Bio Seperation

EDITORIAL ADVISORY BOARD

Steven M. Cramer Rensselaer Polytechnic Institute Troy, New York Milton T. W. Hearn Monash University Clayton, Victoria Australia

William S. Hancock Hewlett Packard Palo Alto, California Brian Hubbard Genetics Institute Andover, Massachusetts

CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors' contributions begin.

Satinder Ahuja (1, 237, 687) Ahuja Consulting, 330 S. Middleton Drive, Suite 803, Calabash, North Carolina 28467 Rakesh Bajpai (365) Department of Chemical Engineering, Department of Biological and Agricultural Engineering, University of Missouri-Columbia, Columbia, Missouri 65211 Egisto Boschetti (535) Life Technologies-BioSepra, 95804 Cergy Saint Christophe, France P. Bowles (633) Kvaerner Process (UK) Ltd., Whiteley, Hants, United Kingdom Douglas L. Cole (511) Development Chemistry and Pharmaceutical Development, Isis Pharmaceuticals, Inc., Carlsbad, California 92008 Catherine E. Costello (299) Mass Spectrometry Resource, Boston University School of Medicine, Boston, Massachusetts 02118 Steven M. Cramer (379) Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180 Ranjit R. Deshmukh (453, 511) Manufacturing Process Development, Isis Pharmaceuticals, Inc., Carlsbad, California 92008

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David E. Garfin (263) Life Science Group, Bio-Rad Laboratories, Hercules, California 94547 Dan Gibson (299) Mass Spectrometry Resource, Boston University School of Medicine, Boston, Massachusetts 02118; and Department of Pharmaceutical Chemistry, School of Pharmacy, Hebrew University of Jerusalem, Jerusalem, Israel Tingyue Gu (329) Department of Chemical Engineering, Ohio University, Athens, Ohio 45701 Rohit Harve (365) Wyeth Ayerst Research, Marietta, Pennsylvania 17547 Milton T. W. Hearn (71) Centre for Bioprocess Technology, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia Alois Jungbauer (535) Institute of Applied Microbiology, University of Agriculture, A-1190 Vienna, Austria Robert M. Kennedy (431) Separations Group, Amersham Pharmacia Biotech, Piscataway, New Jersey 08855 William E. Leitch, II (511) Argyll Associates, Palm Desert, Cahfornia 92210 B. Mattiasson (417) Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Lund, Sweden M. P. Nandakumar (417) Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Lund, Sweden R. M. Nicoud (475) NovaSep, Vandoeuvre-les-Nancy, France Donald O. O'Keefe (23) Macromolecular Structure and Biopharmaceuticals, Bristol-Myers Squibb, Princeton, New Jersey 08543 Randel M. Price (659) Department of Chemical Engineering, University of Mississippi, University, Mississippi 38677 Anand Ramakrishnan (667) Department of Chemical Engineering, University of Mississippi, University, Mississippi 38677 Ajit Sadana (659, 667) Department of Chemical Engineering, University of Mississippi, University, Mississippi 38677 Yogesh S. Sanghvi (511) Manufacturing Process Development, Isis Pharmaceuticals, Inc., Carlsbad, California 92008 Joseph Shiloach (431) Biotechnology Unit, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, Maryland 20892 Abhinav A. Shukla (379) ICOS Corporation, Bothell, Washington 98021 Timothy N. Warner (453) Sartorius Corporation, Edgewood, New York 11717

PREFACE

The commercial success of biotechnology products is highly dependent on the successful development and application of reliable and sensitive bioseparation methods. Bioseparations entail separations of proteins and other materials from biological matrices. This book is planned to serve as a handbook vîth the primary focus on separations of proteins; howêver, separations of other materials of interest, such as nucleic acids and oligonucleotides, are also covered to assist the readers in tackling their particular bioseparation problems. Included in this text is a chapter on the separation of monoclonal antibodies. Monoclonal antibodies and recombinant antibodies have become one of the largest classes of proteins that have received FDA approval as therapeutics and diagnostics. Antisense drugs have been covered because of their unique ability to bind to targeted messenger RNA (mRNA) while avoiding attachment to other proteins. Bioseparations are also helping wîth the development of a large number of drugs for the treatment of a variety of diseases such as cancer, AIDS, rheumatoid arthritis, and Alzheimer's disease. The regulatory considerations applying to bioseparations are discussed in various sections of this book. It is important to remember that the FDA requires a thorough validation program, quality assurance oversight, statistically sound sampling methods, rigorous training, and a comprehensive documentation trail. The guidelines recommended by the International Conference on Harmonization addressing quality, safety, and efficacy have been covered to provide additional insight into this area.XIII

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This book has been broadly divided into three sections: The analytical methodology section covers a variety of methods that are commonly used in bioseparations. Analytical methodology includes an interesting montage of chromatographic methods, capillary electrophoresis, isoelectric focusing, and mass spectrometry. Separation and purification methods provide detailed information on Hquid-liquid distribution, displacement chromatography, expanded-bed adsorption, membrane chromatography, and simulated moving-bed chromatography. This section also provides significant information for process-scale separations. Plant and process equipment, engineering process control of bioseparation processes, economic considerations, and future developments are discussed under the heading of Other Important Considerationsthose elements that are sometimes forgotten but should never be ignored when one is dealing with bioseparations. The chapter on future developments provides some insight into what is coming down the road in the field of bioseparations; to this end, short summaries of various oral presentations made at the Ninth Conference on Recovery of Biological Products (held on May 2 3 - 2 8 , 1999, in Whistler, Canada) have also been included since this conference has become the preeminent meeting in the field of bioseparations. The excellent contributions to the Handbook of Bioseparations are likely to make it an essential reference and guidebook for separation scientists working in the pharmaceutical and biotechnology industries, academia, and government laboratories. February 2000 Satinder Ahuja Calabash, North Carolina

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BIOSEPARATIONS: AN OVERVIEWS. AHUJAAhuja Consulting, Calabash, North Carolina 28467

I. INTRODUCTION A. Regulatory Considerations II. ANALYTICAL METHODOLOGIES A. HPLC B. Capillary Electrophoresis C. Isoelectric Focusing D. Mass Spectrometry E. Methodology Montage III. SEPARATION A N D PURIFICATION METHODS A. Liquid-Liquid Distribution B. Separation of Proteins and Nucleic Acids C. Displacement Chromatography D. Expanded-Bed Adsorption E. Membrane Chromatography F. Simulated Moving Bed Chromatography G. Purification of Oligonucleotides H. Monoclonal Antibodies IV. OTHER IMPORTANT CONSIDERATIONS A. Processing Plant and Equipment B. Engineering Process Control C. Economics of Separations D. Future Developments REFERENCE

I. INTRODUCTION The biotechnology industry has evolved significantly since the introduction in 1982 of human insulin synthesized in Escherichia colithe first Food and Drug Administration (FDA)-approved recombinant therapeutic agent in the United States. Since then, over 75 other recombinant proteins have been introduced. The list is comprised of cytokines, hormones, monoclonal antibodies, and vaccines. There are more than 1100 companies competing for this market, and the current sale of these products comprises approximately 10% of the sales of all therapeutic products sold in the United States. One such product, erythropoietin, an erythropoiesis-stimulating factor also knownSeparation Science and Technology, Volume 2 Copyright 2000 by Academic Press. All rights of reproduction in any form reserved.

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as Epogen, is a circulating glycoprotein that stimulates red blood cell formation in higher organisms and has worldwide sales in excess of 1 billion U.S. dollars. The financial potential of these products is indeed great. This is apparent from the fact that over 500 biotechnology-related drugs are currently in clinical trials. Bioseparations, or separations of biological interest, have played a significant role in the development and growth of the biotechnology industry. These separations have to be performed on both analytical and industrial scalesand everything in between. Bioseparations frequently entail separations of proteins and related materials from biological matrices.^ This book is planned to serve as a handbook of bioseparations, where the primary focus is separations of proteins; however, separations of other materials of interest such as nucleic acids and oligonucleotides are also covered to assist the reader in tackling their particular bioseparation problems. Included in this text is a chapter on the separation of monoclonal antibodies, as these materials have found numerous uses in the biopharmaceutical industry. As a matter of fact, in the last few decades, monoclonal antibodies and recombinant antibodies have become one of the largest classes of proteins that have received FDA approval as therapeutics and diagnostics.

A. Regulatory Considerations The regulatory considerations applying to bioseparations are covered in various sections of this book. It is important to assure that separation and purification methods, when operating within the established limits, produce a product of appropriate and consistent quality. The method and process validations provide assurance that product quality is derived from a careful consideration of various factors such as process design, selection, and control of the process through appropriate in-process and end-process testing.^ Validation studies should be performed through each of the three phases of a product's life span: development, pilot scale, and end-process testing. In addition to validated testing methods and standards, the FDA requires a thorough validation program, quality assurance (QA) oversight, statistically sound sampling methods, rigorous training, and a comprehensive documentation trail. Undeniably, biopharmaceuticals should be safe and effective. This must be demonstrated by effectively planned studies as well as documentation to the satisfaction of regulatory agencies. The young age of this industry is demonstrated by the fact that in 1985, the FDA issued a document entitled "Points to Consider in the Production and Testing of New Drugs and Biologicals Produced by Recombinant DNA Technology." In 1997 a similar document was issued for monoclonal antibodies. Also in 1997, the Center for Biologies Evaluation and Research (CBER) issued guidance on the preparation of a Biologies License Application (BLA). For the first time, manufacturers can file a BLA instead of an Establishment License Application (ELA) and a Product License Application (PLA). The BLA brings the drug and biotechnology therapeutics registration process closer together.

BIOSEPARATIONS: AN OVERVIEW

The CBER was established in 1987 as a spin-off of the FDA's Center for Drugs and Biologies in response to a growing number of applications for new biotechnology products. "Guidelines," "Guidance," "Points to Consider," and other documents are available from CBER (Office of Training and Manufacturers Assistance, HFM-40, Rockville, MD, 20852. Information can be obtained by telephone at 800-835-4709 or by fax at 301-827-3844). It is important to keep current with the latest regulations. Generally, this information can be obtained from the FDA Web site, www.fda.gov/cber/publications.htm. A joint regulatory-industry initiative was taken to provide international harmonization of the drug approval process. The guidelines recommended by the International Conference on Harmonization (ICH) address quality, safety, and efficacy. The ICH issued draft guidelines on analytical validation procedures in 1996 and a document entitled "Draft Consensus Guidelines and Specifications: Test Procedures and Acceptance Criteria for Biotechnological/ Biological Products" in 1998. Further information relating to ICH can be found at the Web site www.ifpma.org of the International Federation of Pharmaceutical Manufacturers Association. The contents of this book have been broadly classified into three sections: Analytical methodologies Separation and purification methods Other important considerations The analytical methodology section covers a variety of methodologies that are commonly used in bioseparations. The section on separation and purification methods covers a broad range of methods, including process-scale separations. Plant and process equipment, engineering process control of bioseparation processes, economic considerations, and future developments are discussed under the heading of other important considerationsthose elements that are sometimes forgotten but should never be ignored when one is dealing with bioseparations. Processing plants and equipment are discussed in this book to assist the scientist or engineer in selecting a method of bioseparation that will be suited to the particular requirements of the process and the product at a commercial scale of operation. A chapter on economics of bioseparations has been included to help evaluate cost considerations prior to the initiation of any project. Finally, the chapter on future developments attempts to provide some insight into what is coming down the pike in the field of bioseparations, a field that is continually evolving and thus defies any fixed descriptive definitions.

II. ANALYTICAL METHODOLOGIES The purity analysis of a recombinant produced product is difficult because the accuracy of protein purity is method-dependent and is influenced by the shortcomings of the analytical procedures (Chapter 2). Proteins are highly complex molecules; therefore, it is generally very desirable to utilize more than one method to define a given protein's purity. To assure the purity of

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the desired product, it is important to evaluate process-related and productrelated impurities (for details, see Chapter 2). Protein purity in excess of 99% is often expected of therapeutic products. Significant impurities, such as host-cell proteins, are expected to be present at no more than trace levels (parts per million). Most proteins can be analyzed by high-pressure, or high-performance, liquid chromatography (HPLC) and electrophoretic methods. These methods are discussed in great detail in this book. A number of analytical methods are discussed at length throughout this book; Chapters 2 - 6 , 10, 14, and 15 offer fairly extensive coverage of analytical methodologies. Chapter 2 provides an excellent coverage of methods primarily used for protein impurities in pharmaceuticals derived from recombinant DNA. Protocols for selected examples are included to assist the reader in carrying out analyses of interest to them. It should be readily recognized that these methods are also useful for purity analysis of proteins as well. Because of the relative importance of analytical methodology, special chapters are devoted to HPLC (Chapter 3), capillary electrophoresis (Chapter 4), isoelectric focusing (Chapter 5), and mass spectrometry (Chapter 6). Chapter 10 covers analytical aspects of expanded-bed chromatography. The variety of methodologies used for the analysis of oligonucleotides and antibodies are covered extensively in Chapters 14 and 15, respectively.

A. HPLC Chapter 3 provides an overview of physicochemical factors that impact analysis and purification of polypeptides and proteins by HPLC techniques. The current status and some of the future challenges facing this major field of separation sciences are considered from both didactic and practical perspectives (Chapter 3). This chapter attempts to provide an overview of terms, concepts, principles, practical aspects, and primary references that underpin the recent developments in this field. Where appropriate, key relationships and dependencies that describe the interactive behavior of polypeptides and proteins with chemically immobilized ligands are discussed. This understanding is central to any subsequent exploration of alternative avenues now available for further research and development into the field of polypeptide or protein purification and analysis. HPLC techniques have occupied a dominant position for over two decades in peptide and protein chemistry, in molecular chemistry, and in biotechnology. These techniques with their various selectivity modes (listed later) can be considered the bridges that link cellular and molecular biology (viz., structural proteomics and atomic biology) and industrial process development associated with the recovery and purification technologies that turn these opportunities into realities. Different dominant interactive modes of HPLC are as follows: Normal phase Ion exchange Reversed phase


Hydrophobic interaction Biospecific and biomimetic affinity This chapter considers the specific physicochemical considerations of various chromatographic modes and provides strategic considerations in HPLC separations as vêll as heuristic approaches and productivity considerations in scale-up operations.

B. Capillary Electrophoresis Electrophoresis is defined as transport of electrically charged particles in a direct-current electric field. The particles may be simple ions or complex macromolecules including proteins, colloids, or particulate matter such as living cells (bacteria or erythrocytes). Electrophoretic separation is based on differential rate migration in the bulk of the liquid phase and is not concerned with any reactions occurring at the electrodes. The highest resolution is obtained when an element of discontinuity is introduced in the liquid phase, such as a pH gradient or the sieving effect of high-density gels. Membrane barriers may also be introduced into the path of migrating particles. Electrophoresis can be classified on the basis of whether it is carried out as a free solution or on the support media. When support media are used, the technique is called zone electrophoresis. Capillary electrophoresis (CE), which is commonly used today, fits into the latter category, and at one time was called capillary zone electrophoresis. Strictly speaking, CE without any of the modifications mentioned below is not a chromatography technique because two phases are not involved in the separation process (Chapter 4). Recall that the two phases in chromatography are designated as the stationary phase and the mobile phase, based on their role in the separation process. Technically, there is no stationary phase in capillary electrophoresis unless the capillary walls are assigned that role. Some chromatographers promote this concept, but it is not entirely correct. In any event, most chromatographers are comfortable using CE because it enjoys a number of similarities to chromatography in that some of the manipulations used to optimize chromatographic separations are also suitable for CE. And symposia on CE are often included in the major chromatographic meetings. In Chapter 4, the following approaches to peptides and proteins separations with CE are discussed: Capillary zone electrophoresis (CZE) Micellar electrokinetic capillary chromatography (MECC) Capillary gel electrophoresis (CGE) Capillary electrochromatography (CEC) Capillary isoelectric focusing, another form of capillary electrophoresis, is covered in Chapter 5 and discussed briefly in Section II.C. Capillary electrophoresis has been found to be quite useful for resolving a very large number of compounds including peptides and proteins. The primary advantage of capillary electrophoresis is that it can offer rapid, high

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resolution of water-soluble components present in small volumes. The separations are based in general on the principles of the electrically driven flow of ions in solution. Selectivity is accomplished by alternation of electrolyte properties, such as pH, ionic strength, and electrolyte composition, or by the incorporation of electrolyte additives. Some of the typical additives include organic solvents, surfactants, and complexing agents. Biomolecules such as proteins, nucleic acids, and polysaccharides are often present in small quantities, and sample sizes are often limited, requiring highly selective and sensitive techniques. Since samples of biological origin are often complex, two or more different yet complementary techniques are often used to perform qualitative or quantitative analysis. The use of complementary techniques provides greater confidence in the analytical results. HPLC and CE that represent chromatography and electrophoresis fulfill this requirement. For example, in reversed-phase HPLC (see Chapter 3), the species are separated on the basis of hydrophobicity; in CE, charge-to-mass ratios play a key role. The difference in separation mechanism is helpful in the characterization or elucidation of the structure of complex molecules of biological origin. Furthermore, these techniques provide fully automated, microprocessor-controlled quantitative assays, as well as high resolution with short analysis time.

C. Isoelectric Focusing Chapter 5 covers isoelectric focusing (IFF), which is one of the commonly used techniques for the separation of proteins. It is a high-resolution method that is well suited for both analytical and preparative applications. IFF fractionations are based on the pH dependence of the electrophoretic mobilities of the protein molecules. Isoelectric focusing, as the name implies, makes use of the electrical charge properties of molecules to focus them in defined zones in the separation medium. It is the focusing mechanism that distinguishes IFF from the other separation processes and makes it unique among the separation methods. In most other separation methods, diffusion and interactions with the medium act to disperse the bands of separated materials. In contrast, the basic mechanism of isoelectric focusing imposes forces on molecules that directly counteract the dispersive effects of diffusion. During the separation process, the molecules in the sample accumulate in specific and predictable locations in the medium, regardless of their initial distribution. The focusing mechanism distinguishes IFF from the other modes of electrophoresis as well. With the other modes of electrophoresis, the applied electrical field moves molecules through the separation media at fixed rates, whereas the applied field in IFF establishes and maintains steady-state distributions of sample molecules. These distributions collapse once the field is discontinued. The basis of the electrofocusing mechanism lies in the properties of the charge-bearing constituents of proteins. The information thus provided by IFF is very useful and complements information obtained for other physical parameters. In comparison to some other separation methods, IFF is easy to


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understand and relatively easy to use. The methodologies are straightforwârd and the results can be readily interpreted. The separations are carried out under nondenaturing conditions in that proteins maintain most of their physical and chemical characteristics. During an lEF separation, proteins are subjected to the simultaneous influences of an electric field and a pH gradient. As proteins migrate electrophoretically through the pH gradient, they gain or lose protons, depending on the local pH. Their net charges assume positive, negative, or zero values according to their positions in the gradient. For every protein, there is a particular pH at which its net charge, and hence, its electrophoretic mobility are zero. This pH is called the isoelectric point (pi). Once a protein migrates to its pi, the net migration of that protein is reduced to zero. The differences in pis account for separation of proteins in lEF. Proteins are positively charged at a pH below^ their pi and negatively charged above their isoelectric points. The net charge on a protein determines its electrophoretic mobility. The key to understanding IFF is the recognition that the net charges carried by proteins are pH-dependent. Furthermore, it is important to note that net charge on a protein is the algebraic sum of all its positive and negative charges. Chapter 5 includes sufficient details on IFF to provide a better understanding of this technique. It also includes a number of applications of various proteins. Isoelectric focusing is applicable only to the fractionation of amphoteric species, such as proteins and peptides, that can act both as acids and bases. Nonamphoteric species, nucleic acids in particular, cannot be resolved by IFF. Both analytical and preparative modes of IFF, included in this chapter, have been developed as valuable tools for studying proteins. D. Mass Spectrometry The advances in technology in the last decade have transformed mass spectrometry from an analytical tool for the study of small and relatively stable molecules to a virtually indispensable technique for studying biomolecules (Chapter 6). The newrly developed ionization methods such as electrospray ionization (FSI) and matrix-assisted laser desorption-ionization (MALDI), coupled wîth advances in instrumentation, laser and computer technologies, and data processing algorithms, enable routine detection and structural analysis of biomolecules. In addition to molecular mass determination of biomolecules, it is nov^ possible to sequence peptides, proteins, oligonucleotides, and oligosaccharides; probe protein folding; and study inter- and intramolecular noncovalent interactions. The new^ commercial mass spectrometers have a large accessible mass range equal to or greater than 300,000 Da; high sensitivity in the lov^-femtomole range; high accuracy, and mass resolution of 1 in 100,000. In contrast wîth some of the older instruments, v^hich required an experienced mass spectroscopist to operate, the new^-generation instruments are user-friendly and can be successfully operated by various researchers in the scientific and medical communities. Unlike other spectroscopic techniques, mass spectrometry (MS) does not require the analytes to possess any special physical properties such as charge, electric or magnetic moments, radioactivity, etc. Furthermore, the short

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measurement times make this technique unique for answering a broad range of questions in a multitude of biological and medical research areas. In addition to its traditional role as an analytical tool used to solve a specific research problem, MS has become an enabling technique in the emerging field of proteomics. Mass spectrometry has played a central role in the attempts to isolate and characterize over 100,000 human proteins. It is increasingly used by biotechnology companies in conjunction with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The goal of Chapter 6 is to familiarize investigators in biological and medical research with mass spectrometry and its potential applications in these fields, with the anticipation that it will encourage and enable them to utilize MS in their research. An attempt has been made to provide a clear basic description of the modern mass spectrometers and of the most relevant types of experiments that they can perform. Discussed also are the advantages and drawbacks of the different methods in the context of biological research with examples of the ability of mass spectrometry to solve problems in this field of research. To benefit general readers, the discussion has been limited to methodologies that are accessible to nonspecialists and that can be carried out on commercially available spectrometers without special modifications. The chapter illustrates the principles of mass spectrometry by demonstrating how various techniques [MALDI, ESI, Fourier transform ion cyclotron resonance (FT-ICR), ion traps, and tandem mass spectrometry (MS-MS)] work. It also provides examples of utilizing mass spectrometry to solve biological and biochemical problems in the field of protein analysis, protein folding, and noncovalent interactions of protein-DNA complexes.

E. Methodology Montage Chapter 2 describes a number of methods that can be useful for analysis of proteins. These methods can be broadly classified as follows: Gel electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Other modes of gel electrophoresis High-performance liquid chromatography Reversed-phase HPLC Hydrophobic interaction chromatography Size-exclusion chromatography Ion-exchange chromatography Capillary electrophoresis Capillary zone electrophoresis Capillary isoelectric focusing Capillary gel electrophoresis Micellar electrokinetic capillary chromatography


Immunoassays Enzyme-linked immunosorbant assay (ELISA) Western blot analysis Immunoligand assay As mentioned before, a number of these methods are discussed at length in this book (see Chapters 2-6). The methodologies for separation of nucleic acids, oligonucleotides, and monoclonal antibodies are covered in Chapters 8, 14, and 15. The followîng chromatographic methods, as they relate to separations of monoclonal antibodies, are discussed in Chapter 15: Ion-exchange chromatography Hydrophobic interaction chromatography Hydroxyapatite chromatography Protein affinity chromatography Thiophilic chromatography Hydrophobic charge induction chromatography Immobilized boronic acid ligand chromatography Dye interaction chromatography Metal chelate affinity chromatography Immunoaffinity chromatography

III. SEPARATION AND PURIFICATION METHODS As mentioned earlier, the biopharmaceutical industry is growîng rapidly, vvîth over IS biotechnology drugs approved for sale in the United States alone and over 500 biopharmaceutical candidates in various phases of clinical trials. In contrast to most of the earlier biotechnology therapeutics that wêre produced on a relatively small scale (kilograms per year), many of the recent products are expected to have production scales on the order of hundreds of kilograms per year. In addition, many biopharmaceuticals are making the transition to generic drugs, with more than one manufacturer competing for market share. Thus, there is an urgent need for the development of efficient, large-scale purification processes in the biotechnology industry. Discussed in this section are various processes used for separation and purification of proteins and other materials of biological interest, such as oligonucleotides and monoclonal antibodies. A. Liquid-Liquid Distribution The International Union of Pure and Applied Chemistry (lUPAC) recommends the use of liquid-liquid distribution rather than the traditional term, solvent extraction. However, solvent extraction is still used commonly in the literature, and that is why it is also being used here interchangeably (Chapter 7). Solvent extraction utilizes the partition of a solute between two practically immiscible liquid phasesone a solvent phase and the other an aqueous phase. Liquid-liquid partitioning methods are important separation tools in modern biotechnology. They have become increasingly popular as part of a

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downstream process for the recovery and purification of biomolecules including alcohols, aliphatic car boxy lie acids, antibiotics, amino acids, and proteins. Solvent extraction has long been established as a basic unit operation for chemical separations. Chapter 7 summarizes the effects of temperature, pH, ion pairs, and solvent selection on solvent extraction for biomolecules. Solvent extraction of fermentation products such as alcohols, aliphatic carboxylic acids, amino acids, and antibiotics are discussed. Enhanced solvent extraction using reversed micelles and electrical fields are also discussed. Solvent-extraction equipment and operational considerations are adequately covered in this chapter. Aqueous tvô-phase partitioning using wâter-soluble polymers and salts has proven to be an effective method in the purification of various biomolecules, especially proteins, which can be denatured by solvents in conventional solvent extraction. The effects of polymer weight and concentration, temperature, salt, and affinity ligands on aqueous two-phase partitioning have been studied. Equipment and operational considerations and large-scale aqueous two-phase partitioning of biomolecules have also been investigated. This chapter also points to sources in the existing literature for both solvent extraction and aqueous two-phase partitioning of biomolecules. Various unit operations are used in the downstream processing of biomolecules. These recovery and purification methods include cell disruption, centrifugation, micro- and ultrafiltration, precipitation, liquid-liquid partitioning, and various forms of liquid chromatography. Among them, liquid-liquid partitioning methods are well established, often inexpensive, and suitable for steady-state large-scale operations. There are two main categories in liquid-liquid partitioning. One is the conventional solvent extraction, which is used for the separations of many metabolites from fermentation, such as alcohols, carboxylic acids, amino acids, and antibiotics. The other is the aqueous two-phase partitioning using water-soluble polymers such as polyethylene glycol (PEG) and dextran, and salts such as potassium phosphate. The latter method is very attractive for the separation of biomolecules, such as proteins and peptides, and including many enzymes that may be denatured by solvents. As the scale of bioseparation processes goes up, liquid-liquid partitioning becomes more and more competitive because it is easy to scale up and it enables continuous steady-state operation. The cost for liquid-liquid partitioning is much lower than that for other more sophisticated bioseparation methods, such as liquid chromatography. This chapter provides a detailed coverage of solvent extraction for bioseparations as well as aqueous two-phase partitioning for bioseparations. B. Separation of Proteins and Nucleic Acids A large number of biologically active molecules are obtained from naturally occurring plants and animal resources. The advances in biotechnology in the past several decades enable the production of many desired compounds under controlled conditions using engineered microorganisms and cells from animals and plants. The recovery of desired products from various sources


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involves sequences of operations with the final aim of obtaining desired products at a prespecified level of purity. The steps involved in the recovery of biological products from their natural environment can be divided into four categories: separation of solubles from insolubles, isolation, purification, and pohshing. A classification of different separation methods based on physicochemical properties is given in Table 1 of Chapter 8. This chapter focuses primarily on unique problems encountered during recovery of intracellularly produced proteins. In a typical recovery and purification process for an intracellular protein, nucleic acids are first removed by precipitation. The isolation and fractionation of undesirable proteins generally follow^ this step. It is vêll known that precipitation of nucleic acids and removal of contaminating proteins can incur large losses of desirable proteins. In this chapter, various methods of removing nucleic acid are reviewed. Also included is a case study that evaluates several precipitation methods and aqueous two-phase extraction for removal of nucleic acids from a cell homogenate of tartrate dehydrogenase (TDH)-producing strain of Pseudomonas putida, C. Displacement Chromatography Displacement chromatography is an efficient mode of preparative chromatography (Chapter 9). Operationally, displacement chromatography is performed in a manner similar to step-gradient chromatography in which the column is subjected to sequential step changes in the inlet conditions (see Chapter 9). The column is initially equilibrated with a carrier buffer in which the feed solutes exhibit a relatively high retention on the chromatographic stationary phase (e.g., low ionic strength in ion exchange, high salt concentrations in hydrophobic interaction chromatography, and low mobile-phase modifier concentrations in reversed-phase chromatography). Following the equilibration step, the feed mixture is introduced into the column, which is then followed by a constant infusion of the displacer solution. The displacer is selected on the basis of the fact that it has a higher affinity for the stationary phase than any of the feed components. Under appropriate conditions, the displacer induces the feed components to develop into adjacent "square-wave" zones of highly concentrated pure material. After the breakthrough of the displacer, the column is regenerated and is reequilibrated with the carrier buffer. The displacer, having a higher affinity than any of the feed components, competes effectively, under nonlinear conditions, for the adsorption sites on the stationary phase. An important distinction between displacement and gradient chromatography is that the displacer front always remains behind the adjacent feed zones in the displacement train, whereas desorbents, e.g., organic modifiers in reversed-phase HPLC, move through the feed zones. It is important to note that displacement chromatography takes advantage of the thermodynamic characteristics of the chromatographic system to overcome many of the shortcomings of preparative elution chromatography. Since preparative chromatography is the single most widely used unit operation for process-scale purification of biologicals, the development of

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more efficient chromatographic operations is assuming increasing importance. This chapter describes the state of the art of displacement chromatography for the downstream processing of biomolecules for purification of products from complex industrial mixtures including selective displacement and the use of retained pH gradients to displace proteins. It also provides a valuable listing, along with suitable references, of high and low molecular weight displacers employed for proteins in the ion-exchange displacement chromatography of proteins. The object of this chapter is to summarize the recent developments in this field and to place in perspective the role that displacement chromatography could play in preparative separations in the years to come. D. Expanded-Bed Adsorption When purifying biomolecules from a complex mixture, a sequence of unit operations is normally needed. Each of these steps involves losses of substance, adding to the overall cost of the process. One way to simplify the situation has been through process integration. This may be done by combining a bioconversion step with one or two steps involving separation. However, it is possible to combine two or more steps in downstream processing and thus reduce the number of different processing steps that are needed. Still another way may be to design processing steps that eliminate the need for a certain treatment. This is the case when the concept of affinity-mediated separation is used (Chapter 10). The use of biospecificity in the interaction replaces several different steps that previously had to be used. A key problem that does not involve difficult theoretical challenges, but rather technical and economical ones, is the need to remove particulate matter before any substantial chromatography can be appfied (Chapter 10). The problem is that particulate matter can clog the column and thereby destroy the separation power. Another option is to use a batch procedure in which an adsorbent is added directly to the feedstock in a stirred tank. The advantage of this method is that the product is captured directly from the unclarified feedstock; however, the disadvantage is that the stirred tank acts as one theoretical plate in a separation process, leading to a long process time because of poor contacting efficiency. One way to circumvent such problems would be to use fluidized-bed adsorption instead of a packed-bed mode of operation. If just the adsorption-desorption of one single entity is wanted, then the fluidized-bed technique may be sufficient. However, there is a constant mixing, and thus extreme band broadening upon passage of a pulse of hquid through such a reactor. The concept of expanded-bed chromatography combines the advantages of good distances between the chromatographic beads when operated in the expanded mode and the adsorption power of the adsorbent particles without severe back mixing. The particles tend to be stored spontaneously with regard to size and density, so that the smaller and lighter particles will be found in the top fraction of an expanded bed that has been operating until equilibrium is established. The particles tend to be sorted spontaneously with regard to size and density, so that the smaller and lighter particles will be found in the top fraction of an expanded bed that has been


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operating until equilibrium is established. The larger and denser particles are found in the bottom section of the bed. It has furthermore been proven that a fraction from the top will be found in the top the next time the column is in operation. It is therefore realistic to state that an expanded-bed column is stable with regard to particle size-particle density distribution. Therefore, back mixing must be less than that found in a fluidized bed. It has, in fact, been shown that expanded beds show a relatively low dispersion, and thus such beds would be useful for separation purposes. The complete process for obtaining pure proteins can be divided into three main steps: capture, purification, and polishing (Chapter 11). The first step is the immobilization of the target protein onto some adsorptive surface, and it can be viewed as a combination of clarification, concentration, stabilization, and initial purification. Because the starting protein solution (feed stock) is usually crude, it is essential to clarify the solution. The traditional or conventional approach involves centrifugation, microfiltration, ultrafiltration, or diafiltration before the target protein solution can be loaded on an adsorbing material, utilizing packed-bed chromatography. The clarification step is a demanding operation and is particularly difficult when processing large quantities of microorganisms, especially disrupted microorganisms. High-speed, large-scale centrifugation and microfiltration are the most common processes used to obtain protein solutions that are suitable for packedbed chromatography; therefore, it is obvious that an approach that eliminates the clarification step can significantly simplify and improve the overall purification process. Direct adsorption of the protein not only eliminates the clarification step, but also produces a concentrated and partially purified product ready for the next purification step (see Fig. 1 in Chapter 11). Several protein capture procedures, such as batch adsorption, solvent extraction, and expanded-bed adsorption, do not require centrifugation and filtration. This chapter describes the expanded-bed adsorption approach for capturing target proteins. In the expanded-bed mode, the starting protein solution is pumped through a bed of adsorbent beads that are constrained by a flow adapter. As a result of the upward flow and the properties of the beads, the bed expands as spaces open up between the beads. If the physical properties of the beads are significantly different from those of the particles in the feed stock, the particles can pass through the bed without being trapped. An effective process depends on parameters such as viscosity, ionic strength, sofid content, and pH of the feed stock as well as the linear flow rate. A number of applications are given in this chapter that detail the capture and recovery of intracellular proteins including recombinant proteins. The initial protein recovery steps, regardless of the source, are usually associated with large volumes and crude solutions, requiring removal of particles and reduction of volume before their purification can take place. Centrifugation, filtration, precipitation, solvent extraction, and batch adsorption are common unit operations involved in the preliminary steps of protein recovery. Expanded-bed adsorption, as described here, is an approach for the initial protein recovery that eliminates the need for clarification and volume reduction. In this process, a crude starting solution is pumped directly onto an

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adsorbent matrix, which is in an expanded state in a special column. This expanded state provides enough space for contaminating particles to move through, and at the same time, it enables the interaction between the targeted protein and the matrix. Following this capturing step, the protein can be eluted from the matrix, which at this stage is in an unexpanded state. The result of this process is volume reduction and partial purification. The chapter lays out the principles of expanded-bed adsorption, it describes the columns and the matrices that are used, and it provides examples for recovering various proteins from various sources. As mentioned before, the use of chromatographic particles in fluidized beds often gives poor separation because of back mixing. However, when a heterogeneous population of chromatographic particles (with regard to size and density) is used, an ordered arrangement is detected, where the denser and larger particles are found at the bottom and the smaller and lighter particles at the top of the bed. Such beds are called expanded beds. When expanding the bed, distances are introduced between the individual particles in the bed. This fact forms the basis for the ability of expanded beds to be used in connection with particle-containing material, e.g., fermentation broths. When applied in downstream processing, expanded-bed chromatography offers possibilities to recover products without previous separation of cell mass or cell debris. This new concept is often mentioned as capturing. E. Membrane Chromatography Membrane chromatography is gaining wider interest and acceptance in the process bioseparation industry (Chapter 12). Better understanding of membrane materials, large-scale availability, and identification of niche applications have promoted this new technology. The focus of Chapter 12 is on aspects of membrane adsorbers (MAs) that have the greatest impact on large-scale preparative chromatography applications in the bioindustry. It also addresses a few novel technologies, such as thin columns, monolithic matrices, and innovative media, which seem to cross the classical definition of chromatography media. The general technology is fairly well known. This chapter attempts to provide a state-of-the-art look at the various MA separation modes, discusses commercially available technology, and provides guidelines to develop large-scale applications based on this technology. MAs are membranes with chemically functionalized surface sites for chromatography. The appeal of the membrane-based chromatographic surface stems from the fact that it is an ideal monolithic support, i.e., it is a uniformly distributed chromatographic surface with convection-enhanced separation. On the practical side, membrane absorbers can be in modular form, leading to easy and convenient use. The scale-up for MA builds on the knowledge base gained in scale-up of technologies of both chromatography and membrane filtration. To rival traditional chromatography, MA technology must address the age-old chromatography problems. The ligand chemistry should allow high dynamic binding capacity, as well as very low nonspecific binding. The development of membrane housings should consider appropriate fluid distribution to take advantage of the high resolution. The


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ultimate challenge is to develop usable membrane devices capable of good chromatography. A viable alternative to bead-based chromatographic supports should be able to deliver consistent results after repeated cleaning cycles and should be able to fulfill all FDA regulatory guidelines. Table 1 in Chapter 12 lists the commercially manufactured modules and their available formats and chemistries. The geometries reflect the vîdely used formats in membrane filtration: primarily, flat sheets in filter holders, flat sheets wound in spiral or cylindrical configuration, and hoUow^-fiber membranes. Syringe filters are popular housings containing flat sheet MAs. These enable easy lov^-pressure chromatography vîth low^ cost, disposability, and easy connectivity of units for series operation. These may also be connected to chromatographic vôrkstations. These syringe-type filters can function in place of small chromatographic columns and are wêll suited for quick method development. The hallmark of membrane separations is its speed; membranes are capable of flow^ rates 10- to 100-fold faster than classical chromatography. Furthermore, they offer good resolution and capacity. The rate-limiting step in the mass transfer is the diffusion of solutes due to the functional groups. Discussion covers these topics as well as scale-up and a variety of interesting applications that include purification of a recombinant vaccine protein, reduction of viral DNA and endotoxin under good manufacturing practice (GMP) conditions, monoclonal antibody purification, and purification of oligonucleotides. F. Simulated Moving Bed Chromatography A number of different products are now purified by chromatographic processes, from the laboratory scale (gram quantities) up to the industrial pharmaceutical scale (a few tons per year). Among the possible technologies, elution HPLC technology (sometimes with recycle) has taken a very important part of the small-scale (10 tons/year) market during the previous decade. And simulated moving bed (SMB) technology has been extensively used for very large scale fractionation of sugars and xylenes for the last 30 years (Chapter 13). Presently, there is considerable interest in the preparative applications of liquid chromatography, even though it is often considered expensive. To make the chromatographic process more attractive, attention is focused on the choice of the operating mode in an effort to minimize eluent consumption and to maximize productivity, which is of key importance when expensive packings are used. Among the alternatives to the classical process (elution chromatography), much attention is paid to SMB. Although SMB is well known as a process that is able to maximize productivity and minimize eluent consumption in some industries, it has been ignored in the pharmaceutical and fine chemical industries during the last 30 years. The reasons may be the patent situation and the complexity of the concept. Recently, separations of pharmaceutical compounds have been performed using SMB technology. It is now considered a real production tool (for instance, the Belgian pharmaceutical company U.C.B. Pharma recently announced the use of SMB for performing multiton-scale separation of

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optical isomers). Small plants are now commercially available for fine chemical industry, the pharmaceutical industry, and biotechnologies. The basic idea of a moving bed system is to promote a countercurrent contact between the solid and the hquid phases. The concept and principles of SMB are discussed at length in Chapter 13, and applications of protein purifications and other complex molecules are given. G. Purification of Oligonucleotides There is a great interest in nucleic acids and oligonucleotides because of recent developments in biochemistry, genetic engineering, genomics, and antisense therapeutics. Oligonucleotides are primarily used as diagnostic tools in biochemical research, and they are being developed as therapeutic agents (Chapter 14). The purification problem in these cases is different in the amount of oligonucleotides required. For example, in one case a large amount of a fewer number of compounds is required in stringent therapeutic quality, whereas, in the second case, a large number of compounds is required with very high throughput in small quantities. However, the general application of separation techniques is similar in both cases. There are presently more than 12 antisense oligonucleotides in human clinical trials. Recently, Fomivirsen (Isis Pharmaceuticals) became the first antisense drug approved by the U.S. FDA. Further success of such compounds is likely to spur greater innovation and development in all facets of oligonucleotide manufacturing and purification. Chapter 14 focuses on two areas: Application of various modes of separations for purifications of these compounds State-of-the-art large-scale technologies for purification of therapeutic antisense oligonucleotides In general, antisense oligonucleotides are short single-strand DNA or RNA analogues. The current therapeutic candidates in clinical trials are mostly within 30 nucleotides in length. Many of these compounds are phosphorothioate analogues, where the nonbridging oxygen of the DNA backbone is replaced by a sulfur atom. This chemical modification improves the stability of the oligonucleotide from degradation by cellular nucleases. There are many other chemical modifications of DNA molecules in the literature, under development, and in human clinical trials. Chapter 14 deals primarily with phosphorothioate-modified oligonucleotides because of their rapid progress in clinical trials and the possibilities of NDA (New Drug Application) submission to the FDA for approval as drugs. In recent years, DNA synthesis technology has advanced significantly because of the need for large-scale oligonucleotides for human clinical trials. Gene machines that barely made 1 mg of oligonucleotide now have been scaled up with advanced technology to synthesize almost a kilogram of oligonucleotide per synthesis campaign. Solid-phase oligonucleotide synthesis technology has progressed further up to now compared with solution-phase synthesis. Various column geometries and fluid contact mechanisms have been tested, but packed axial flow columns have been most successful and are


I 7

commonly used at the largest scales. The reagents are pumped in through automated process computer-controlled protocols. The use of a packed column affords optimal solvent efficiency and better process control. Purification strategies and large-scale purification of therapeutic oligonucleotides are also discussed in Chapter 14. H. Monoclonal Antibodies The FDA has approved monoclonal antibodies and recombinant antibodies as therapeutics and diagnostics relatively recently (Chapter 15). Monoclonal antibodies have now become one of the largest classes of proteins currently in clinical trials. This success has resulted from the large intellectual and capital investment that has been made in them. As a result, significant progress has been made in understanding antibody function, host-defense mechanisms, the role of antibodies in cancer, and substantial improvement of production and purification technology. The development of protein-free culture media, continuous production of animal cells in perfusion culture, genetically engineered "humanized" antibodies, single-chain antibodies, phage display, and cellsurface display libraries have been important steps in this dynamic discipline. The design of antibodies according to the special needs for therapy, diagnosis, and purification technology is now^ possible. Specific properties for in vivo behavior such as defined pharmacokinetics and tumor targeting are simply achieved by combining various fragments with desired properties. These represent a few examples of recent progress. Antibodies are expressed by hybridoma cells formed by cell fusion of sensitized animal or human B lymphocytes with myeloma cells, or they are generated by EBV (Epstein-Barr virus) transformation of sensitized B lymphocytes. Other heterologous expression systems such as bacteria, yeast, insect cells, and mammaHan cells have also been used for expression of antibodies and their fragments. However, because of renaturation problems, glycosylation, and expression levels, mammalian cells are mostly used for the expression of monoclonal antibodies. More recently, technologies have been extensively developed for the expression of antibodies in transgenic animals and transgenic plants. Intact antibodies with biologically active glycosylation profiles, crucial for the effector functions, require eukaryotic expression {in vitro or in vivo). These circumstances have inspired many scientists to find effective methods for production, as well as methods for the selection of the best extractionpurification methods. Purity, safety, potency, and cost-effectiveness are some of the main factors that should be considered when designing an expression method and, more importantly, when defining the purification processes. The purification of antibodies was most likely initiated with the separation of proteins, mainly paraproteins, several decades ago. A plethora of protocols have now been described involving precipitation with a variety of chemical agents, electrophoretic separation, membrane methodologies, and liquid chromatography. The latter probably represents the most popular technique because of the ease of implementation, the capability to play on the selectivity, and the

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level of purity that can be achieved. Specific liquid chromatography methodologies and resins have been especially developed for this purpose. To date, monoclonal antibodies and immunoglobulins vîth all their derivatives represent by far the largest class of produced and purified proteins in numbers and mass. Chapter 15 focuses mainly on antibody purification by chromatographic means. Numerous sorbents have been developed for protein separation, and they are based on a variety of adsorption-desorption principles. Selection of suitable materials and principles depends on the properties of the particular immunoglobulins to be separated and on the composition of the impurities that constitute the feedstock. Antibodies are very diverse in molecular properties, chemical characteristics, and biological activity. Purification strategies are therefore also diverse, since they are based on a large variety of molecular interactions. Antibodies have several common properties that are frequently exploited from the initial feedstock. Knov^ledge about the nature and concentration of impurities is the key to success. Therefore, the initial composition of feedstock is important when designing the separation process. The expression system can be selected to simplify the extraction-purification procedures. Chapter 15 provides an in-depth coverage of various chromatographic methods, such as ion exchange, hydrophobic interaction, affinity, ligand, immunoaffinity, gel filtration, etc., that can be used for separations of antibodies by liquid chromatography.

lY. OTHER IMPORTANT CONSIDERATIONS The selection of appropriate processing plants and equipment, economics, and future developments are important considerations that are discussed in this section.

A. Processing Plant and Equipment The diversity of industries that involve bioseparations has led to the development of a wîde range of techniques and unit operations for the efficient processing of biological materials. Chapter 16 is planned to aid the scientist or engineer in selecting a method of bioseparation that wîll be suited to the particular requirements of the process and the product at a commercial scale of operation. The complexity of biological processes generally requires many stages to produce a final, purified product from a particular composition of rav^ materials. Although a typical bioprocess consists of twô main parts, upstream fermentation and downstream product recovery, it is not unusual to have betvêen 10 and 20 steps in the overall process. This reflects the complex nature of a typical fermentation broth, v^hich wîll consist of an aqueous mixture of cells, intracellular or extracellular products, unreacted substrates, and by-products of the fermentation process. From this mixture, the desired


I 9

product must be isolated at a given purity and specification, and all of the unwânted contaminating materials must be removed. The choice of a bioseparation technique wîll depend on a number of factors, including the initial location of the product inside or outside the cell, as wêll as the product size, charge, solubility, chemical or physical affinity to other materials, and so on. Economic factors also come into play, including the value of the product, the regulatory environment in w^hich the product is manufactured, and the balance betwêen the capital cost of the bioseparation equipment and the operating cost of running it. In moving from laboratory- or pilot-scale processing to full-scale manufacturing, it can be difficult to scale up certain types of bioseparation equipment easily; for example, high " g " centrifuges are available as benchmounted units (using test tubes), but an equivalent industrial machine vîth a similar g force is unlikely to be a cost-effective solution, even if it were possible to build a suitable unit. It wôuld not be realistic to consider 10 or 100 identical units as a realistic alternative. Compromises are therefore required as a process is commercialized, to ensure that the process remains technically and economically feasible. Chapter 16 provides guidance relating to the choice of industrial bioseparation equipment that is available and the issues that must be taken into account when selecting a suitable system to meet both technical and economic objectives. B. Engineering Process Control Chapter 17 deals briefly with the engineering process control, which primarily involves measurement of a product property and comparison to a desired value. The process operation can be thus immediately adjusted to reduce deviation from the specifications. This feedback procedure can be used to adjust the process whenever the product deviates from the set point and can be used to change operating points and to reject the effect of outside disturbances. C. Economics of Separations Valuable products are being produced increasingly by biotechnological methods. By the year 2000, the worldwide sales of these biotechnological products will be around $100 billion (Chapter 18). The Western hemisphere countries, particularly the United States, Germany, France, and England, are the leading players. For example, diabetic and thrombolytic drugs have a very large market throughout the world; hence it would be desirable to review the sales of the above-mentioned classes of drugs. In the United States, the estimated market for diabetes drugs is $1.8 biUion, including $800 million for insulin. The estimated market for thrombolytic drugs is $355 million. Biotechnology products include not only pharmaceutical drugs but also other biological macromolecules of interest. One must separate the biological macromolecule of interest; and herein lies a very significant cost of the entire manufacturing process. For different processes, the fraction of the entire cost of the process

20

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required for bioseparation will vary. The purification and recovery costs may be as high as 80% of the total manufacturing costs. These costs may be higher if ultrahigh purity DNA-involved products are manufactured. Recognize that during processing, one may have to purify products at 99.9% levels with virtually complete removal of DNA, viruses, and endotoxins. The key to cutting production costs is to emphasize improvements in downstream processing. Traditionally, all the steps occurring in the fermenter that result in the production of the desired biological macromolecule can be considered as upstream processes. All the other processes occurring after the fermentation and which result in the separation, purification, concentration, and conversion of the biomolecule to a form suitable for its intended final use can be classified as downstream processes. Thus, it is helpful to better analyze and understand the different facets involved during downstream processing. Better physical insights are required and are continuously being obtained in downstream processes, and these will eventually lead to a more efficient and economical process. Note that upstream processes are already well understood. Even though further improvements in upstream processes are possible, they do not have the potential of making as significant an impact on production costs as improvements in downstream processes. Also, one should not treat the upstream and downstream processes separately, but should integrate the downstream processes with the upstream processes. For instance, any change or improvement envisaged in an upstream process should also consider the possible effect of this change on the downstream process. Minor changes in upstream conditions may have a significant impact on downstream processes. Thus, early in the design of processes, one must consider the impact of upstream processes on downstream processing. One technique where different possible "what-if" scenarios may be analyzed is with the development of an appropriate model for the process. The importance of computer-aided process simulation, and the early necessity of providing a workable process flow sheet can not be overemphasized. These activities should be carried out during the early stages of process development and can serve as an important tool to help optimize the process expeditiously. Considering the high stakes that are involved in getting a drug to the market and the fierce competition involved, it seems appropriate to get as much useful information on a process as early as possible during the development process. This also explains the extreme secrecy involved in the research and development of key steps in the bioprocessing of a highly marketable and valuable product. Chapter 18 emphasizes that drug manufacturing is a high-risk, high-gain business that requires economic analysis at each stage of the developmental process to minimize costs. Several examples are provided to assist the reader in evaluating the economics of bioseparation of a given process.

D. Future Developments The field of bioseparations is very dynamic. As a result, new developments are constantly being made in the techniques discussed here. At the same time.


2 I

new techniques are also evolving that wîll have an impact on this field in the future. Chapter 19 attempts to address this topic. The important point to recognize is that all future developments are targeting larger separations in the shortest possible time, vîth the objective of lowêring costs so that these processes become economically more feasible.

REFERENCE1. Sadana, A. (1998). In "Bioseparation of Proteins" (S. Ahuja, ed.). Academic Press, San Diego, CA.

ANALYSIS OF PROTEIN IMPURITIES IN PHARMACEUTICALS DERIVED FROM RECOMBINANT DNADONALD O. O'KEEFEBristol-Myers Squibb, Macromolecular Structure and Biopharmaceuticals, Princeton, New Jersey 08543

I. INTRODUCTION A. The Regulatory Environment B. Purity Analysis of Recombinant Pharmaceuticals C. Sources and Types of Impurities D. Levels and Identification of Impurities II. PROTEIN IMPURITY ANALYSIS A. Gel Electrophoresis B. High-Performance Liquid Chromatography C. Capillary Electrophoresis D. Immunoassays E. Identification of Host-Cell Protein Impurities III. SUMMARY IV. CASE STUDIES A. Identification of a Host-Cell Protein Impurity in Recombinant Acidic Fibroblast Growth Factor B. Selective Resolution of a Protein Impurity Using RP-HPLC and Fluorescence Derivatization C. Detection of N-terminal Variants Using Peptide Mapping and Fluorescence Detection REFERENCES

INTRODUCTION Recombinant DNA methodology has come of age. It has spawned a growing industry that seeks to commerciahze products derived from this technology. Most notable among these biotechnology products are those for human therapeutic use, and of these there are two categories: proteins and nucleic acids. The research, development, and commercialization of therapeutic proteins derived from biotechnology is far more advanced than that of nucleic*To reproduce or otherwise use this article in whole or in part, permission must be obtained from Bristol-Myers Squibb, the author, and Academic Press.

Separation

Science and Technology,

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Copyright 2000 by Academic Press. All rights of reproduction in any form reserved.

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acids and therefore the former will be the focus of this chapter. In 1982 the Food and Drug Administration (FDA) approved human insulin synthesized in Escherichia coli as the first recombinant therapeutic in the United States. Since then over 75 other recombinant proteins have achieved this same status.^'^ Among these are hormones, cytokines, vaccines, and monoclonal antibodies. Simultaneous vîth this success has been the proliferation of biotechnology companies. In the United States alone there are more than 1100 such companies,^ and the economic impact of this grov^th is substantial. Current annual revenues of biotechnology drugs in the United States alone total $8 billion and account for nearly 10% of all therapeutic sales in the United States.^ Significant among these sales is that of recombinant erythropoietin, which in 1997 had worldwide sales in excess of $1 billion. This financial potential partially explains the approximately 500 biotechnology drugs currently in clinical trials.^ The increased onslaught of biotechnology drugs in the last two decades has challenged both the regulatory agencies responsible for approving new drugs and the biotechnology industry, which must consistently produce a definable and safe product.

A. The Regulatory Environment As with traditional drugs, biopharmaceuticals must be safe and effective to be approved by national regulatory agencies. From the very beginning, it was intuitive that a high degree of purity is elemental to the safety of recombinant therapeutics. But what level of purity is sufficient? Initially there were no predetermined guidelines. This led to extensive and in-depth discussions between regulatory agencies and the first companies attempting to develop recombinant proteins as human therapeutics.^'^ Thereafter, the FDA issued working draft guidelines to industry for producing and testing these biologicals in the form of "Points to Consider" documents.*''^"^ These guidelines are not binding requirements but instead are recommendations on how to direct the clinical development of a biopharmaceutical. Likewise, other national and international agencies have done the same.^~^ Fundamentally, all these agencies have the same responsibility, i.e., assuring the quality (purity), safety, and efficacy of biopharmaceuticals. Over time, however, the technical and clinical standards for biopharmaceuticals under development have diverged among the national regulatory agencies to the point that independent studies and licensing applications must be made in the individual countries. The drawbacks here are obvious, and they have led to a regulatory renaissance at the start of the 1990s. In 1990 a joint regulatory-industry initiative was conceived to provide international harmonization of the drug approval process. The result has been the ongoing International Conference on Flarmonization (ICH). The ICH is charged with developing harmonized guidelines on technical issues relating to drug development. The ICH is attempting to codify consensus guidelines for obtaining market approval of drugs on a worldwide scale. The* These documents, as well as others produced by the FDA, can be obtained through the Internet at the website of the FDA at www.fda.gov.

PROTEIN IMPURITY ANALYSIS

25

guidelines recommended by the ICH are listed under the four separate topics of quality, safety, efficacy, and multidisciplinary.* Within the quality topic are specifications for testing biotechnology products.^^ This guideline and others proposed by the ICH appear to be the future for international drug development. Therefore, these documents will provide the boundaries for any discussions regarding purity, impurities, and contaminants.

B. Purity Analysis of Recombinant Pharmaceuticals An important fact inherent in the purity analysis of a recombinant pharmaceutical is that the absolute purity of any protein is an elusive, if not an unobtainable, measurement. For biopharmaceuticals, purity is a relative term. Protein purity is method-dependent and is defined by the shortcomings of the analytical procedure. Also, unlike small traditional drugs, proteins are highly complex molecules. For these two reasons, more than one method must be utilized to define a protein's purity. The greater the number of methods used in the purity analysis, the greater the assurance is that the product is pure. Furthermore, the purity determined by an analytical method can only be properly interpreted based on the method's validation. Analytical methods vaUdation is critical to and inseparable from purity determinations. A detailed discussion on analytical methods validation is beyond the scope of this chapter but other sources of information are available for the interested reader.ii-i^ Purity analysis of therapeutic recombinant proteins generally occurs at two stages of the production process. The first material tested is the drug substance, or bulk material, which is the final purified active product prior to formulation. Upon dilution to the final dose, the addition of excipients, and possibly lyophilization, the protein preparation is referred to as the drug product or finished product. Any constituent within these two preparations that is not the active product or an excipient, excluding contaminants, is an impurity. ^^ At these two stages, the purity assays of the bioanalyst are selected based on the potential impurities that are found in the drug substance or the drug product.

C. Sources and Types of Impurities There are two categories of impurities: process-related impurities and product-related impurities. Process-related impurities are components derived from the manufacturing process. Included here are fermentation ingredients, host organism components, and process additives to Hst a few. Product-related impurities are variants of the desired protein product that do not have the desired biological activity, safety, or efficacy. Examples of these impurities might be aggregates, degradates, or misfolded isomers of the protein. Product-related impurities can also arise during storage and are an indication of* ICH topics and guidelines can be obtained at the Internet website of the International Federation of Pharmaceutical Manufacturers Association (IFPMA) at www.ifpma.org.

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the instability of the drug substance or the drug product.* Product-related impurities are distinguishable from product-related substances. The later are molecular variants that have activity, safety, and efficacy indistinguishable from the desired product.^^ An example of product-related substances might be different glycosylated forms of a glycoprotein, for example, recombinant tissue plasminogen activator.^ The analysis of product-related substances will not be presented in this chapter, but many of the analytical techniques for their testing are the same as those used for impurity analysis. A compilation of important process- and product-related impurities is given in Table 1 along vîth some techniques commonly used for their analysis. Little attention in this chapter will be given to contaminants, which are distinct from impurities. A contaminant is any entity that adventitiously enters the production process, drug substance, or drug product. This includes viruses, mycoplasma, bacteria, fungi, and their products. The control of contamination relates to process validation issues and will not be discussed here.* Protein stability, and the techniques for its analysis, is a related but separate topic from this chapter and will not be covered in depth, but references are available for the interested reader.i'^'i^

TABLE 1 Important Impurities in Recombinant Pharmaceuticalsimpurity Product-related impurities Aggregates (including dimers) Denatured forms Degradates Deamidations Oxidations (methionine sulfoxides) Amino acid substitutions Misfolded conformers (S-S isomers) N-terminal variants Fragmented products Process-related impurities Culture derived Culture media proteins Amino acids Inducers Antibiotics Downstream derived Solvents Protein denaturants Reducing agents Column leachables (antibodies, protein A) Trace metals Enzymes (nucleases) Host cell derived Host proteins DNA (genomic and vector) Endotoxin and other pyrogens Common methods of analysis

SEC; PAGE; CGE HIC lEF; CIEF; lEX; peptide mapping; chromatofocusing Peptide mapping Peptide mapping HIC; peptide mapping RP-HPLC; peptide mapping SDS-PAGE; RP-HPLC; HIC

Immunoassay RP-HPLC HPLC HPLC Gas chromatography HPLC HPLC Immunoassay Atomic absorption spectroscopy Immunoassay SDS-PAGE; HPLC; CE; immunoassay Hybridization LAL; rabbit test for pyrogens


27

The analytical measurement of process-related impurities is not always routine. Some of these impurities are well defined and therefore detecting and quantitating these are straightforward. Examples here include reducing agents, chaotropes, detergents, eluent components used in chromatography, or media additives to benefit the growth of the host organism. The same is generally true for product-related impurities. Conversely, other process-related impurities are not well defined, and therefore their detection and quantitation can be more difficult. Herein Hes the challenge facing the bioanalyst. Included in this category are nucleic acids and host-cell proteins. Of these two types of potential impurities, host-cell proteins are the most difficult to address because of the large diversity of proteins that exists. In E. coli, the vanguard of recombinant organisms, the sequenced genome suggests there may be greater than 4200 different proteins in this organism, each one a potential impurity. ^^ Moreover, other recombinant hosts, such as the yeast Saccharomyces cervisiae^ mammalian cell lines, and transgenic animals, are eucaryotes and their greater complexity leads to ever more potential protein impurities. D. Levels and Identification of Impurities Well-documented guidelines do exist for the analysis of impurities in traditional small molecular weight drugs.^^ This analysis includes the identification, quantitation, and qualification of all impurities. Once the analysis is complete, initial limits are set for each impurity based on its known toxicity profile. For biopharmaceuticals, on the other hand, the allowable levels and the identification of impurities are less standardized, despite the progress of the ICH. The required purity level for a recombinant pharmaceutical is dependent on several factors. Possible factors considered by regulatory agencies include the size and the frequency of the dose, the route of administration [e.g., topical versus intra muscular (IM)], the duration of the administration (chronic or short term), the intended use of the drug (therapeutic versus prophylactic), the seriousness of the disease (risk versus benefit assessment), the patient population (elderly versus young), and the results of preclinical studies. Impurities in recombinant pharmaceuticals have been commonly categorized by quantity. Those in excess of 0.5% are considered major impurities and, if possible, their toxicity, immunogenicity, and pharmacology should be evaluated if they cannot be eliminated. Impurities less than 0.5% are minor but still should be identified.^^ Major impurities are often product-related impurities, while minor impurities are generally process-related impurities. It is the process-related impurities that are the most worrisome from a potential health hazard perspective and their levels should be reduced to parts per million (ppm, nanogram impurity per milligram recombinant product) or less. Potential hazards due to impurities can include oncogenicity (both protein and DNA), unwanted immunological responses that create anaphylactic or allergic reactions, different pharmacology or antigenicity of product-related impurities, and general or specific toxicity. Furthermore, impurities might adversely affect the protein pharmaceutical by altering either its activity or its stability prior to administration. With these potentially

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deleterious effects, proteins derived from recombinant DNA are expected to be of high purity. Protein purities in excess of 99% are not uncommon, and are often expected for therapeutics. A complete purity analysis, however, not only reports the purity as a percentage (generally weight-to-vêight) but also reports the level of impurities. Significant impurities such as host-cell proteins are reduced to ppm levels vîth today's sophisticated purification techniques. Yet even at 99.99% purity (100 ppm of impurities) a 0.1 m g / k g dose for a 70 kg patient exposes the subject to 0.7 fig of impurities. With repeated dosing over a long time, the cumulative health effect of these impurities might be significant. Therefore, it might be necessary to identify these minor impurities to assure greater safety of the drug product. This might appear to be a daunting task for the bioanalyst, but as the sophistication and the sensitivity of both analytical instruments and procedures continues to increase this challenge will be met. When is a recombinant therapeutic pure enough? What levels of impurities are acceptable? The foregoing discussion makes it apparent that such questions are not easily answered. The regulatory agencies have generally agreed with this assessment and therefore have adopted a policy of considering each therapeutic protein on a case-by-case basis. In this regard, information on the potential patient population and the proposed therapy, results of preclinical and clinical studies, and a complete analytical package are indispensable. The latter, of course, should include a thorough and complete purity and impurity analysis, both qualitative and quantitative. It is apparent that impurity analysis in recombinant pharmaceuticals is a broad topic. This chapter is not intended to be either an all inclusive or comprehensive treatise on the subject. Such a chapter would be impossible, given the diversity of both recombinant products and the processes used for their production. Hence, there is no generic protocol or blueprint for performing impurity analysis. Therefore, this chapter will focus on presenting and discussing the methodology most commonly used to analyze process- and product-related protein impurities in therapeutic proteins derived from recombinant DNA. It is hoped that this chapter will provide a starting point for these analyses and expose the reader to many available options. Further in-depth pursuits can be satisfied by consulting the list of references at the end of the chapter.

II. PROTEIN IMPURITY ANALYSIS Protein impurities are either process- or product-related impurities. Processrelated impurities include proteins added to the culture medium, proteins used during purification, such as nucleases and chromatography ligands, and proteins from the host organism. Product-related impurities include degradates, aggregates, and conformational isomers of the recombinant drug product. Eliminating all protein impurities in a recombinant pharmaceutical is realistically impossible. In fact, proteins are the most common impurity in


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recombinant drugs and they also may be potentially the most deleterious. Furthermore, protein impurities, as a class, are more complex compared to other potential impurities. This complexity makes the use of a single method for impurity analysis unsuitable. Methods that separate based on different physicochemical properties need to be utilized jointly. All these factors explain both the effort and the number of techniques for protein impurity analysis. The major techniques to analyze protein impurities are gel and capillary electrophoresis, high-performance liquid chromatography, and immunoassays.

A. Gel Electrophoresis i. Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis

Polyacrylamide gel electrophoresis (PAGE) of proteins is a high-resolution separation technique for purity analysis. Proteins, which are multicharged macromolecules, migrate in an electric field. When proteins in a porous polyacrylamide gel are subjected to an electrical current, they migrate based on their total charge and molecular size, i.e., their charge-to-mass ratio. When the anionic detergent sodium dodecyl sulfate (SDS) is added to the gel, the detergent binds uniformly to proteins at a ratio of 1.4 g SDS per 1.0 g of protein.^^ SDS imparts an overall negative charge to proteins, giving each one an identical charge-to-mass ratio. Hence, SDS-PAGE separates proteins based solely on their mass. Although protein electrophoresis exists in many forms,^^ the best methods utilize a discontinuous system. In these methods different components comprise the buffers for the gel, the sample, and the reservoir chambers. Upon application of an electric current, a steep potential gradient is created that causes the proteins to undergo the stacking that is responsible for the high resolution of discontinuous systems. SDS-PAGE is the most common electrophoretic method used for impurity analysis and the protocol adopted from Laemmli is the standard.^^ This method is often the first step employed to analyze a protein's impurity profile because of its ease of use, and it requires little development time. The ICH recommends that SDS-PAGE impurity analysis be done under both reducing and nonreducing conditions with increasing amounts of purified protein.^^ The actual amounts of protein analyzed will depend on the staining technique used after electrophoresis (see later). The protocol given next has been found suitable for a broad range of recombinant proteins.Protocol I: SDS-PAGE

Suitable glass plates are assembled to produce a resolving gel of 14 cm X 15 cm X 1.0 mm. The Protean II electrophoresis apparatus from BioRad has worked well in the author's laboratory but other equipment can be used. The resolving gel is prepared according to the following recipe:

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SDS'PAGE Resolving GelPercentage polyacrylamide 4% 8% 10% 12% 14% 16%

3.0 M Tris-HCl,pH 8.8 7.5 7.5 7.5 Deionized water 17.9 13.9 11.9 30% acrylamide 0.8% bis-acrylamide 4.0 8.0 10.0 Degas under vacuum for at least 5 min 10%SDS 0.6 0.6 0.6 10% ammonium persulfate 0.2 0.2 0.2 TEMED 0.02 0.02 0.02

7.5 9.9 2.0 0.6 0.2 0.02

7.S 7.9 14.0 0.6 0.2 0.02

7.5 5.9 16.0 0.6 0.2 0.02

Recipe notes: All volumes are in milliliters. Ammonium persulfate is made fresh before use. TEMED is N, N, N\ N'-tetramethylethylenediamine. Once the resolving gel is poured between the sealed glass plates, it is overlaid with water-saturated isobutanol and allowed to polymerize for approximately 30 min. Afterward, the water-saturated isobutanol is removed and the 4% polyacrylamide stacking gel mixture is poured on top of the resolving gel and an appropriate sample comb is inserted.SDS-^PAGE Stacking Gel

2.5 mL 0.5 M Tris-HCl, pH 6.8 5.8 mL deionized water 1.3 mL 30% acrylamide-0.8% bis-acrylamide Degas under vacuum for at least 5 min 0.2 mL 10% SDS 75 fiL 10% ammonium persulfate 75 ^tL 1% bromophenol blue 10 fiL TEMED After the stacking gel has polymerized for at least 30 min, the sample comb is removed and the upper and lower reservoir chambers are filled with Electrode Buffer (25 m M Tris-HCl, 192 m M glycine, 0.1% SDS, pH 8.3). The samples are prepared by mixing four parts of sample with one part of 5X Sample Buffer (175 m M Tris-HCl, pH 6.8, 1 1 % SDS, 0.14% bromophenol blue, 55% glycerol, 2 M DTT). The samples are heated at 95100 C for 2 - 5 min. Each sample well is rinsed with Electrode Buffer before applying the samples to individual wells. The gel's electrodes are then connected to the

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