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