Protein Purification. From Molecular Mechanisms to Large-Scale Processes

285 Pages·1990·5.53 MB·English

by  Michael R. Ladisch

ACS SYMPOSIUM SERIES 427
Protein Purification
From Molecular Mechanisms
to Large-Scale Processes
Michael R. Ladisch,E DITOR
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00 Purdue University
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0-04 Richard C. Willson, EDITOR
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19 University of Houston
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102 Chih-duen C. Painton, EDITOR
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99 Stuart E. Builder, EDITOR
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1 Genentech, Inc.
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ati Developed from a symposium sponsored
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ubli by the Division of Biochemical Technology
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at the 198th National Meeting
of the American Chemical Society,
Miami Beach, Florida, September 10-15, 1989
American Chemical Society, Washington, DC 1990
Library of Congress Cataloging-in-Publication Data
Protein purification: from molecular mechanisms to large-scale
processes
Michael R. Ladisch, editor ... [et al.].
p. cm.—(ACS symposium series, ISSN 0097-6156; 427)
"Developed from a symposium sponsored by the Division of
Biochemical Technology at the 198th National Meeting of the
American Chemical Society, Miami Beach, Florida, September
10-15, 1989."
Includes bibliographical references.
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w0 ISBN 0-8412-1790-4
7.f 1. Proteins—Purification—Congresses. 2. Proteins-
2 Biotechnology—Congresses. I. Ladisch, Michael R., 1950- .
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0 II. American Chemical Society. Division of Biochemical
90- Technology. III. Series
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k- TP248.65.P76P765 1990
b 660'.63—dc20 90-35551
21/ CIP
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0 | The paper used in this publication meets the minimum requirements of American
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2, Materials, ANSI Z39.48-1984.
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ACS Symposium Series
M. Joan Comstock, Series Editor
1990 ACS Books Advisory Board
01 Paul S. Anderson Michael R. Ladisch
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7.fw Merck Sharp & Dohme Research Purdue University
2 Laboratories
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0- John L. Massingill
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19 V. Dean Adams Dow Chemical Company
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b Tennessee Technological
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02 University Robert McGorrin
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0. Kraft General Foods
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oi: Alexis T. Bell
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9 Berkeley University of Iowa
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2,
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ne Malcolm H. Chisholm Eisa Reichmanis
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e: J Indiana University AT&T Bell Laboratories
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on Natalie Foster C. M. Roland
cati Lehigh University U.S. Naval Research Laboratory
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G. Wayne Ivie Stephen A. Szabo
U.S. Department of Agriculture, Conoco Inc.
Agricultural Research Service
Wendy A. Warr
Mary A. Kaiser Imperial Chemical Industries
Ε. I. du Pont de Nemours and
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University of Connecticut
Foreword
The ACS SYMPOSIUM SERIES was founded in 1974 to provide a
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00 medium for publishing symposia quickly in book form. The
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04 IN CHEMISTRY SERIES except that, in order to save time, the
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Preface
IROTEIN PURIFICATION: From Molecular Mechanisms to Large-Scale
Processes had its genesis during informal discussions in Houston, Texas,
in April 1989, based upon Richard Willson's suggestion of this project
1 The thought was that a relevant cross-disciplinary treatment of large-
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pr0 scale protein purification could be possible given the rapid progression
27. of several recombinant protein products from laboratory to large-scale,
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0-0 and the willingness of industry to present some of the fundamental
99 aspects of parameters that have an impact on practical considerations of
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k- protein purification. This was reflected, in part, by the papers which had
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21/ already been submitted and organized as part of the Miami Beach pro
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0.1 gram (Jim Swartz, Genentech, Program Chairman) of the Division of
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oi: Microbial and Biochemical Technology, now known as the Division of
0 | d Biochemical Technology. Specifically, two sessions on separations:
9 Large-Scale Protein Purification (M. R. Ladisch and C-D. Painton,
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2, chairpersons) and New Advances in Protein Purification (R. G Willson
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e and S. E. Builder, chairpersons) included approximately 50% of the
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ate: reached as many as 250 people.
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n Upon embarking on this project, Dr. Chih-duen Painton (of Mal-
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ati linckrodt) and Dr. Stuart Builder (of Genentech) were quickly enlisted
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contributors was tremendous, the response of the reviewers impressive,
and the assistance of the ACS Books Department outstanding. The
result is that we can bring this volume to you in a timely fashion.
MICHAEL R. LADISCH
Purdue University
West Lafayette, IN 47907
February 12, 1990
vii
Chapter 1
Large-Scale Protein Purification
Introduction
Richard C. Willson1 and Michael R. Ladisch2
1Department of Chemical Engineering, University of Houston,
Houston, TX 77204
2Laboratory of Renewable Resources Engineering and Department of
Agricultural Engineering, Purdue University, West Lafayette, IN 47907
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ch Large scale protein purification is the final production step, prior to product
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2 packaging, in the manufacture of therapeutic proteins, specialty enzymes,
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0-0 and diagnostic products. The art and science of protein purification evolves
99 from laboratory scale techniques which are often adapted and scaled up to
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k- satisfy the need for larger amounts of extremely pure test quantities of the
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0 als, and, finally, full scale commercialization. Development of appropriate
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2, 1 well as rigorous quality control of manufacturing practices embodied in
1 current good manufacturing practices (cGMP's), provide further challenges
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n to the scale-up of protein purification. Analysis of electrokinetic, chromato
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at yield recovery is paramount, documented purity is critical, and both must
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Pu mone, and bovine growth hormone illustrate conceptual approaches used in

successful industrial processes.
Bio-separation processes have a significant impact on the economics of producing proteins
for animal and human health care products (i). The recovery sequence for isolating product
from a fermentation broth has changed little over the last ten years and consists essentially of
(2):
1. removal of insolubles;
2. primary product isolation;
3. purification; and
4. final product isolation.
0097-6156/90/0427-O001$06.00/0
© 1990 American Chemical Society
2 PROTEIN PURIFICATION
The advent of manufacture of recombinant proteins and peptides for pharmaceutical, diagnos
tic, and agricultural applications has, however, changed the way in which separation tech
niques are chosen, purification strategies are developed, and economics applied to
purification scale-up (see Chapters 2, 3, and 14). Purification costs are particularly important
determinants in production costs of diagnostic reagents, enzymes and animal care products.
Even the most promising human pharmaceuticals are ultimately subject to cost constraints.
Within this context, purity and product activity are still the primary goal.
Removal of trace contaminants that are difficult to detect is becoming a key issue as new
recombinant and cell culture production technologies are phased in (Chapter 2). Examples of
such contaminants include pyrogens, viruses, and transforming DNA, inaccurately translated
or glycosylated forms of the protein, degradation and oxidation products, aggregates and con
formational isomers which are similar to the desired product. Process validation is therefore
quite complex and requires many different types of analytical procedures as shown in Table I.
The detection of trace contaminants also presents many challenges (see, for example,
Chapters 2,11, 14, and 15). It is particularly important that any fractionation-based analyti
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00 cal method used in product characterization employ a separation mechanism different from
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c those already used in the purification process. Otherwise, a contaminant which has co-
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42 purified with the product through the preparative process could escape detection. This con
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9 where several purification steps based on different principles would be used (7). For exam
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1/ raphy (HIQ, and affinity chromatography. Technical issues for each of these steps are the
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10 effect of overload on protein retention (Chapter 7) attaining high throughput at reasonable
10. pressure drop (Chapter 8), prediction of protein retention in HIC as a function of salt type
oi: (chaotrope vs. kosmotrope) and salt concentration (Chapter 6), and selection of appropriate
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9 11).
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2, Novel affinity methods can reduce the number of separation steps by enabling highly
e 1 selective separation from a relatively impure starting materials. Recent developments in this
un context include chelating peptide-immobilized metal affinity chromatography for fusion pro
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Dat in two phase extraction (Chapter 10).
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ionic charge, solubility and density as illustrated in Figure 1 (from reference J). This applies
to both small molecule and protein separations. Recovery and separation of proteins covers
this entire range: i.e., microbial cells (ca. 1 to 5 microns), inclusion bodies (ca. 0.1 to 0.5
microns), protein aggregates (ca. 10 nm to 200 nm), as well as proteins and peptides them
selves (less than ca. 20 nm). This book emphasizes protein properties and purification and
consequently focusses on chromatography and partitioning in liquid systems. Analogies
between traditional chemical separation principles and those applicable to proteins are
apparent (Chapter 3). However, the structure and function of proteins results in product
molecules which differ from variants by as little as one amino acid out of 200. Separations
also need to accomplish removal of other macromolecules (such as DNA and RNA) which
could compromise product efficacy at trace levels. This requires a long list of special
analytical techniques, many of which are based on use of recombinant technology, to validate
product purity and process operation (Table I) (3,4).
—
Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch001 Microfilters · -Cloth and Fibre Filters-^ Screens • Ultrafiltration -^ *nd Strainers -Gel Chromatography - - Reverse Osmosis — Dialysis — —Electrodialysisn. • Ion Exchange" > Distillation/Freese Concentration- • Solvent Extraction ^ • Foam and Bubble Fractionation · • Ultracentrifuges - -4 "Centrifuges - Liquid Cyclones ' • Gravity Sedimentation - 100-1000- 100 · 1000 Micron Fine Coarse Macromolecular Ionic MParticle - • Particle -• Particle -Range"" Range Range Range Range 1. Downstream processing unit operations as a function of size, and physical properties (reprinted with permission, from reference 5, copyright of The Nature Press, MacMillan Publishers, Ltd., all rights reserved).
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Primary Factor Affecting Separation Sise Diffusivity Ionic Charge apour Temperature Pressure Solubility Surface Activity Density Angstroms (A) — 1 Microns(yu) —
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Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch001 Example: Assessment of Product Purity and Processing Conditions Objective Procedure Detect contaminating proteins Carry out electrophoresis (examples for bovine growth hormone in Chapter 2, human serum albumin and hemoglobin in Chapter recombinant IFN and IL-1, IL-2 in Chapter 11, signal peptide in Chapter 14; principles discussed in Chapters 15 and 16). Use Coomassie blue. For more sensitive detection follow with destaining, and silver staining to detect protein bands. Also combine silver staining and immunoblotting. Detect contaminating proteins Select appropriate stationary phase (recall orthogonal separation principle), inject samples look for more than one peak (examples for insulin, insulin A, insulin Β in Chapter 7; IgM in Chapter 14). Detect host proteins Enzyme Linked Immunosorbent Assay. Competitive binding assay (see ref. 3 for monoclonal antibodies; Chapter 14 for IgM (human) monoclonal antibodies). Based on spécifie antigen-antibody reactions (discussed in Detect contaminants (i.e., antibodies leached from affinity columns); small Chapters 2,11, and 14). epitope size makes antibodies poor for variants ("see" small area). Determine terminal sequence of a Carry out controlled hydrolysis of protein. Analyze for amino newly produced protein for acids or peptides using appropriate liquid chromatography comparison against a standard techniques. (See Chapter 12 for discussion of lutenizing hormonreleasing hormone (LHRG) and His-Trp proinsulin.)
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Methods NALYTICAL Electrophoresis Liquid chromato ELISA Immunoassays Amino acid seqcomposition
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oi: 10.1021/bk-1990-0427.ch001 Protease digestion. Reverse phase chromatography of resulting peptides (See Chapter 13 for discussion of proteases used for site-specific cleavage applied to fusion proteins). Binding of complementary nucleic acid sequences to find specific sequences of DNA or RNA (see reference 4 for background on techniques). These cover a wide range of analytical techniques discussed in biochemistry and biology textbooks (see, for example, ref 4). Standard Assay. Check for production of anti-virus antibodies by pathogen free mice immunized with product samples. Host organism, not producing the recombinant products, put through purification process at full production scale, followed by isolation of contaminants (if any) not eliminated by the purification process. Add radiolabeled contaminants, viruses to crude product to demonstrate their elimination after the product is processed through the purification train. Run product through the purification sequence many times, Detect proteins, eluting from column, based on activity, protein content using methods based on different detection principles (see Chapter 7 for example with β-gal/BSA separation).
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Publication Date: June 12, 1990 | Map protein based on analysis and identification of peptide fragments Detect transforming DNA sequences Characterization of biophysical properties, and state of aggregation Detect presence of pyrogens Detect viral contamination Detect contaminants due to host Demonstrate elimination of certain contaminants Detect if changes occur during puri- fication due to product/stationary phase interactions and other purification operations Verify that protein recovery is complete
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Tryptic mapping Hybridization NMR, MS, Light Scattering, Analytical UltracentrifugatioSize Exclusion Chromatogra Rabbit pyrogen test Antibody Production Test ROCESS Blank Runs Tracer Studies Repeated Processing of product Protein and activity material balance of eluting peaks from chromatography syste
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