Downstream Processing and Bioseparation. Recovery and Purification of Biological Products PDF
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ACS SYMPOSIUM SERIES 419 Downstream Processing and Bioseparation Recovery and Purification of Biological Products Jean-François P. Hamel, EDITOR Massachusetts Institute of Technology Jean B. Hunter, EDITOR Cornell University Subhas K. Sikdar, EDITOR National Institute of Standards and Technology Developed from a symposium sponsored by the Division of Industrial and Engineering Chemistry, Inc., at the Third Chemical Congress of North America (195th National Meeting of the American Chemical Society), Toronto, Ontario, Canada, June 5-11, 1988 American Chemical Society, Washington, DC 1990 In Downstream Processing and Bioseparation; Hamel, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990. Library of Congress Cataloging-in-Pubtication Data Downstream processing and bioseparation: recovery and purification of biological products Jean-François P. Hamel, editor, Jean B. Hunter, editor, Subhas K. Sikdar, editor. p. cm.—(ACS Symposium Series, 0097-6156; 419). "Developed from a symposium sponsored by the Division of Industrial and Engineering Chemistry. Inc., at the Third Chemical Congress of North America (195th National Meeting of the American Chemical Society), Toronto, Ontario, Canada, June 5-11, 1988." Includes bibliographical references ISBN 0-8412-1738-6 1. Separation (Technology)—Congresses. 2. Biotechnology—Technique—Congresses. I. Hamel, Jean-François P., 1958- II. Hunter, Jean B., 1955- . III. Sikdar, Subhas Κ. IV. American Chemical Society. Division of Industrial and Engineering Chemistry. V. Chemical Congress of North America (3rd: 1988: Toronto, Ont.) VI. Series. TP248.25.S47D68 1990 660'.2842—dc20 89-49336 CIP The paper used in this publication meets the minimum requirements of American National Standard for Information Sciences—Permanence of Paper for Printed Library Materials, ANSI Z39.48-1984. Copyright © 1990 American Chemical Society All Rights Reserved. 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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 Downstream Processing and Bioseparation; Hamel, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990. ACS Symposium Series M. Joan Comstock, Series Editor 1990 ACS Books Advisory Board Paul S. Anderson Michael R. Ladisch Merck Sharp & Dohme Research Purdue University Laboratories V. Dean Adams Dow Chemical Company Tennessee Technological University Robert McGorrin Kraft General Foods Alexis T. Bell University of California— Daniel M. Quinn Berkeley University of Iowa Malcolm H. Chisholm Elsa Reichmanis Indiana University AT&T Bell Laboratories Natalie Foster C. M. Roland Lehigh University U.S. Naval Research Laboratory 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 Company Robert A. Weiss University of Connecticut In Downstream Processing and Bioseparation; Hamel, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990. Foreword The ACS SYMPOSIUM 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 ADVANCES IN THE CHEMISTRY 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 lished papers are no accepted report research are acceptable, because symposia may embrace both types of presentation. In Downstream Processing and Bioseparation; Hamel, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990. Preface THE RECENT ADVANCES IN GENETIC ENGINEERING AND CELL CULTURE that have spawned the new biotechnology industry have also stimulated new thinking and research in downstream processing. This new research and development, which focuses on separation and purification of biological materials, is welcome and much needed, in view of the central role of bioseparation engineering in the process economics of biotechnology. Downstream processin steps: broth conditioning and removal of insolubles; isolation of the desired product (including clarification and extraction); purification with high-resolution techniques; and polishing. Of these steps, isolation and purification currently enjoy the most attention from researchers. The authors of this book have made further progress in their respective research programs since the symposium on which this book is based. These revisions and new data are included in this book. Most chapters include data that have not been published before. Moreover, each chapter has received two reviews by relevant experts. The aim of this book is not to provide an exhaustive treatise on all areas of isolation and purification of biotechnology products, but to present the spectrum of current thinking and activities on bioseparations, specifically of large molecules such as proteins and polysaccharides. The chapters are divided into three categories: extraction and membrane processes, processes using biospecific interaction with proteins, and novel isolation and purification processes. An overview chapter by Hamel and Hunter presents the state of the art of research on bioseparations. Extraction processes using biphasic aqueous systems, liquid membranes, reversed-micellar systems, and membrane processes are all being actively studied. Significant advances in these topics, including predictive mathematical models, are presented in the first section. The second section includes several papers on affinity and other interaction techniques that are finding uses in protein purification. In the last section, we offer several reports that delineate advances in isolation and purification processes such as electrophoresis and chromatography. vii In Downstream Processing and Bioseparation; Hamel, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990. We gratefully acknowledge the assistance of our reviewers, whose insight and guidance have enlightened the editors and authors alike. We thank the authors for their special assistance generously extended. Finally, we are indebted to Cheryl Shanks of the ACS Books Department for her patience and many helpful hints during the preparation of this book. JEAN-FRANÇOIS P. HAMEL Massachusetts Institute of Technology Cambridge, MA 02139 JEAN B. HUNTER Cornell University Ithaca, NY 14853 SUBHAS K. SIKDAR National Institute of Standards and Technology Boulder, CO 80303 November 6, 1989 viii In Downstream Processing and Bioseparation; Hamel, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990. Chapter 1 Modeling and Applications of Downstream Processing A Survey of Innovative Strategies Jean-François P. Hamel1 and Jean B. Hunter2 1Department of Chemica of Technology 2Department of Agricultural and Biological Engineering, Cornell University, Ithaca, NY 14853 Downstream processing is playing an increasingly important role in the biochemical industry, especially since the advent of recombinant DNA technology. The use of recombinant DNA technology not only enables improvements in the production efficiency of therapeutic and industrial proteins, but it also permits the modification and improvement of protein structure and thus function. However, the commercial application of such technology was initially accompanied by concerns over product safety. Quality criteria have been made especially stringent for products derived from genetically-modified microorganisms. The establishment of strict quality guidelines was the result of early concern about the oncogenic potential related to products contaminated by DNA sequences of the host mammalian cells (1). The quest for high quality has created a growing need for high-resolution techniques at the process scale as well as for novel strategies for the isolation and purification of bioproducts. Since the typical environment for producing biologicals is a complex one and quality criteria need to be strict, primary recovery techniques are typically implemented in a purification scheme prior to (or in conjunction with) high-resolution techniques. The most sophisticated and useful schemes take advantage of both the different physical and chemical properties of the components in complex mixtures and of the interactive nature of the downstream processing techniques (see Figure 1). 0097-6156/90/0419-0001$09.75/0 © 1990 American Chemical Society In Downstream Processing and Bioseparation; Hamel, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990. 2 DOWNSTREAM PROCESSING AND BIOSEPARATION Purification Scheme of a Bioproduct | Diluted Cell Material Centrifugation Clarified Filtration Aqueous Aqueous-Two phase Phase Cell Concentrate Disruption: • Mechanical • Extraction • Physical • Precipitation • Chemical • Adsorption • Ion Exchange Cell Homogenate • Filtration Centrifugation Filtration Micelle Aqueous-Two phase • Liquid Emulsion Membrane Clarified Homogenate Precipitation (Affinity) Filtration Reversed-Micelle Liquid Emulsion Membrane Chromatography Electrophoresis Affinity HPLC Figure 1. Bioproduct Purification Chart Intracellular Bioproduct Route Extracellular Bioproduct Route W Since proteins are polymers of amino acids, the chemical nature of the amino acid side chains and the order of the amino acids play an important role in establishing the biological properties of the active protein. Proteins may differ from each other according to size, charge density, shape and biological activity. Similarly, protein purification schemes require a similar diverse combination of separation techniques based on the various physicochemical properties of proteins. Typically, protein is lost at every purification step and one normally wishes to reduce the number of steps. An added advantage of fewer steps for some unstable proteins is faster processing time and thus, improved quality of the desired protein when time is critical to maintain In Downstream Processing and Bioseparation; Hamel, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990. 1. HAMEL & HUNTER Modeling and Applications of Downstream Processing 3 stability. However, the number of physicochemical properties are limited; so are the number of purification techniques developed from them. In non-genetically engineered microorganisms or cells, the protein of interest often represents a small fraction of total cellular or extracellular protein. Several strategies have been developed, using the techniques of molecular biology (e.g. gene dosage, leader sequence), which permit the design of efficient and simple purification schemes. For example, the overexpression of cloned genes in Escherichia coli or animal cells is an increasingly used strategy to produce eukaryotic proteins. Overexpression in bacteria often results in the formation of insoluble protein aggregates which are usually not in an active form. In some cases, the desired protein is already relatively pure and may represent up to 25% of total cell protein. An initial isolation step involving a combination of a disruptio therefore produce a relatively pure product. By comparison, if that same protein were produced as a soluble protein, its initial purity would likely be significantly lower. Thus, an integrated view of each process is of critical importance. Whether the protein produced is soluble or insoluble, the isolation of intracellular proteins typically requires the use of disruption techniques. High-pressure homogenization is an effective technique to free intracellular products. The detailed mechanisms by which the cells are disrupted are not known, and the parameters for determining the degree of disruption can only be determined empirically (2). Then, such knowledge would be likely to impact the design of equipment. In the last ten years, for example, major efforts have been devoted to homogenizer valve design and to configurations permitting higher pressure (>600 bar) operation, with the rationale that such conditions produce more efficient disruption - in terms of amount of product released per pass. Since the relationships between pressure and particle size distribution are poorly understood, there is a possibility that increasing the homogenizer operating pressure produces decreasing particle size. Smaller particles, in turn, may have a negative impact further downstream, in that their removal during clarification operations may be more difficult. Often, high-resolution techniques like electrophoresis or affinity chromatography cannot be used readily on a complex mixture. However, in most isolation/purification processes of proteins, chromatography will appear in one form or another. Affinity chromatography has received considerable attention in the last ten years since it is one of the most powerful tools for separating biological products. This technique has been largely researched at the small-scale and only recently have large- scale studies been detailed in the literature. For example, the use of a monoclonal antibody column was recently reported to have provided major purification in a single step of interferon a-2a from extracts of recombinant Escherichia coli cells (3). As a result, a process using affinity chromatography may permit the reduction of the number of steps In Downstream Processing and Bioseparation; Hamel, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990. 4 DOWNSTREAM PROCESSING AND BIOSEPARATION compared to processes based on other techniques like centrifugation and filtration. Overall however, there are few published reports of large-scale processes based on affinity separations, and in this context aqueous two-phase systems and membrane technology have imposed themselves (4). This book includes detailed studies by Cabezas £t al. (5), Forciniti and Hall (6), Szlag et aL (7), Dall-Bauman and Ivory (8), Guzman et aL (9) and Sheehan e_t aJL (10) based on such technologies as well as many others still confined to the laboratory scale. The contributions are varied in that: 1) some are theoretical, some experimental and some are both, 2) the authors represent both the academic and the private sectors, 3) there are several attempts to describe large-scale processes. The remainder of this introductory chapter focuses on downstream processing and bioseparation relevant to the chapters presented in this book. Thus phase systems, membrane separation, centrifugation and adsorption techniques, electrophoresis, chromatography, and affinity separations. MULTI-PHASE SYSTEMS FOR THE RECOVERY OF PROTEINS Aqueous Biphasic System More than 70 years elapsed between the first report of aqueous two-phase systems (11) and their subsequent applications to biochemical systems (12). In the last ten years in particular, there have been several innovative applications of aqueous two-phase systems (13). Aqueous two-phase systems consist of two immiscible fluids in a bulk water solvent. In such systems, the percentage of water in both phases is high, i.e. between 75 and 95%. As a consequence, the surface tension between the two immiscible phases may be as low as 0.1 dyne/cm so that a gentle mixing is sufficient to produce and maintain an emulsion (14). One of the best characterized systems involves mixtures of dextran and polyethyleneglycol (PEG). In such a system, biological substances ranging from soluble proteins to particulate materials (cells or organelles) will partition preferentially in one of the phases. In order to characterize the separation of a substance of interest in an aqueous two-phase system, it is convenient to define a partition coefficient as the ratio of this substance's relevant property in the top and bottom phases. For example, for a protein with biological activity: K = ACT /ACT act top b ottom where: K is the partition coefficient of the protein, act ACT p is the activity in the top phase, and t0 ACTbottom is the activity in the bottom phase. In Downstream Processing and Bioseparation; Hamel, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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