimum system a r c h i t e c t u r e . T h i s system a r c h i t e c t u r e s h o u l d i n c o r p o r a t e t h e segmented column design that i s necessary f o r a maximally f l e x i b l e u n i t separation p r o c e s s i n a p r o d u c t i o n environment. Segmented column d e s i g n a l l o w s column sequence c o n t r o l w i t h i n t h e system. Thus, one can get a more c o n t i n u o u s r e c o v e r y o f p r o d u c t o r a h i g h e r o p e r a t i n g e f f i c i e n c y out o f the system. And, because one i s segmenting t h a t column, one can use more o f t h e column bed f o r an o p e r a t i o n a t any s i n g l e time t h a n would be a b l e t o be done w i t h a s i n g l e l a r g e column. S c h e m a t i c a l l y ( F i g u r e 5 ) , one can compare a s e p a r a t i o n o f two components—one on a l o n g column and one on a s h o r t o n e — a n d one can see t h a t , w i t h a segmented d e s i g n one can p u l l out i n t o the p r o d u c t stream a m a t e r i a l t h a t i s p a r t i a l l y p u r i f i e d o r m a t e r i a l t h a t i s f u l l y p u r i f i e d , and the p a r t i a l l y p u r i f i e d m a t e r i a l i s t h e n sequenced onto a n o t h e r column segment f o r f u r t h e r p u r i f i c a t i o n . T h i s m i n i m i z e s t h e amount o f dead time between sample stream a d d i t i o n s and a l s o a l l o w s one t o v i e w what's g o i n g on w i t h i n t h e column a t a h i g h e r f r e q u e n c y r a t e t h a n one i s a b l e t o do on a l a r g e r column. Through p a r a l l e l column segment o p e r a t i o n ( F i g u r e 6 ) , one can c o n t i n u a l l y l o a d p r o d u c t onto t h e columns a t s h o r t e r c y c l e s , t h u s g e t t i n g a h i g h e r product stream o u t p u t . I f one i s g o i n g t o c o n t r o l column sequence, t h i s a l l o w s one to u t i l i z e complex s o l v e n t sequences a s w e l l ( F i g u r e 7 ) . F o r example, f o r c e r t a i n samples one w i l l have t o put on a sample; a f t e r t h a t s e p a r a t i o n i s c o m p l e t e , one w i l l have t o f l u s h t h a t bed s t r u c t u r e and t h e n one w i l l have t o r e - e q u i l i b r a t e t h a t bed s t r u c t u r e b e f o r e the next sample can be added. I f one uses a segmented column system, one c a n o p e r a t e each column i n d e p e n d e n t l y i n terms o f t h e o p e r a t i o n sequence, and o p t i m a l l y g e t t h e downstream s i d e o f t h e system f u n c t i o n i n g a t i t s maximum because, at each s t e p , t h e r e w i l l be a product coming out o f t h e bed. I n a t h r e e column system, on Column One the column o p e r a t i o n a l sequence w i l l be sample, f l u s h , e q u i l i b r a t e , and t h e n on Column Two one w i l l have t o e q u i l i b r a t e , sample, f l u s h ; and t o set up Column Three i n sequence, one i s g o i n g t o e q u i l i b r a t e t h r o u g h t h e f i r s t two s t e p s and t h e n l o a d sample and t h e n f l u s h . Note t h a t t h e p r o d u c t i s c o n t i n u o u s l y coming out o f t h e system a f t e r each sequence. T h i s i s d i f f e r e n t t h a n i f one had a s i n g l e column and one had t o w a i t f o r l o n g e r p e r i o d s o f t i m e between e v e n t s . I t a l l o w s optimum u t i l i z a t i o n o f downstream r e c o v e r y equipment w i t h l e s s b a t c h o p e r a t i o n . Dynamic column l e n g t h c o n t r o l and column sequence c o n t r o l a l s o o f f e r t h e a b i l i t y t o f r a c t i o n a t e complex m i x t u r e s by t h e use of v a r i o u s column segments t o p e r f o r m t h e s e p a r a t i o n t a s k s d i f f e r e n t l y . F o r example: I n the c o n f i g u r a t i o n i n F i g u r e 8, one can f l u s h t h e e a r l y e l u t e r s from a complex m i x t u r e , and t h e n shunt the p a r t i a l l y p u r i f i e d p r o d u c t onto t h e s e p a r a t i o n segments o f t h e system. And s i m u l t a n e o u s l y , f l u s h t h e l o n g e l u t e r s o f f t h a t f i r s t column segment a s w e l l . T h i s g i v e s one some f l e x i b i l i t y i n o p e r a t i o n by a l l o w i n g t h e system t o h a n d l e b o t h complex and s i m p l e s e p a r a t i o n p r o c e s s e s on the same equipment.

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

PURIFICATION OF FERMENTATION PRODUCTS

A

>

A B A B A B A

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch005

B

< F i g u r e 5.

A B PRODUCT STREAM

Column segmentation f o r optimum column

SAMPLE

FLUSH

utilization.

EQUILIBRATE

" PRODUCT STREAM FLUSH

EQUILIBRATE

SAMPLE

"9 Q EQUILIBRATE

SAMPLE

PRODUCT STREAM FLUSH

pu. 3 1

gure 6.

^ PRODUCT STREAM

Column sequence c o n t r o l ( f o r complex s o l v e n t sequences.)

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch005

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SAMPLE FLUSH EQUILIBRATE SAMPLE

101

Process Scale Chromatography

EQUILIBRATE SAMPLE FLUSH EQUILIBRATE

EQUILIBRATE EQUILIBRATE SAMPLE FLUSH

PRODUCT PRODUCT PRODUCT PRODUCT

F i g u r e 7. Column sequence c o n t r o l ( f o r complex s o l v e n t sequences.)

INLET FEED

FLUSH LONG ELUTERS

PRODUCT STREAM ""FLUSH EARLY ELUTERS

PRODUCT STREAM ' INLET FEED

F i g u r e 8. Dynamic column l e n g t h c o n t r o l and column sequence c o n t r o l ( f o r complex m i x t u r e s i m p l i f i c a t i o n . )

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

F i g u r e 9.

Segment 1

Segment

2

PrepLC 5Û0A

P2I

Begin Injection

Load: 15 gm Flow Rale: 500 ml/min Column: 57 mm χ 30 cm silica

-ι 4 3 2 1 0 Time (min.)

KILOPRFP Flow Schematic (Dynamic Column Length Control "Recycle")

Sample

segment-2

Begin Injection segmenM

150 gm 4 Umin (15 cm χ 60 cm) χ 2 (14 χ PrepPAK Cartridge) χ 2

Unit Separation Process System

Comparison o f column performance a t e q u i v a l e n t l o a d i n g .

t

Time (min.) Mobile Phase

KILOPREP Load: Flow Rate: Column:

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch005

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch005

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Process Scale Chromatography

103

T h i s t e c h n o l o g y has been b u i l t i n t o the Waters p i l o t p l a n t u n i t c a l l e d t h e KILOPREP P r o c e s s Chromatography System. I t ' s the f i r s t o f a s e r i e s of i n c r e a s i n g l y l a r g e r chromatographs i n c o r p o r a t i n g r a d i a l compression technology i n t o the system. The performance o f t h i s l a r g e r system can be p r e d i c t e d from the d a t a g e n e r a t e d i n an e q u i v a l e n t l a b o r a t o r y d e v i c e as i n F i g u r e 9. T h i s i s i m p o r t a n t t o p r o c e s s development programs because i t m i n i m i z e s the amount of product and s o l v e n t needed to work out a development program. The t e c h n o l o g y of h i g h performance l i q u i d chromatography has been s u c c e s s f u l l y extended from the a n a l y t i c a l s c a l e t o the p r o c e s s s c a l e . The a b i l i t y t o c o n t r o l the v a r i o u s o p e r a t i o n parameters t o s c a l e up d i r e c t l y from t h e l a b o r a t o r y t o the p i l o t p l a n t and beyond to the p r o d u c t i o n environment has been developed. T h i s t e c h n o l o g y can be combined w i t h o t h e r s e p a r a t i o n s t e c h n o l o g i e s , such a s membrane s e p a r a t i o n s , t o p r o v i d e p a r t i c l e - f r e e s o l v e n t s , u l t r a p u r e p r o d u c t s , and c o n c e n t r a t e d product streams. T h i s w i l l g i v e the o p p o r t u n i t y t o d e a l w i t h f u t u r e s e p a r a t i o n s problems of the chemical process i n d u s t r y . R E C E I V E D October 4, 1984

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

6 Immunosorbent Chromatography for Recovery of Protein Products J O H N P. H A M M A N and GARY J. C A L T O N

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch006

Purification Engineering, Inc., Columbia, M D 21046

The recovery of protein products from fermentation processes by immunosorbent chromatography can be economically attractive. The amount of immunosorbent required is a major cost factor. The proper selection of antibody, matrix, immobilization method and elution conditions can allow high throughput for a given volume of immunosorbent. The throughput depends mainly on the flow rates through the column and cycle half-life. Genetic engineering has provided a cost effective method for the production of large quantities of pharmaceutical proteins. The next major challenge is the isolation of these proteins in a highly purified form in high yield at low cost. The development of a cost effective isolation procedure involves chosing methods for the release of the protein from the cells, separation of the desired protein from other soluble proteins and isolation of the protein in a form required for stability and use. Except in cases where the protein is secreted by the cells, the initial steps of cell concentration, cell disruption and removal of cellular debris are common to all protein isolation schemes, and will not be considered here. A major cost decision will be the selection of a method for separation of the desired protein from other soluble proteins. Classical methods for protein purification based on charge, size, mass and solubility are individually non-specific and must be used in appropriate combinations as experimentally determined. These multi-step procedures increase capital costs, labor costs and the time required for the purification. For labile proteins the decreased yield of material due to degradation during the relatively long time required for purification is a major cost factor. The use of molecular recognition as exemplified by the immunological complex formed by antigen and antibody, has definite advantages for protein purification. The application of chromatographic methods using immobilized monoclonal antibodies can effect the purification of a protein from a complex mixture in a single step (1-6). Cost savings would result from reduced capital and labor costs and increased isolation yields. 0097-6156/ 85/ 0271 -0105506.00/ 0 © 1985 American Chemical Society In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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C r i t e r i a f o r S e l e c t i n g an Immunosorbent The i n d u s t r i a l a p p l i c a t i o n o f t h i s technique r e q u i r e s the c a r e f u l s e l e c t i o n of a n t i b o d y , i m m o b i l i z a t i o n method and i n s o l u b l e m a t r i x . The g e n e r a l r e q u i r e m e n t s f o r an immunosorbent p u r i f i c a t i o n method and t h e f a c t o r s t h a t a f f e c t these r e q u i r e m e n t s a r e l i s t e d i n T a b l e I.

T a b l e I . Immunosorbent Requirements

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch006

REQUIREMENT Absorb p r o t e i n from m i x t u r e Quanitative elution Adequate c a p a c i t y Retains capacity after repeated c y c l i n g Adequate f l o w r a t e s

FACTORS AFFECTING REQUIREMENTS a n t i b o d y , m a t r i x , feed s t o c k p r o t e i n , antibody, immobilizat i o n method m a t r i x , i m m o b i l i z a t i o n method matrix, antibody, immobilizamethod, feed s t o c k matrix, antibody

Only monoclonal a n t i b o d i e s a l l o w one t o s e l e c t the s p e c i f i c i t y , a f f i n i t y and s t a b i l i t y of an a n t i b o d y r e q u i r e d f o r the s p e c i f i c a b s o r p t i o n o f t h e p r o t e i n , the q u a n t i t a t i v e e l u t i o n o f the p r o t e i n under c o n d i t i o n s which r e t a i n i t s a c t i v i t y and the c a p a c i t y a f t e r repeated c y c l i n g . The i m m o b i l i z a t i o n method i s chosen t o r e t a i n a h i g h percentage of the a n t i b o d y a c t i v i t y a f f e c t i n g t h e c a p a c i t y o f the immunosorbent. Some methods of i m m o b i l i z a t i o n form c h e m i c a l bonds between the a n t i b o d y and m a t r i x t h a t a r e u n s t a b l e i n s o l u t i o n s t h a t may be used f o r l o a d i n g o r e l u t i o n . The a n t i b o d y can then b l e e d o f f the m a t r i x , thus a f f e c t i n g t h e p u r i t y of t h e p r o t e i n and the c a p a c i t y of the immunosorbent a f t e r repeated c y c l i n g . A m a t r i x i s chosen f o r i t s a b i l i t y t o support h i g h f l o w r a t e s , i t s l a c k of n o n - s p e c i f i c a b s o r p t i o n s i t e s f o r o t h e r p r o t e i n s i n the s o l u t i o n , and r e s i s t a n c e to mechanical, p r o t e o l y t i c or m i c r o b i a l degradation. Immunosorbent C a p a c i t y A f t e r t h e a p p r o p r i a t e monoclonal a n t i b o d y , i m m o b i l i z a t i o n method and m a t r i x have been chosen a c c o r d i n g to t h e c r i t e r i a d i s c u s s e d above and methods p r e v i o u s l y d e s c r i b e d (7,8) t h e major f a c t o r i n d e t e r m i n i n g t h e c o s t of t h i s p u r i f i c a t i o n method i s t h e amount o f a n t i b o d y r e q u i r e d . The amount of a n t i b o d y r e q u i r e d i s d e t e r mined by the c a p a c i t y per c y c l e of the immunosorbent and the number of c y c l e s t h a t can be u t i l i z e d i n a g i v e n p r o c e s s . The c a p a c i t y per c y c l e f o r t h e immunosorbent i s g i v e n by E q u a t i o n 1.

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

107

Immunosorbent Chromatography

6. HAMMAN AND CALTON

E q u a t i o n 1. Immunosorbent C a p a c i t y

per Cycle

6 9 3 η

η 1 /

C - Α χ Υ χ M.W.P/E.M.W.A. χ V χ ~ ° · / 2 C » Capacity/cycle (g) A « T o t a l Antibody Immobilized ( g l T ) Y Immobilization Y i e l d M.W.P. M o l e c u l a r Weight o f P r o t e i n E.M.W.A. = E q u i v a l e n t M o l e c u l a r Weight o f A n t i b o d y V = Column Volume ( L ) η = C y c l e Number nV2 = Number o f C y c l e s U n t i l C a p a c i t y = 0.5 β

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch006

1

As shown i n E q u a t i o n 1, t h e c a p a c i t y p e r c y c l e i s d i r e c t l y p r o p o r ­ t i o n a l t o t h e amount o f a n t i b o d y i m m o b i l i z e d , t h e i m m o b i l i z a t i o n y i e l d , the M.W. o f t h e p r o t e i n and t h e column volume and an e x p o n e n t i a l f u n c t i o n o f the number o f c y c l e s . The amount o f a n t i ­ body i m m o b i l i z e d w i l l u s u a l l y be l e s s than 10 gL"*. H i g h e r a c t i v a ­ t i o n o f the m a t r i x r e q u i r e d f o r g r e a t e r than 10 g L ~ l l o a d i n g r e s u l t s i n a decrease i n t h e i m m o b i l i z a t i o n y i e l d . The maximum i m m o b i l i z a t i o n y i e l d i s 1.0 (100%) w h i l e 0.8 (80%) i s n o t d i f f i c u l t t o o b t a i n . The M.W. o f t h e p r o t e i n t o be i s o l a t e d i s f i x e d . The o n l y way t o i n c r e a s e t h e c a p a c i t y p e r c y c l e s i g n i f i c a n t l y i s t o i n c r e a s e the volume o f t h e immunosorbent o r i n c r e a s e t h e number o f c y c l e s p r i o r t o r e a c h i n g 50% o f i n i t i a l c a p a c i t y ( c y c l e h a l f - l i f e ) . I n c r e a s i n g t h e volume o f immunosorbent i n c r e a s e s t h e amount o f monoclonal antibody r e q u i r e d . S i n c e t h e c o s t o f t h e a n t i b o d y i s the major c o s t , i n c r e a s i n g the volume o f immunosorbent i s t h e most e x p e n s i v e way t o i n c r e a s e c a p a c i t y p e r c y c l e . The l e a s t e x p e n s i v e way t o i n c r e a s e t h e c a p a c i t y o f t h e i s o l a t i o n system i s t o i n c r e a s e t h e number o f c y c l e s . The number o f c y c l e s t h a t c a n be o b t a i n e d i n any g i v e n p u r i f i c a t i o n i s dependent on t h e c y c l e h a l f - l i f e and time-volume c o n s t r a i n t s . The t o t a l amount o f p r o t e i n t h a t can be i s o l a t e d i n a g i v e n number o f c y c l e s i s g i v e n i n E q u a t i o n 2.

-0.693 n

n

V 2

T o t a l p r o t e i n i s o l a t e d - Co -0.693 n V 2

Where Co - c a p a c i t y o f 1 s t c y c l e η - number o f c y c l e s V2 ™ c y c l e h a l f - l i f e n

I n η = A χ n y c y c l e s , where A i s a c o n s t a n t , t h e t o t a l amount p u r i f i e d i s d i r e c t l y proportional to n y . For a given n l / t h e t o t a l amount p u r i f i e d i s an e x p o n e n t i a l f u n c t i o n o f n. The amount o f p r o t e i n t h a t can be p u r i f i e d w i t h a g i v e n n y number o f c y c l e s i s shown i n T a b l e I I . 2

2

2

i

n

n

2

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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PURIFICATION OF FERMENTATION PRODUCTS

Table I I .

T o t a l Amount of P r o t e i n P u r i f i e d as a F u n c t i o n of C y c l e H a l f - L i f e and C y c l e Numbers

Cycle h a l f - l i f e (nl/ ) 5 10 20 40 80 160 320 2

T o t a l P r o t e i n P u r i f i e d i n η Cycles η = 3 nl/2 η = η η = 2 ni/2 5.79 6.76 3.86 13.07 11.20 7.46 25.69 22.02 14.68 50.94 43.66 29.11 101.44 86.95 57.96 202.44 173.52 115.67 404.45 346.66 231.10

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch006

Factors A f f e c t i n g Cycle H a l f - L i f e The c y c l e h a l f - l i f e i s a f u n c t i o n o f the i m m o b i l i z a t i o n method, the r e a g e n t s used t o d i s s o c i a t e t h e a n t i g e n / a n t i b o d y complex, p r o ­ t e o l y t i c and d e n a t u r i n g agents i n the p r o c e s s stream and t h e r m a l d e n a t u r a t i o n w i t h time. We have i m m o b i l i z e d the F a b fragment of a p o l y c l o n a l goat anti-human IgG a n t i b o d y as a model system. Immunosorbent columns were prepared a t 7 gL~^ and 1 gL~^ a n t i b o d y l o a d i n g . P e r i o d i c a l l y , 50 mg o f human IgG was loaded on the columns i n 10-50 mL o f PBS o r o c c a s i o n a l l y human serum. A f t e r washing w i t h 2 column volumes o f PBS, they were e l u t e d w i t h 0.2 i l a c e t i c a c i d c o n t a i n i n g 0.15 M N a C l . The human IgG i n t h e pass t h r o u g h and e l u a n t was r o u t i n e l y assayed by UV a b s o r p t i o n . As a c h e c k , a q u a n t i t a t i v e ELISA a s s a y f o r human IgG was a l s o used p e r i o d i c a l l y . F i g u r e 1 shows the amount of human IgG bound as a f u n c t i o n of c y c l e number. Between c y c l e s over a p e r i o d o f up t o 195 days t h e immunosorbent was l e f t on t h e bench a t room tem­ p e r a t u r e i n PBS c o n t a i n i n g 0.2% sodium a z i d e . F o r immunosorbent A, (7 gL"" ) c y c l e d 30 t i m e s over a 65 day p e r i o d , n y " cycles. F o r Β (1 mg m l " * ) , c y c l e d 50 times over a 195 day p e r i o d , V 2 ~ ^ c y c l e s . F i g u r e 2 shows the c a p a c i t y of immunosorbent Β above c y c l e d 40 times over a p e r i o d o f 3 d a y s . I n t h i s case no change i n t h e c a p a c i t y was observed as a f u n c t i o n o f c y c l e number. T h i s model showed t h a t a n t i b o d y l o a d i n g and time between c y c l e s had a g r e a t e r e f f e c t on c a p a c i t y as a f u n c t i o n o f c y c l e number than p r o c e s s stream o r e l u t i o n s o l v e n t . However, every system i s not e x p e c t e d t o p a r a l l e l t h i s example. 1

1

1 9

2

n

E f f e c t o f M a t r i x on C y c l e

Time

A l a r g e c y c l e h a l f - l i f e and many c y c l e s i n the p r o c e s s w i l l a l l o w the use of a lower immunosorbent volume ( l e s s a n t i b o d y ) i f the c y c l e s a r e s h o r t enough. Under chromatographic c o n d i t i o n s w i t h t h e p r o p e r " a f f i n i t y s e l e c t e d " i m m o b i l i z e d a n t i b o d y , the f o r m a t i o n o f the i m m u n o l o g i c a l complex i s q u i t e r a p i d . The l i m i t i n g f a c t o r i s the volume t h a t can be passed over t h e immunosorbent i n a g i v e n time. Assuming proper column d e s i g n , t h e l i m i t i n g f a c t o r i s t h e m a t r i x on which t h e a n t i b o d y i s i m m o b i l i z e d . Gels (agarose or

In Purification of Fermentation Products; LeRoith, D., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

HAMMAN AND CALTON

Immunosorbent

Chromatography

109

100

CP

Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch006

Ε

=> Ο DÛ

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