Handbook of Bioseparations (Separation Science and Technology, Volume 2)

HANDBOOK OF BIOSEPARATIONS

This Is Volume 2 of SEPARATION SCIENCE A N D T E C H N O L O G Y A reference series edited by Satinder Ahuja

HANDBOOK OF BIOSEPARATIONS Edited by

Satinder Ahuja Ahuja Consulting Calabash, North Carolina

ACADEMIC PRESS A Harcourt Science and Technology Company

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Academic Press Harcourt Place, 32 Jamestown Road, London NWl 7BY, UK http://www.hbuk.co.uk/ap/ Library of Congress Catalog Card Number: 00-102786 International Standard Book Number: 0-12-045540-4 PRINTED IN THE UNITED STATES OF AMERICA 00 01 02 03 04 05 QW 9 8 7 6

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CONTENTS

PREFACE

xiii

I Bioseparations: An Overview S. AHUJA

I. II. III. IV.

Introduction 1 Analytical Methodologies 3 Separation and Purification Methods Other Important Considerations 18 Reference 21

2 Analysis of Protein Impurities in Pharmaceuticals Derived from Recombinant DNA DONALD O. O'KEEFE

I. Introduction 23 II. Protein Impurity Analysis 28 III. Experimental Summary 57

VI

CONTENTS

IV. Case Studies 58 References 64

3 Physicochemical Factors in Polypeptide and Protein Purification and Analysis by High-Performance Liquid Chromatographic Techniques: Current Status and Challenges for the Future MILTON T. W. HEARN

I. II. III. IV. V. VI. VII. VIII. IX. X. XI.

Introduction 72 Basic Chromatographic Terms and Concepts Id The Chemical Structure of Polypeptides and Proteins 79 Physicochemical Factors That Underpin Ligate Interactions with Polypeptides and Proteins in HPLC Separation Systems 84 Strategic Considerations behind the HPLC Separations 107 Specific Physicochemical Considerations on the Individual Chromatographic Modes 117 The Effect of Temperature and the Thermodynamics of Polypeptide- or Protein-Ligate Interactions 135 Factors That Control Performance and Efficiency 156 Scaling-Up Possibilities: Heuristic Approaches and Productivity Considerations 172 Effect of Mass Transfer Resistances in Preparative HPLC of Polypeptides and Proteins 178 Summary 218 References 222

4 Capillary Electrophoresis of Compounds of Biological Interest S. AHUJA

I. II. III. IV. V. VI. VII. VIII. IX. X.

Introduction 238 Capillary Zone Electrophoresis 239 Migration Behavior of Peptides and Proteins 243 Modifications of Fused Silica Capillaries 247 Effect of Temperature on Separations 252 Strategy for Protein Separations 252 Capillary Gel Electrophoresis 253 Micellar Electrokinetic Chromatography 255 Capillary Electrochromatography 255 Applications 256 References 262

CONTENTS

VII

5 Isoelectric Focusing DAVID E. GARFIN

I. II. III. IV. V. VI. VII. VIII.

Introduction 263 The Principles of Isoelectric Focusing 264 Analytical Isoelectric Focusing 276 Two-Dimensional Gel Electrophoresis 285 Preparative Isoelectric Focusing 287 Capillary Isoelectric Focusing 291 Summary 292 Appendix 292 References 295

6 Mass Spectrometry of Biomolecules DAN GIBSON AND CATHERINE E. COSTELLO

I. Introduction 299 II. Examples of Applications of Mass Spectrometry to Biological Research 309 III. Conclusions 325 References 325

7 Liquid-Liquid Partitioning Methods for Bioseparations TINGYUE GU

I. II. III. IV.

Introduction 329 Solvent Extraction for Bioseparations 330 Aqueous Two-Phase Partitioning for Bioseparations Summary 360 References 361

8 Separation of Nucleic Acids and Proteins ROHIT HARVE AND RAKESH BAJPAI

I. II. III. IV. V. VI.

Introduction 365 Precipitation of Nucleic Acids 368 Nuclease Treatment 370 Aqueous Two-Phase Extraction 370 A Case Study 372 Conclusions 376 References 376

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CONTENTS

9 Bioseparations by Displacement Chromatography ABHINAV A. SHUKLA AND STEVEN M. CRAMER

I. Introduction 380 II. Purification of Amino Acids and Peptides by Displacement Chromatography 382 III. Purification of Proteins by Displacement Chromatography 383 IV. Alternative Modes of Displacement Chromatography 390 V. Methods Development for Displacement Chromatography 393 VI. Displacement Chromatography for the Purification of Biomolecules: Industrial Case Studies 400 VII. Design of Lov^ Molecular Weight Displacers 406 VIII. Conclusions 410 References 412

10 Physicochemical Basis of Expanded-Bed Adsorption for Protein Purification B. MATTIASSON AND M. P. NANDAKUMAR

I. Introduction 417 II. Typical Procedure to Operate an Expanded-Bed Chromatographic System 418 III. Ligand Selection 423 IV. Applications 424 V. Conclusion 427 References 428

I I Expanded-Bed Adsorption Process for Protein Capture JOSEPH SHILOACH AND ROBERT M. KENNEDY

I. II. III. IV. V. VI. VII.

Introduction 431 Principles of Expanded-Bed Operation Experimental Strategy 435 Instrumentation 437 Matrices 438 Applications 438 Discussion and Conclusions 449 References 450

433

12 Adsorptive Membranes for Bioseparations RANJIT R. DESHMUKH AND TIMOTHY N. WARNER

I. Introduction 454 II. Comparison of Membrane Chromatography to Traditional Chromatography 458

CONTENTS

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III. Scale-Up of Chromatography Membranes 460 IV. AppUcations of MA to Preparative Bioseparations V. Conclusions 470 References 471

464

13 Simulated Moving-Bed Chromatography for Biomolecules R. M. NICOUD

I. II. III. IV. V. VI.

Introduction 475 Basic Principle 476 Operating Conditions 482 Main Applications and Developments 490 Practical Application: Separation of Sugars 499 Conclusion 506 References 508

14 Large-Scale Chromatographic Purification of Oligonucleotides RANJIT R. DESHMUKH. WILLIAM E. LEITCH II, YOGESH S. SANGHVI, AND DOUGLAS L COLE

I. Introduction 512 II. General Purification Strategies for Oligonucleotides 516 III. Large-Scale Purification of Therapeutic Oligonucleotides 519 IV. Purification of Related Molecules—DNA Fragments, Plasmids, Ribozymes, and RNA 529 V. Economics of Oligonucleotide Purification 530 VI. Summary 531 References 531

I 5 Separation of Antibodies by Liquid Chromatography EGISTO BOSCHETTI AND ALOIS JUNGBAUER

I. II. III. IV. V. VI. VII.

Introduction 536 Antibodies: An Overview 538 Biological Starting Material 546 Prepurification 551 Purification of Antibodies by Liquid Chromatography 556 Regulatory Considerations 612 General Conclusion on Antibody Separation Technologies 620 References 621

CONTENTS

16 Processing Plants and Equipment p. BOWLES

I. II. III. IV. V.

Introduction 633 Industries Using Bioseparations 634 Process-Scale Bioseparations 636 Process-Scale Considerations 653 Summary 656 References 657

17 Engineering Process Control of Bioseparation Processes RANDEL M. PRICE AND AJIT SADANA

I. Need for Process Control in Bioseparations 660 II. Brief Overview of Current Control Methods 661 III. Application Examples 662 IV. Opportunities for Continuing Development 664 References 664

18 Economics of Bioseparation Processes ANAND RAMAKRISHNAN AND AJIT SADANA

I. Introduction 667 II. Drugs Market and Sales 670 III. Applications of Models and Flow Sheets in Bioseparation Economics 673 References 684

19 Future Developments S. AHUJA

I. II. III. IV. V. VI. VII.

Introduction 687 The Partnership of Proteins and Nucleic Acids Biotech Drugs 690 Assuring Production and Purity 694 Genomics 698 Lab on Chip 699 Recovery of Biological Products 700 References 710

INDEX

713

690

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 XI

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CONTRIBUTORS

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 v^ith the primary focus on separations of proteins; how^ever, 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 w^ith 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

XIV

PREFACE

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 Considerations—those 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

EDITORIAL ADVISORY BOARD

Steven M. Cramer Rensselaer Polytechnic Institute Troy, New York

William S. Hancock Hewlett Packard Palo Alto, California

Milton T. W. Hearn Monash University Clayton, Victoria Australia

Brian Hubbard Genetics Institute Andover, Massachusetts

This Page Intentionally Left Blank

I

BIOSEPARATIONS: AN OVERVIEW S. AHUJA Ahuja 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 coli—the 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 known Separation 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 scales—and 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 considerations—those 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

BIOSEPARATIONS: AN OVERVIEW

• 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 v^ell 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

BIOSEPARATIONS: AN OVERVIEW

/

understand and relatively easy to use. The methodologies are straightforw^ard 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 w^ith 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 w^ith 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

BIOSEPARATIONS: AN OVERVIEW

• 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 follow^ing 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 grow^ing rapidly, vv^ith 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 w^ere 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 phases—one 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 tv^o-phase partitioning using w^ater-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 v^ell 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 v^idely 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 v^ith low^ cost, disposability, and easy connectivity of units for series operation. These may also be connected to chromatographic v^orkstations. These syringe-type filters can function in place of small chromatographic columns and are w^ell 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

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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 v^ith 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 w^ide 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 w^ill 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 tw^o main parts, upstream fermentation and downstream product recovery, it is not unusual to have betv^een 10 and 20 steps in the overall process. This reflects the complex nature of a typical fermentation broth, v^hich w^ill 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

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product must be isolated at a given purity and specification, and all of the unw^anted contaminating materials must be removed. The choice of a bioseparation technique w^ill depend on a number of factors, including the initial location of the product inside or outside the cell, as w^ell 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 betw^een 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 v^ith a similar g force is unlikely to be a cost-effective solution, even if it were possible to build a suitable unit. It w^ould 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

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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.

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new techniques are also evolving that w^ill 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, v^ith the objective of low^ering costs so that these processes become economically more feasible. REFERENCE 1. Sadana, A. (1998). In "Bioseparation of Proteins" (S. Ahuja, ed.). Academic Press, San Diego, CA.

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ANALYSIS OF PROTEIN IMPURITIES IN PHARMACEUTICALS DERIVED FROM RECOMBINANT DNA« DONALD O. O'KEEFE Bristol-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.

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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 v^ith 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.

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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.

26

DONALD O. O'KEEFE

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 v^ith 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 Pharmaceuticals impurity 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

PROTEIN IMPURITY ANALYSIS

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

28

DONALD O. O'KEEFE

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-v^eight) but also reports the level of impurities. Significant impurities such as host-cell proteins are reduced to ppm levels v^ith 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

PROTEIN IMPURITY ANALYSIS

29

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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DONALD O. O'KEEFE

SDS'PAGE Resolving Gel Percentage polyacrylamide

4%

8%

10%

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

12%

7.5 9.9 2.0 0.6 0.2 0.02

14%

7.S 7.9 14.0 0.6 0.2 0.02

16%

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 95°100° 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 power supply and the gels are run at a constant current of 40 mA per gel until the tracking dye reaches the bottom of the gel ( ^ 4 hr). An alternative to Protocol 1 is to use a resolving gel containing a polyacrylamide gradient. Such a gel allows for the analysis of a wider range of molecular weight impurities and yields higher resolution. A 4 - 1 6 % polyacrylamide gel is prepared by placing 15 mL of the 4% polyacrylamide resolving gel solution in the forward chamber of a gradient mixer and an equal volume of the 16% polyacrylamide resolving gel solution into the rear chamber. A small amount of bromophenol blue is added to the rear chamber.

PROTEIN IMPURITY ANALYSIS

31

enabling the analyst to check the gradient after it is formed. The gradient is formed using a peristaltic pump to drive the solutions through a long needle fully inserted between the assembled gel plates. After the solutions are completely poured, the needle is withdrawn, the gel is overlaid with watersaturated isobutanol, and then it is allowed to polymerize for a minimum of 30 min prior to adding the stacking gel as already outlined. ii. Other Modes of Gel Electrophoresis

Additional electrophoretic methods of impurity analysis exist besides that of SDS-PAGE. The resolution of proteins smaller than 20 kDa is better when tricine is used as the trailing ion instead of glycine. The method of Schagger and von Jagow describes a discontinuous denaturing PAGE system utilizing tricine.^^ Native PAGE does not incorporate a denaturing step during sample preparation and SDS is not included in any of the gel solutions or sample buffers. The same procedure outlined in Protocol 1 can be used except SDS is removed from all the solutions and replaced with an equimolar amount of sodium sulfate. Also, the samples are not heated to 95°-100° C but instead are applied directly to the gel in the modified sample buffer. This method suffers from an inability to detect the more basic proteins because their charge does not allow them to migrate under these conditions. Two-dimensional SDS-PAGE has the ability to separate proteins based on their size and charge. The method of O'Farrell is often used.*'^^'^"* Methods of discontinuous urea PAGE have the ability to separate proteins based on charge, size, and conformation.^^ Electrophoretic separations based on several physicochemical properties might resolve impurities that are not detected by one-dimensional SDS-PAGE, i.e., those that comigrate with the recombinant protein, but their general application for impurity analysis has not been widespread. Isoelectric focusing gel electrophoresis (lEF) separates proteins based entirely on their charge.^ A large-pore polyacrylamide gel is prepared containing small charged molecules called ampholytes. When an electric current is applied to the gel, the charged ampholytes migrate rapidly and create a pH gradient in the gel. Simultaneously, proteins will migrate in the gel, although more slowly, to their isoelectric points, where the protein's total charge is zero and migration ceases. lEF gels are stained with Coomassie Blue as described in the next subsection but they do not provide quantitative results. lEF provides the analyst with a qualitative assessment of product-related impurities for highly pure proteins such as deamidated variants of the product, i.e., the amide side chain of glutamine or asparagine residues is converted to a carboxyl side chain. However, this analysis is complicated if the recombinant protein contains covalent carbohydrates with terminal sialic acid residues. Isoelectric focusing in capillary electrophoresis is starting to replace lEF, and this will be discussed later in the chapter. * A detailed protocol for analytical two-dimensional gel electrophoresis is found on the Internet at http://expasy.hcuge.ch/ch2d/technical.info.html. Detailed protocols for lEF are given in Ref. 24 and 26.

32

DONALD O. O'KEEFE

iii. Detection in Gel Electrophoresis

Proteins separated electrophoretically in gels are detected by staining. Most commonly, Coomassie Brilliant Blue R-250 and silver stains are used. The mechanism of protein staining by either remains uncertain. The anionic form of Coomassie Blue is believed to interact electrostatically with arginine, lysine, and histidine residues.^^'^^ Furthermore, van der Waals forces and hydrophobic contacts with aromatic amino acids might enhance dye binding.^^'^^ Despite this uncertainty, the advantage of this stain is the nearly universal quantitative binding of Coomassie Blue to proteins. Davis showed that the staining of 26 proteins in solution with the dye varied by less than twofold.^^ A simple staining solution of Coomassie Brilliant Blue R-250 consists of the dye dissolved in 50% methanol-10% acetic acid at 1 mg/mL. Gels are placed in this solution for > 1 hr and then destained using washes consisting of 50% methanol/10% acetic acid. In recent years, more sensitive methods of Coomassie Blue staining have evolved and these take advantage of the colloidal properties of Coomassie Brilliant Blue G-250 (also known as Xylene Cyanine Brilliant G). One such procedure the author has found useful is the GelCode® Blue Stain Reagent made by Pierce Chemical Company. The procedure is fast, the background staining is minimal, and the sensitivity can be as low as 10 ng. SDS-PAGE gels stained with Coomassie Blue are both a qualitative and a quantitative method of impurity analysis. Coomassie Bluestained gels are made quantitative after image analysis such as with a scanning densitometer. In these instances, the author has found the assay with GelCode® Blue to be linear from 25 ng to 1 ^tg. However, the dynamic range of the assay is greater when larger protein loads are analyzed. For example, an impurity detected at 25 ng, as interpolated from a standard curve, with a total protein load of 2 fig yields a single impurity level of 1.2% (12,000 ppm). Staining proteins with silver often yields a method 100-fold more sensitive than that of Coomassie Blue, reportedly detecting proteins at the subnanogram levels.^^'^^ The argument has been, however, that silver staining methods are significantly more variable than Coomassie Blue staining meth^jg 32,33 ^j^g mechanism of protein silver staining is understood less than that of Coomassie Blue. Lysine residues, negatively charged residues, and the sulfur-containing residues of cysteine and methionine have all been suggested to bind silver cations.^"^ Paradoxically, however, some proteins do not stain at all with silver.^^'^^ Reports have also appeared suggesting that silver staining of proteins is method-dependent.^"^ Acidic-based methods stain acidic proteins more intensely, while basic proteins are detected better in an alkaline environment. Despite all these shortcomings, regulatory agencies recommend silver staining of SDS-PAGE gels as a method of purity analysis'^. In comparing numerous silver-staining protocols, the author has found the method of Wray et al. to be suitable in many circumstances and it is presented in Protocol 2?"^ Note, however, that silver staining of SDS-PAGE gels can at best be considered a qualitative form of impurity analysis. The ICH also acknowledges that silver staining methods are less quantitative than

PROTEIN IMPURITY ANALYSIS

33

Coomassie methods.^"^ Silver staining also suffers from its potential ability to detect nonprotein impurities including nucleic acids, carbohydrates, and lipids."^ Protocol 2: Silver Staining of Polyacrylamide Gels

• Gloves should be worn throughout this procedure to prevent fingerprints from appearing on the developed gel. The gel is agitated moderately during the entire protocol. • The electrophoresed gel is fixed for 1 hr in 50% methanol with solution changes after 20 and 40 min. • Prepare solution A by dissolving 1.6 g of silver nitrate in 8 mL of deionized water. • Prepare solution B by mixing 42 mL of 0.36% (w/v) NaOH with 2.8 mL of 14.8 M NH4OH. • Prepare solution C by adding solution A dropwise to solution B while stirring vigorously. If a brown precipitate persists the solutions should be discarded and prepared again. Bring the final volume to 200 mL with deionized water. Solution C must be used immediately. Dispose of any remaining solution C properly because the decomposition product is explosive. • Place the gel in solution C for 15 min. • Rinse the gel with deionized water. Wash the gel for 5 min in deionized water with one change after 3 min. • Prepare solution D by dissolving 25 mg of citric acid in 2.5 mL of deionized water. Add 250 /xL of 38% formaldehyde and mix. Bring the final volume to 500 mL with deionized water. Prepare this solution fresh. • Place the gel in solution D. Bands should appear within 10 min. Develop the gel until the appropriate sensitivity is achieved. If the color is developing too quickly then the addition of methanol to 5-10% can slow its progress. If solution D begins to turn brown then remove it, rinse the gel with deionized water, and add fresh solution D. • Stop development by removing solution D, rinsing with deionized water, and placing the gel in 4 5 % methanol/10% acetic acid. • After 15 min, equilibrate the gel in deionized water. Staining methods other than silver and Coomassie Blue do exist for detecting proteins in polyacrylamide gels. Often these methods involve labeling the proteins with fluorescent probes or radioisotopes.^^ In both cases, the labeling is based on definable properties, i.e., the labels target specific protein functional groups. The most notable targets for fluorescent probes are protein thiols and amine groups. The advantages and disadvantages of these methods have been discussed previously.^^ In general, these methods are not universal detection methods for protein impurities but they might have application under defined circumstances.^^ Recently though, a new series of fluorescent dyes, SYPRO® Red and SYPRO® Orange (Molecular Probes), has become available for labeling proteins in SDS-PAGE gels.^^'^^ These dyes label protein-SDS complexes noncovalently and preferentially. Since all proteins bind equivalent amounts of SDS based on their mass, it is beheved that these

34

DONALD O. O'KEEFE

dyes will bind to protein-SDS complexes equally, and therefore should be quantitative. Their sensitivity is reportedly comparable to that of silver staining with as little as 1 ng of protein detectable.^^ This sensitivity, along with the reported universal quantitative binding of these dyes, might lead to their replacing silver staining as a standard method for protein impurity analysis. B. High-Performance Liquid Chromatography High-performance liquid chromatography (HPLC) is the primary quantitative technique for purity and impurity analysis of recombinant proteins. Recommended by regulatory agencies, this high-resolution technique routinely detects impurities at the level of 0.1% (1000 ppm).^^ Reversed-phase has been the HPLC method of choice ever since the efficiency of protein separations increased with the development of small particle (3-5 /im), wide pore (300 A) silica bonded to C4 through C^g ligates. Additional modes of HPLC are receiving increased usage for impurity analysis, but in general, their application is more specific and limiting. These include ion-exchange chromatography, hydrophobic interaction chromatography, and size-exclusion chromatography. Each one will be discussed briefly. i. Reversed-Phase High-Performance Liquid Chromatography In reversed-phase high-performance liquid chromatography (RP-HPLC), protein hydrophobic regions interact with the alkyl or aromatic ligates of the stationary phase until a critical concentration of organic solvent in the mobile phase comes along and releases the protein from the column."^^ These hydrophobic regions are surface exposed or internal to the protein. The latter are rendered accessible if the protein is denatured by contact with either the stationary phase or the mobile phase. In either case, the hydrophobic region that dictates retention on the column is the contact region. It is differences in the contact region among the many proteins that affects separation in RP-HPLC. There are three types of organic modifiers commonly used in the mobile phase of RP-HPLC methods. They are isopropanol, acetonitrile, and methanol listed in their decreasing ability to elute proteins. These solvents are generally combined with water in a gradient of increasing organic. Other aqueous solutions, such as acetate or phosphate buffers, have replaced water on occasion but these are generally for more specific applications. The mobile phase might also include a small amount of an ion-pairing agent, generally 0.1% trifluoroacetic acid (TFA) is the most common. Other acids used include phosphoric, hydrochloric, perchloric, and heptafluorobutyric acid. A larger amount of a weaker acid, such as acetate or formate, can also be used. These acids maintain the pH of the mobile phase below 3.5, where uncapped silanol residues on silica columns are prevented from interacting with the proteins and giving rise to mixed mode separations. These unwanted interactions can also be minimized by increasing the ionic strength of the mobile phase or by adding a competing cation such as triethylammonium phosphate. The pH of the mobile phase can be adjusted to achieve the desired separation

PROTEIN IMPURITY ANALYSIS

35

and to ensure protein solubility in the organic phase but pH values above 8 are not compatible with columns composed of silica particles. The use of polymeric supports eliminates this problem and enables the analyst to perform RP-HPLC at alkaline pH. However, RP-HPLC with a wateracetonitrile gradient in 0.1% TFA is the most widely used mobile phase system for protein analysis. Protocol 3 is a general method for analyzing the purity and impurity profile of a protein by RP-HPLC. Based on initial results, the analyst should modify this method to achieve optimal separation. Protocol 3: Reversed-Phase HPLC

• Column Vydac 214 TP (C4 ligate)* 300 A pore size 150 X 4.6 mm i.d. 5 jum particle size • Mobile phases A: 0.1% TFA in water B: 0.05% TFA in acetonitrile • Detection UV at 210 nm • Linear gradient 0 to 100% B over 100 min • Flow Rate 1.0 mL/min • Injection 50 /jiL

The lower concentration of TFA in mobile phase B partly counteracts the increased background absorbance normally seen when using a wateracetonitrile gradient containing TFA. Protocol 3 should be considered as a starting point for protein impurity analysis by RP-HPLC. Modifications will have to be made based on the particular sample undergoing analysis. For example, a more hydrophobic protein might be difficult to elute and require a stationary phase that absorbs the protein less strongly, such as one containing a phenyl ligate."*^ A stationary phase intended for hydrophobic interaction chromatography can be operated under re versed-phase conditions for extremely hydrophobic proteins. Alternatively, a more hydrophobic mobile phase might be employed to elute the strongly adsorbed protein, for instance, one containing isopropanol or a mixture of isopropanol and acetonitrile. An important advance in the RP-HPLC impurity analysis of proteins has been the advent of short columns containing micropellicular stationary phases."^^ These short columns contain nonporous particles of the order of 2 fim that are capable of attaining high flow rates ( ^ 4 mL/min) at elevated temperatures ( ^ 80° C). Such a configuration leads to very rapid and highly • Separations in RP-HPLC can be highly dependent upon the column utilized. Corran"^^ and Johns"^"^ list many commercially available reversed-phase columns suitable for protein analysis.

36

DONALD O. O'KEEFE

efficient protein separations using conventional HPLC equipment. Separations in less than 5 min are not uncommon.'^^''^^ As with any method of protein impurity analysis, RP-HPLC does have its limitations. Certain impurities might not adsorb to the column, v^hile others might not elute from the column under one set of conditions. These impurities will either not be quantitated accurately or they will not be detected, respectively. It is critical to remember that more that one method of analysis must be employed to give assurances about the level of impurities. With RP-HPLC, however, analysis can be carried out using two different solvent systems or different columns to help provide more assurance. ii. Hydrophobic Interaction Chromatography

Columns for hydrophobic interaction chromatography (HIC) are similar to those for RP-HPLC in that both types of stationary phases consist of hydrophobic ligates. A critical difference, however, is the density of these bonded groups. RP-HPLC ligates are generally very densely packed for re versed-phase particles leading to a hydrophobic surface that may be denaturing to a protein. On the other hand, the hydrophobic ligates on HIC columns are much less densely packed with the intention of having them interact with only surface hydrophobic regions on proteins. This, along with the use of more aqueous phases of high ionic strength, makes HIC generally a nondenaturing technique that primarily separates proteins based on their surface hydrophobicity. Because of this property, HIC is widely used for separating product-related impurities of highly pure recombinant proteins such as conformational isomers. HIC is generally carried out near neutral pH with a starting mobile phase high in salt, often (NH4)2804, that promotes protein interaction with the stationary phase. As the salt concentration is lowered, the less hydrophobic proteins are eluted first. The pH of the mobile phase can affect separation and it should be adjusted if results prove dissatisfying at neutral pH. Protocol 4 is a general procedure for performing HIC analysis of proteins. Isopropanol can be included in both mobile phases for the analysis of more hydrophobic proteins. If these conditions are not sufficient, then the analyst can adjust the pH (6.5 to 8.0) or increase the amount of (NH4)2S04 up to 2.0 M. Protocol 4: Hydrophobic Interaction Chromatography

• Column TSKgel Phenyl-5PW(TosoHaas)* 1000 A pore size 75 X 7.5 mm i.d. 10 ^tm particle size • Mobile phases A: 1 M (NH4)2S04 100 m M Na2P04 pH adjusted to 7.0 (±5% isopropanol) • Different column ligates can dramatically affect the separation. A list of several commercially available HIC columns suitable for protein analysis is given in Ref. 44.

37

PROTEIN IMPURITY ANALYSIS

• • • •

B: 100 m M Na2P04 pH adjusted to 7.0 ( ± 5% isopropanol) Detection UV at 220 nm Linear gradient 0 to 100% B over 15 min Flow rate 1.0 mL/min Injection 50 fiL

iii. Size-Exclusion Chromatography

Size-exclusion chromatography (SEC) is the most straightforward of the HPLC techniques presented. Separation is based on the protein's hydrodynamic volume and its interaction with the porous stationary phase. The greater the hydrodynamic volume, the greater the probability that the protein will be excluded from the interior of the particles and the sooner it will elute. Smaller proteins enter the particles more frequently and thus are retained longer. The mobile phases are aqueous buffers containing a sufficient amount of salt to prevent protein retention due to ionic adsorption. The mobile phase utilizes an isocratic gradient. The technique has only moderate resolution and it is mostly used to detect aggregates of the recombinant product, which can be immunogenic. Often two size-exclusion columns are placed in tandem to increase resolution but with a concomitant increase in back pressure. Protein size standards are injected onto the column separately from the protein drug samples for calibration. Protocol 5: Size-Exclusion Chromatograptiy

• Column Bio-Sil SEC 250-5 (Bio-Rad)* o

• • • •

250 A pore size 300 X 7.8 mm i.d. 5 ^tm particle size Mobile phase: 20 m M Na2PO4-150 m M NaCl-pH 7.0 Detection UV at 210 nm Flow rate 1.0 mL/min Injection 50 IJLL

iv. Ion-Exchange Chromatography

Ion-exchange chromatography (lEX-HPLC) is intended to separate proteins based on differences in their total charge. Both anionic and cationic exchange modes are available. This technique can potentially detect and • Additional columns are described by Johns."^"^

38

DONALD O. O'KEEFE

quantitate product-related impurities, such as deamidated proteins, and product-related substances, such as heterogeneously glycosylated proteins. The resolution of this technique is generally only moderate. lEF provides much greater resolution for detecting these variants. lEX-HPLC may prove to be useful in some impurity analyses, but the method must be specifically tailored to a particular application. Numerous variables must be estabhshed for the separation, and therefore lEX is often the most difficult HPLC method to develop."^^ As such, it is difficult to give a brief generic starting protocol. Interested readers are encouraged to look at references at the end of this chapter for more information on this technique."^^'"^^ V. Detection in HPLC

The most common mode of detection in HPLC impurity analysis is UV absorbance monitoring. Generally, UV v^avelengths in the 210-220 nm range are used. At these wavelengths, the major chromophore in proteins is the peptide bond. The absorbance signal for a given protein is proportional to the number of peptide bonds. Therefore, protein impurities can be quantitated with reasonable accuracy using a known protein as a standard. However, slight errors will occur because the side chains of several amino acids (trp, phe, his, tyr, gin, asn) also contribute to absorbance in the low UV range.'*^ Using low UV wavelengths, sensitivities for detecting protein impurities can be as low as 20 ng, depending on the particular detector. Utilizing an upper linear limit of 49 jjig for a recombinant protein in a valid RP-HPLC assay, the detection limit for protein impurities can be as low as 0.04% (400 ppm)."^^ Caution should be taken in selecting the exact wavelength because certain organic modifiers, such as isopropanol, do absorb in the low UV range. An inherent problem in using a single low UV wavelength is its inability to discriminate a protein impurity from a nonprotein impurity. Monitoring for absorbance at 257, 275, or 280 nm can circumvent this lack of specificity. At these wavelengths, the predominant protein chromophores are phenylalanine, tyrosine, and tryptophan, respectively. If possible, two wavelengths are monitored simultaneously to assess a peak's purity and identity. The ratio of the absorbance of a protein at two wavelengths is equivalent to the ratio of the protein's molar absorptivites at those wavelengths. This ratio is independent of protein concentration. Hence, this ratio should be the same throughout a chromatographic peak if that peak represents a pure protein. If an impurity coelutes precisely with the recombinant protein of interest, then this technique will fail if a standard of the pure protein is unavailable. Also, different conformational isomers of the same protein might have different absorbance ratios, so this technique might be limited to RP-HPLC where the protein is mostly denatured. This method of purity analysis can be extended further using photodiode array detection (PDA) where the entire UV spectrum can be monitored simultaneously.^^ Although PDA detection is a very powerful technique for assessing the purity of protein pharmaceuticals, its sensitivity is often lower than that of absorbance at a single UV wavelength. Increased sensitivity of detection in HPLC can often be gained by monitoring the column effluent for intrinsic protein fluorescence by excitation

PROTEIN IMPURITY ANALYSIS

39

at 280 nm and emission at 310-350 nm. This sensitivity can be increased further by excitation at 220 nm. The major fluorophore in proteins is tryptophan with tyrosine contributing a minor signal. Monitoring impurities for fluorescence at these wavelengths almost assuredly identifies them as proteins, however, quantitation of impurities by this method is unlikely. Proteins may not be detected fluorescently because they lack tryptophan residues or those impurities that do contain these residues have them in unknown numbers. Furthermore, the quantum yield of all tryptophan residues will not be identical owing to their diverse molecular microenvironments in their native proteins. Hence, a standard curve from a known unrelated protein is not useful. Another sensitive technique for monitoring HPLC of proteins is to utilize fluorescent derivatization. These methods are often based on the reaction of small fluorescent compounds with specific functional groups in proteins.^^'^^ Most often protein amines and sulfhydryl groups are the targets of these agents.^^ Amine-reacting reagents are more popular since the rarity of proteins lacking amine groups is less than those that lack sulfhydryls. Fluorescamine is a common reagent for this purpose. A stock solution of fluorescamine is dissolved in acetone and added to the protein in an alkaline buffer (pH 9).^"* The reagent labels the a-amino group at the N terminus and the e-amino group on lysine residues. The reaction is completed in less than 1 min and unreacted fluorescamine shows little fluorescence. The labeled products can then be separated by RP-HPLC. Post-column-derivatization procedures are also used.^^'^^ However, amine groups are not exclusive to proteins and the detection of a fluorescent impurity peak does not guarantee the impurity is a protein. Furthermore, quantitation is difficult for the same reasons discussed earlier in the chapter regarding tryptophan fluorescence. Nevertheless, fluorescently labeling proteins in HPLC impurity analysis has found some applications (see the Case Studies at the end of the chapter).^^ vi. Peptide Mapping

The most powerful method available for evaluating primary structural variants of recombinant proteins is peptide mapping. This technique is applicable to the analysis of product-related impurities. Variants that are amenable to this type of analysis include deamidated asparagine or glutamine residues, oxidized methionine residues, N-terminal variants, disulfide isomers, substituted amino acids due to mistranslation or point mutation, and protein variants chemically modified during processing. Although many of these modifications are detectable by other techniques, such as IFF and lEX chromatography, peptide mapping by RP-HPLC is generally used to identify the site of modification. The peptide containing the modification is first identified by a shift in retention time compared to that of a reference peptide map. However, the technique is generally only capable of detecting a modification at the 2 - 5 % level. The methodology of peptide mapping by RP-HPLC is straightforward. The protein is first denatured and reduced with urea and dithiothreitol (DTT), respectively, to unfold the protein. Cysteine residues are then blocked

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DONALD O. O'KEEFE

with a small sulfhydryl-reactive reagent such as iodoacetamide. This minimizes the number of peaks in the chromatogram from peptides containing cysteine residues. Alternatively, DTT can be omitted if disulfide isomers need to be examined. Next, the protein is fragmented completely. Generally, this is carried out using proteolytic enzymes that cleave specific amino acid sequences. Trypsin, which cleaves after arginine and lysine residues, and V8 protease from Staphylococcus aureus, which cleaves after glutamate residues, are most often used. Chemical cleavage methods can also be employed. Cyanogen bromide cleaves proteins after methionine residues,^^ o-iodosobenzoic acid cleaves after tryptophan residues,^^ and hydroxylamine^^ cleaves between asparagine and glycine residues. The exact method of protein fragmentation will depend on the sequence of the recombinant protein under study. The objective is to fragment the protein into peptides no greater than 25 amino acids in length. The fragmented protein is then separated by HPLC. Reversed-phase is the mode most often chosen because of its high resolving capabilities. However, these separations can be difficult because of the enormous number of peaks that must be separated. For example, the peptide map of recombinant tissue plasminogen activator has over 50 chromatographic peaks after trypsin digestion. A multiphasic linear gradient was employed to achieve this separation.^^ Protocol 6 for peptide mapping utilizes trypsin to fragment the protein enzymatically.^^ The trypsin used should be tosylphenylalanylchloromethyl ketone (TPCK) treated to eliminate residual chymotrypsin activity that, if present, would cleave the recombinant protein after phenylalanine, tryptophan, and tyrosine residues. The fragmented protein is then chromatographed via RP-HPLC using a gradient of acetonitrile containing TFA Replacing TFA with 50 m M sodium phosphate as the ion-pairing agent reportedly has given better resolution of peptide peaks.^^ It is likely that the gradient given in the following will have to be modified to achieve optimal separation of a particular peptide mixture. Protocol 6: Tryptic Peptide Mapping Peptide Mapping—Trypsinolysis

• Samples should be prepared in triplicate including a reference standard, if available, and a buffer blank. • Dry separate 200 jx g samples of the protein under vacuum (i.e., using a Savant Speed Vac). Once dried, the samples can be stored at — 20° C. • Resuspend the dried protein in 50 /xL of fresh 8 M urea-80 m M methylamine-0.5 M ammonium bicarbonate. Avoid heating this buffer during preparation. Heat increases the formation of cyanate in the urea that can lead to the carbamylation of the e-amino group of lysine residues. Methylamine helps eliminate cyanate in urea. • Add 5 JJLL of 45 m M DTT and mix thoroughly. Heat the samples at 50°C for 5 min. • After cooling to room temperature, add 5 /xL of 100 mMiodoacetamide to each sample. Incubate for 15 min.

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PROTEIN IMPURITY ANALYSIS

• Bring each sample to 200 ^LL with 0.5 M ammonium bicarbonate and one unit of TPCK-treated trypsin. The TPCK-treated trypsin stock is prepared in 0.5 M ammonium bicarbonate and can be frozen in single use aliquots at -20°C. • Incubate the samples at 37°C for approximately 18 hr. • Stop the proteolysis by adding 60 juL 2% TFA. The samples are now ready for chromatography or they can be stored at — 20°C. Peptide

Mapping—Cliromatograpliy

• Column Vydac 218 TP (Cig Hgate) 300 A pore size 150 X 4.6 mm i.d. 5 iJLtn particle size • Mobile phases A: 0.1% TFA in water B: 0.05% TFA in acetonitrile • Detection UV at 210 nm • Linear gradient 0 to 100% B over 100 min • Flow rate 1.0 mL/min • Injection 80 fjiL

The detection modes for a peptide map are the same as those described earlier for HPLC. Absorbance at low UV wavelengths is most often used. Absorbance at other wavelengths, tryptophan fluorescence, or fluorescence after derivatization might provide additional information on the location of a modification (see Section IV). The use of electrospray mass spectrometry on-line with HPLC is a powerful tool for peptide mapping that can identify the modification without further analysis.^^

C. Capillary Electrophoresis

One of the most powerful separation techniques for purity and impurity analysis for the bioanalyst is capillary electrophoresis (CE). However, it is a relatively new analytical tool and its methodology is evolving at a rapid pace, so there is limited reference to its application to protein impurity analysis in the literature. Nonetheless, this is only a temporary respite. In the future, CE will become a standard and routine analytical technique for the analysis of protein impurities in recombinant pharmaceuticals. Capillary electrophoresis combines the separation principles of gel electrophoresis with the throughput and detection methods of HPLC. It overcomes the disadvantages of slab gel electrophoresis, including slow and labor intensive procedures and the difficulty and inaccuracies of quantitation. CE is

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DONALD O. O'KEEFE

a high-resolution technique. Its resolution surpasses that of slab gel electrophoresis and in many cases that of HPLC. In practical terms it is able to separate closely related protein species present as impurities including those having aspartic acid in place of asparagine (deamidated species). The speed and high resolution of CE is a direct result of the high voltages that are applied. The higher the voltage, the faster the separation, v^hile resolution increases with the square root of the applied voltage. The high heat created by these voltages is effectively dissipated by the use of narrow diameter capillaries of 50-100 /xm. The capillaries are most often made of fused silica surrounded by a polyimide sheath. The internal walls of the capillary will bear a negative charge at pH 3 or greater due to the ionization of the silanol groups. When an electric current is applied to the capillary, cations in the electrolyte move past the immobilized silanol anions towards the cathode buffer and cause a bulk movement of the buffer termed electroendoosmotic flow (EOF). Regardless of charge, all analytes will eventually migrate past the detector due to the EOF. The EOF, in addition to electrophoretic mobility, influences the separation of the analytes, but it can be modified to varying degrees by adjusting the p l i or by using either a coated capillary or buffer additives. The choice of pH in CE separations is critical. It may be the single most important variable in a CE separation. Lowering the pH to 2 or less neutralizes the silanol residues and prevents ionic interactions with proteins. Alternatively, using a pH higher than the isoelectric point (pi) of the most basic protein in the analysis creates repulsion between the negatively charged proteins and the silanol residues. Unfortunately, for analysis of host protein impurities the pi of the most basic protein is unknown so that can limit this approach. Furthermore, a higher pFi leads to higher EOF, and a lower pH might protonate all proteins thus sacrificing differences for their separation. Some widely used buffers and their respective pK^ values include formate (3.75), acetate (4.75), MES (6.15), imidazole (7.00), HEPPSO (8.00), morpholine (8.49), borate (9.24), and CHAPSO (9.60).^^ If pH extremes cannot be utilized, then either a coated capillary or buffer additives can be used. Without either of these, proteins might adsorb to the inner capillary wall. Adsorption will affect the separation and in some circumstances this may be beneficial. Additives that are often added to CE buffer systems include surfactants, zwitterionic salts, ethylene glycol, methylcellulose, organic modifiers, and quaternary amines. Capillary coatings include polyacrylamide, polyethyleneglycol, polyvinylpyrrolidone, and methylcellulose. Mazzeo and KruU^^ discuss capillary coatings and buffer additives further. One significant limitation of CE regarding protein impurity analysis is its inability to be a routinely preparative technique owing to the small injections of sample (-^2-10 nL). Generally, numerous runs must be performed to gather enough samples for subsequent analysis. The use of a fraction collector to automate such a system can be problematic if consecutive runs are not precisely reproducible, which is not an uncommon feature of CE. However, one interesting system was developed by Eriksson et aL^^ The authors designed a moving polyvinylidene difluoride (PVDF) membrane that collected

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43

proteins and peptides as they eluted from the capillary past the detector. The membranes were stained with common protein reagents and they were also identified immunologically with antibodies. This approach can be a significant advance for the separation and identification of minor protein impurities. CE has many separation modes that are beneficial to protein impurity analysis. Within the many thousands of potential protein impurities in a recombinant product there will be several that have only minor physicochemical differences from the drug product. The application of different CE modes can potentially resolve these impurities. CE methods can be divided into four principle modes that are applicable to recombinant protein impurity analysis: capillary zone electrophoresis, capillary isoelectric focusing, capillary gel electrophoresis, and micellar electrokinetic capillary chromatography. Each mode will be discussed briefly. Since the technology is so young and still very exploratory, CE methods are developed empirically for specific separations. It is difficult to provide standard protocols for CE impurity analysis. Instead, protocols that can be used as a starting point for impurity analysis will be provided as well as the citation of examples of impurity analyses from the literature to provide additional sources of protocols for interested readers. i. Capillary Zone Electrophoresis

Capillary zone electrophoresis (CZE), also known as free-solution CE, is the most widely used mode of CE essentially because of its versatility. Protein separation in CZE is based on the differential electrophoretic mobility of the analytes. This mobility is primarily dependent on a protein's size and net charge, the charge-to-mass ratio. Solvent properties that influence the size and charge of a protein include pH, ionic strength, viscosity, and dielectric constant.^^ Manipulation of these properties, most notably pH, dictates the selectivity in CZE. Maximizing the charge difference between two proteins via pH modification optimizes their separation. CZE is an attractive method for the analysis of process-related protein impurities because its mechanism of separation is different from that of RP-HPLC. The former is based on a charge-to-mass ratio, while the latter is based on hydrophobicity. CZE has been used to monitor the impurity profile of recombinant proteins during downstream processing. This includes recombinant hepatitis B surface antigen^^ and recombinant hirudin.^^ However, for impurity analysis, CZE has most often been employed to analyze productrelated impurities, such as deamidated products. Examples include recombinant growth hormone,''^''^^ recombinant insulin^^ recombinant tumor necrosis factor beta,^^ and recombinant interleukin-4.^^ Nielsen et al7^ separated recombinant insulin from its deamidated degradate (desamido-A21) and two incorrectly processed impurities. However, the misprocessed impurity desthreonine-B30 was not resolved by this method but was separated by RP-HPLC. Likewise, these researchers used this same method to separate recombinant growth hormone from its deamidated derivatives but were unsuccessful in resolving a methionine sulfoxide degradate. The latter was also resolved by RP-HPLC. These results underscore the complementary

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DONALD O. O'KEEFE

nature of RP-HPLC and CE, both of which exploit different protein physicochemical properties to affect separation. CZE is a method for the rapid analysis of peptide mixtures and therefore has particular application to peptide mapping and identification of productrelated impurities/"^ Unlike peptide mapping by RP-HPLC, trypsinolysis might not be the method of choice for fragmentation because most of the peptides generated have a net charge of -h 2 at low pH. Alternative methods of fragmentation should be considered or separations utilizing higher pH buffers. Peptide mapping of recombinant proteins by CZE include the following along with their method of fragmentation: growth hormone (chymotrypsin, V8 Protease, trypsin),^^'''^'^^ insulin-like growth factor (trypsin),""^ and interleukin-4 (trypsin).""^ Protocol 7 for CZE was adapted from Gordon et al7^ These authors used this protocol to analyze a variety of proteins including a complex protein mixture. It may provide a good starting point for protein impurity analysis. If insufficient separation is achieved then the analyst is advised to change the pH by using one of the other buffers discussed above. Such an approach showed markedly different separations for a number of recombinant proteins and product-related impurities.^^'""^ Protocol 7: Capillary Zone Electrophoresis

• Running buffer: 50 m M sodium borate, pH 10.0 • Sample preparation: in 20 m M sodium borate, pH 4.0, containing 20% ethylene glycol • Capillary: 37.5 cm X 75/>tm i.d. fused silica; length to detector: 30.5 cm • Voltage: 10 kV • Temperature: 20°C • Injections: variable • Detection: 200 nm • Wash the capillary between runs with 0.1 N NaOH (5 min), then deionized water (3 min), followed by running buffer (5 min) ii. Capillary isoelectric Focusing

Capillary isoelectric focusing (CIEF) separates proteins based on differences in their isoelectric points. CIEF has been used to separate product-related impurities of recombinant proteins, mainly deamidated species, such as those of human growth hormone.''^ There are two basic forms of CIEF that differ based on the method for mobilizing the focused proteins. One method of CIEF is to fill a coated capillary with the sample and the ampholytes and then apply the voltage. Estabhshment of a pH gradient occurs quickly and subsequent focusing of the proteins at their pi values soon follows. The focusing generally takes 15-20 min. Since it is a coated capillary the EOF is negligible and the focused proteins must be mobilized to move past the detector. Mobilization is accomplished by reapplying the voltage after either replacing the acidic anolyte with base or the alkaline catholyte with acid or adding salt to either the anolyte or the catholyte.^^ As with slab gel IFF, the focused proteins can

PROTEIN IMPURITY ANALYSIS

45

precipitate, but incorporating urea, detergents, or ethylene glycol into the running buffer can minimize this problem/^ A second method does not eliminate the EOF but instead uses it to force the focused proteins past the detector.^^ Hence, a postfocusing mobilization step is not required. Meaningful results can be obtained only if protein focusing is faster than the EOF. Protocol 8 uses this one-step method and it is adapted from that of Mazzeo and KruU.^^ Due to run-to-run variability, standards of known pi should be included in the sample. Protocol 8: Capillary Isoelectric Focusing

• Catholyte: 20 m M NaOH • Anolyte: 10 m M phosphoric acid • Sample preparation: wash the capillary with 10 m M phosphoric acid and then fill it with sample (100 /ig/mL) and standards containing 5% ampholytes (Pharmalyte, pH 3-10, Pharmacia), 0.1% methylcellulose, 1% TEMED • Capillary: uncoated fused silica 60 cm X 75 fim i.d., 40 cm from anode to detector • Vohage: 30 kV • Detection: 280 nm (the ampholytes absorb at low UV wavelengths) iii. Capillary Gel Electrophoresis

The separation principle in capillary gel electrophoresis (CGE) is the same as that of slab gel electrophoresis. Most often CGE is used in a denaturing mode with the incorporation of SDS and is referred to as SDS-CGE. As such, separation is based on the protein's molecular mass and, due to the sieving mechanism of the gel, smaller proteins migrate past the detector first. The use of a gel material and SDS decreases the EOF and eliminates protein adsorption to the capillary walls further ensuring that migration is based on molecular mass. This precludes the need for additives and coated capillaries. One of the earliest successes with CGE was that of Tsuji in the analysis of recombinant proteins.^^ In that study, acrylamide gels were polymerized inside and attached to a fused-silica capillary measuring 7 cm X 50 /^m i.d. Utilizing a running buffer of 300 m M Tris, pH 8.8, 0.1% SDS with ethylene glycol and a sample buffer consisting of 15.6 m M Tris, pH 6.8, 0.1% SDS essentially created a discontinuous electrophoresis system. In two recombinant protein preparations, this system detected a dimer in one and a 1500 Da smaller impurity in the other.^^ In recent years, however, gel polymerization within capillaries has been abandoned. Instead linear polymer "gels" have been developed for CGE that eliminate the difficulty and tedium of preparing polymerized gels within capillaries. These gels also provide longer column lifetimes. In this method, a viscous linear polymer solution, or a mixture of polymers, is incorporated into the running buffer. The running and sample buffers of these methods are similar to those of the discontinuous Laemmli SDS-PAGE system. During electrophoresis, protein migration in the capillary is retarded based on the protein's molecular mass. When the technique is used without SDS under

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DONALD O. O'KEEFE

nondenaturing conditions it provides a means of assessing the aggregation state of a recombinant protein. Polymers consisting of dextran, linear polyacrylamide, polyethylene oxide, and polyethylene glycol have been utilized at concentrations of 1-6%. Benedek and Thiede^^ utilized polyethylene oxide (PEO) polymers to analyze recombinant proteins such as erythropoietin, interferon, granulocyte stimulating factor, brain-derived neurotrophic factor, and platelet-derived growth factor. Protocol 9 describes an SDS-CGE method adapter from their work. Protocol 9: SDS ~ Capillary Gel Electrophoresis

• A PEO (M^ = 100,000) stock solution is made in 0.1% ethylene glycol and passed through a 5.0 fim membrane filter • Running buffer: 100 m M Tris-2-(N-cyclohexylamino)ethansulfonic acid (CHES), pH 8.5, 0.1% SDS, 3 % (w/v) PEO • Sample preparation: The samples are placed in a buffer with a final composition of 60 m M Tris-HCl, pH 6,6, 5% (v/v) j8mercaptoethanol, 1% (w/v) SDS, with a trace amount of Orange G as a tracking dye and heated at 95°-100°C for 2 - 5 min. • Capillary: 27 cm X 100 /xm i.d. (20 cm to the detector) fused-silica washed with 1 M NaOH, then deionized water, and then 1 M HCl before conditioning with the running buffer • Voltage: 8.1 kV • Injections: variable • Detection: 214 nm (the detector is placed at the anode end) • Wash the capillary between runs with 1 M HCl, then deionized water, followed by running buffer. Separations in Protocol 9 are comparable to those of SDS-PAGE but each sample run is less than 20 min and more accurate quantitation can be achieved. Limits of protein impurity detection in CGE systems with absorbance detection at a low UV wavelengths are approximately 0 . 1 % (1000 ppm). This is about 10-fold lower than the detection limit of SDS-PAGE. Commercially available linear polymers and CGE kits are available. The chemical composition of some of these polymers is proprietary but others are not. Companies marketing these linear polymers for use in CGE include Bio-Rad,^"^ Perkin Elmer/ABI (linear polyacrylamide),^^ and Beckman Instruments (PEO).^^ The Beckman SDS-Protein Gel Column Kit includes many of the supplies and reagents required for Protocol 9. iv. Micellar Electrokinetic Capillary Chromatography

CE can also separate neutral analytes via micellar electrokinetic capillary chromatography (MEKC). This mode of CE is similar to CZE except a micellar solution, often anionic in nature (e.g., SDS), is incorporated into the running buffer. Neutral analytes partition between the micelles and the running buffer based on their relative hydrophobicity, which greatly influences the separation. Charged analytes also partition according to their relative hydrophobicity but their charge also affects their separation. Charged analytes can potentially interact with the anionic micellar surface to affect

PROTEIN IMPURITY ANALYSIS

47

their separation further. In many ways MECC is analogous to RP-HPLC, and as such it has been mostly used for peptide mapping with rare uses for proteins. For further information and applications, the interested reader is directed to the review by Mazzeo.^^ V. Detection in CE Detection methods in CE are similar to those used in HPLC. Much of the discussion presented early for detection in HPLC can be applied to CE. Low UV absorbance detection is the most popular form of detection in protein CE. Other modes of protein detection in CE include photodiode array detection, mass spectrometry, both intrinsic and derivatized protein fluorescence, and indirect fluorescence. In indirect fluorescence the capillary is filled with a small fluorescent compound having the same charge as the analytes. The analytes are detected as a nonfluorescent band within a fluorescent electrolyte. The sensitivity of this detection is between that of UV absorbance and direct fluorescence .^^ One additional mode of detection in CE showing great promise and increasing usage is laser-induced fluorescence (LIE) detection. This mode almost always involves derivatization of the protein so that it can be excited by the wavelength of the laser. Helium-cadmium lasers emit at 325 and 442 nm while argon-ion lasers emit at 458, 488, and 514 nm. These wavelengths dictate the type of fluorophore used in the derivatization. Fluorophores commonly used to tag protein amines and their excitation maxima include o-phthaldialdehyde (OPA) (340 nm), naphthalenedialdehyde (440 nm), 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA) (456 nm), 4-chloro-7-nitrobenz-2-oxa-l,3-diazole(470 nm), and fluorescein isothiocyanate (488 nm).^^ Detection limits with LIE are reported to be as low as the attomole and zeptomole range .^^'^^ As with all modes of fluorescence detection, LIE only has the potential to be a qualitative technique for the analysis of unknown impurities. The mode of detection selected for a CE method is significant in determining the sensitivity of the method. In CZE using UV absorbance detection at 200 nm, Nielsen and Rickard^^ conservatively estimated the minimum and maximum protein detection levels of their linear range to be 0.1 and 25 ng, respectively. This translates to an impurity sensitivity of 0.4% or 4000 ppm. Coupling CE with electrospray ionization mass spectrometry (MS) can potentially be a very powerful tool for detecting and identifying product-related impurities in recombinant pharmaceuticals. Proteins can be detected in the femtomole range with this mode. Conceivably, the bioanalyst could perform a peptide map with CZE-MS and detect, identify, and sequence aberrant peptides derived from degradates. D. Immunoassays Immunoassays are the most specific and sensitive techniques available for detecting protein impurities. There are two classes of protein impurities that are most often analyzed with these techniques: host-cell proteins and protein additives, both of which are process-related impurities. Although protein additives are known entities and therefore amenable to other quantitative

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analyses such as HPLC, the use of immunoassays is more sensitive. There are two separate formats commonly employed for immunodetecting protein impurities: enzyme-linked immunosorbent assays (ELISA) and Western blot analysis. Both have advantages and disadvantages and both suffer from the potential difficulty of obtaining a suitable antiserum. Both methods v^ill be discussed in turn. With a properly prepared antiserum, immunoassays are capable of detecting protein impurities in the lov^ ppm range. More significantly though, they can do this in the presence of milligram quantities of the recombinant pharmaceutical. For known protein impurities, such as serum proteins, nuclease additives, and chromatography ligands (e.g., monoclonal antibodies or protein A) commercial ELISA or antisera are generally available and these will not be discussed further. For host-cell protein impurities, however, achieving a ppm level of detection requires an appropriate reference standard comprised of potential host protein impurities to use as an immunogen. The reference standard is generally obtained from a mock manufacturing run of the production process. Host cells containing a plasmid identical to the production strain, but lacking the gene for the recombinant product, are grown and processed through the normal purification procedure. Two important assumptions are made regarding this approach. First, the absence of recombinant protein expression does not alter the expression of host proteins. Evidence suggests that there is little difference from the normal process.^^ Second, the absence of the recombinant protein does not alter the behavior of host proteins in the purification steps. Throughout the mock process, fractions are combined, as they would be if the recombinant product was present. At the process step where the product is normally 9 5 - 9 9 % pure, the material in the pooled fractions is collected and this comprises the reference standard.^^ After concentrating this material, either rabbits or goats are immunized. It is best to immunize several animals with the preparation to reduce the effects of immunological diversity among animals of the same species. Immune antiserum is collected, pooled, and an immunoglobulin fraction is prepared by ammonium sulfate precipitation. Passing it through an affinity column made from the reference standard preparation processes this fraction further. The resulting fraction contains antibodies more selective for potential host-cell protein impurities and thus increases the sensitivity of the assay.^^ Anicetti and coworkers^^"^^ and Eaton^^ provide in depth discussions on the preparation of these host-cell protein antibodies. i. ELISA For use in an ELISA, a portion of this antibody preparation is conjugated to a moiety that is capable of eliciting a detectable signal, generally an enzyme, such as alkaline phosphatase. The format of a host-cell protein ELISA is a double-antibody sandwich. The assay begins by coating the bottom of a 96-well microtiter plate with an excess of the purified antibodies to capture the protein impurities. After blocking the remaining binding sites on the plate surfaces, the recombinant product is added. Without the initial antibody layer and subsequent blocking most of the plate's surface would be coated with the recombinant protein and very little binding would be avail-

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49

able for the impurities. A calibration curve is also constructed simultaneously using the impurity reference standard prepared above. After the incubation, the wells are washed and the conjugated antibody is added. The plate is then washed and processed to record the detectable signal. Extensive details on the methodology of host-cell protein ELISA are found in references by Anicetti and coworkers.^^'^^ Host-cell ELISA have been used to detect low ppm protein impurities in a number of recombinant proteins including human growth hormone (18 ppm) [95] and human insulin ( < 4 ppm).^^ Protocol 10 is a host cell ELISA modified from that presented by Anicetti et al.^^ Protocol 10: Host-Cell Protein ELISA

• Add 100 /x,L of 2.5 ^ig/mL of the purified anti-host-protein antibody in 10 m M carbonate buffer, pH 9.6, to the wells of a 96-well microtiter plate and incubate overnight at 4°C. • Wash the wells twice with Tris-buffered saline containing 0.05% Tween 20. • Add 200 fjiL of Tris-buffered saline containing 0.1% gelatin and 0.05% Tween 20 to each well to block the remaining binding sites on the plate surfaces and incubate for 1 hr at 37°C. • Wash the wells twice with Tris-buffered saline containing 0.05% Tween 20. • Add 100 IJLL of the serially diluted samples or calibration standards to individual wells and incubate for 2 hr at room temperature. • Wash the wells twice with Tris-buffered saline containing 0.05% Tween 20. • Add 100 fjiL of the antibody-alkaline phosphatase conjugate (at 0.3 ^tg/mL) and incubate for 1 hr at 37°C. • Wash the wells twice with Tris-buffered saline containing 0.05% Tween 20. • Add 100 /xL of p-nitrophenyl phosphate at 1 mg/mL in 50m M Tris-HCl, pH 9.0, containing 0.5 m M MgCl2. Incubate for 30 min at room temperature. • Add 25 /xL of 3 M NaOH per well to stop color development. • Read the absorbance at 405 nm for each well using a microtiter plate reader. • Determine the concentration of host-cell proteins from the calibration curve. If the samples do not show linear dilution then the capture antibody was not in excess and the assay results are invaUd. ii. Western Blot Analysis

Unlike ELISA, which can detect and quantitate host-cell proteins as a group. Western blots detect single protein impurities. Western blot analysis starts with an SDS-PAGE protocol but does not include a final colorimetric staining step. Instead after electrophoresis the proteins are electrotransferred (blotted) from the gel onto a thin membrane. Membranes made of nitrocellulose or PVDF are most often used. Once the transfer is complete, the membrane is incubated with a nonspecific protein solution to saturate and to

50

DONALD O. O'KEEFE

block the membrane. The blocked membrane is then probed with the specific antiserum (the primary antibody) followed by a secondary antibody that recognizes the primary antibody. The secondary antibody is conjugated to a moiety capable of producing a detectable signal after development. Reports document as little as 30 pg of a protein can be detected by this method.^^ If the total protein analyzed by this method was 10 jitg, then the sensitivity of the assay for a single protein impurity can be as low as 3 ppm depending on the quality of the antiserum employed. Nevertheless, Western blotting is not a quantitative technique. It is a qualitative technique and useful for comparing the protein impurity profile from lot to lot, but in some circumstances it can provide a semiquantitative estimate of a protein impurity if a standard is available (see Section IV).^^ Western blot analysis of host-cell protein impurities should not stand alone. Using recombinant human growth hormone, Gooding and Bristow found £. coli protein impurities with greater sensitivity by Western blotting then with silver-stained polyacrylamide gels but in lesser numbers.^^ For this reason, regulatory agencies insist that immunoassays will not replace silver-stained polyacrylamide gels, but will complement them."* A ppm level of sensitivity is not universal for all host-cell protein impurities in Western blots. It is limited by the shortcomings of the antiserum preparation discussed previously and the Western blot protocol. The efficiency of transfer from the gel to the membrane is variable for different proteins. Electrotransfer from the gel to the membrane is inversely related to the size of the protein, i.e., smaller proteins transfer much more efficiently than larger ones. Including 0 . 1 % SDS in the blotting buffer or partial proteolysis of the protein within the gel facilitates the transfer of larger proteins.^^^ For smaller proteins, the membrane might bind them less effectively and may be permeable to some proteins, causing them to escape detection. Using a membrane with a smaller pore size might ameliorate this, while others have reported overcoming this phenomenon by using crosslinkers to trap the proteins on the membrane.^^^ The electrotransfer described in Protocol 11 is a modification of Burnette's method^^^ as adapter from Towbin et al}^^ Protocol 11: Western Blot Analysis SDS-PACE

• Samples are prepared and electrophoresed as described in Protocol 1 except mini SDS-polyacrylamide gels are used that measure approximately 5.5 cm X 9 cm X 0.75 mm. Suitable gels are supplied by Bio-Rad for use with the Mini Protean II electrophoresis apparatus. • The gels are electrophoresed at 20 mA/gel until the tracking dye reaches the bottom of the gel. Electrotransfer • The electrophoresed gel is washed in transfer buffer (192 m M glycine, 25 m M Tris, 20% methanol, 0.1% SDS) for 10 min to remove protein not within the gel. This lowers the background signal once the membrane is developed.

PROTEIN IMPURITY ANALYSIS

51

• A piece of nitrocellulose membrane (pore size 0.45 fim^ Schleicher and Schuell) is cut to the size of the gel and then equilibrated in transfer buffer. Always wear gloves when handling nitrocellulose to reduce the background on the developed membrane. • The gel is overlaid with the nitrocellulose membrane and any trapped air bubbles are removed. • The gel and the membrane are then sandwiched between sheets of Whatman 3MM paper and the entire assembly is placed in a Genie Electrophoretic Blotter (IDEA Scientific) according to the manufacturer's instructions. • The Genie Electrophoretic Blotter is filled with transfer buffer and connected to the power supply. Transfer is for 1 hr at 24 A with a Schauer battery charger (IDEA Scientific). Other electroblotters can be used but the author has obtained unparalleled success with the Genie. Proteins are completely transferred to membranes in 30-60 min.^^"^ Immunodetection

• All steps are performed at room temperature with gentle agitation. • The nitrocellulose membrane is remove from the Genie Electrophoretic Blotter and incubated for 1 hr in buffer A [10 m M Tris-HCl, pH 8.0, containing 50 m M NaCl, 2 m M EDTA, 1% (w/v) bovine hemoglobin]. • Add the appropriate dilution of the primary antibody to the blocked nitrocellulose membrane. Generally, 1:1000 to 1:5000 dilutions are suitable. Incubate overnight. • Remove the primary antibody and wash the nitrocellulose membrane three times with buffer B [100 m M Tris-HCl, pH 8.0, containing 200 m M NaCl, 1% (v/v) Igepal CA 630 (Sigma Chemical Co.)] for 5 min each time. • After the third wash, place the nitrocellulose membrane in buffer A. • Add the secondary antibody, which recognizes the primary antibody and is coupled to alkaline phosphatase, to a final dilution of 1:3000. Incubate for 2 hr. • Remove the secondary antibody solution and wash the nitrocellulose membrane three times with buffer B for 5 min each time. • Prepare the color development solution by adding the BCIP [25 mg of 5-bromo-4-chloro-3-indolyl phosphate, p-toluidine salt (Sigma Chemical Co.) in 2.5 mL of N,N'-dimethylformamide] and NBT (50 mg of nitro blue tetrazolium in 5 mL of 50% N,N'-dimethylformamide) solutions to 100 mL of 1.0 M Tris-HCl, pH 9.0. • Incubate the nitrocellulose membranes in the color development solution for 10-30 min. • Stop the color development solution by removing the solution and thoroughly rinsing the nitrocellulose membrane in 1 m M EDTA. Certain circumstances might arise where the bioanalyst wants to examine a recombinant protein preparation for low level protein impurities but a suitable antiserum is unavailable and the variability and nonspecificity of silver staining is unacceptable. An additional assay would be a Western blot analysis after derivatization of the protein mixture with Sanger's reagent

52

DONALD O. O'KEEFE

(2,4-dinitro-l-fluorbenzene,DNFB). DNFB reacts with protein amine groups. The subsequent DNP-protein derivatives are detected in a Western blot using anti-DNP antibodies as the primary antibody. How^ever, this procedure suffers from the disadvantage of also detecting the recombinant protein, w^hich is in excess, and producing a strong band at its location. Silver staining polyacrylamide gels has the same drav^back. Nevertheless, the author has detected bacterial protein impurities at levels less than 0.1 ng using this method. Protocol 12 describes this procedure in further detail. Protocol 12: Western Blot Analysis after Derivatization with Sanger's Reagent Derivatization

Prepare the follov^ing solution in an amber-colored tube: 100 fiL protein at 2 mg/mL in phosphate-buffered saline 32 fjiL 0.5 M sodium bicarbonate 160 jLtL DMSO 32 IJLL 0.1% DNFB in DMSO Incubate at room temperature in the dark for 10 min then add 36 ixL 0.1 M lysine SDS-PAGE

Samples are prepared and electrophoresed as described in Protocol 11. Electrotransfer

The proteins separated by SDS-PAGE are transferred to nitrocellulose as described in Protocol 11. Immunodetection

DNP-derivatized proteins are detected on the nitrocellulose membrane as described in Protocol 11 except the primary antibody is a 1:200 dilution of rabbit anti-DNP antibodies (Sigma Chemical Co.). Protocol 12 w^as adapted from Wojtkov^iak et aL w^ho used a similar procedure to react the proteins w^ith DNFB after the electrophoresed proteins w^ere bound to a nitrocellulose membrane.^^^ A disadvantage to derivatization prior to SDS-PAGE is the potential of altering the migration of some proteins or causing aggregation that prohibits protein entry into the gel. This potential problem is avoided w^ith postelectroblotting derivatization using the method of Wojtkov^iak et aL A similar procedure using other reagents is described in detail by Kittler et al}^^ Hi. Immunoligand Assay

In addition to ELISA and Western blots for detecting host-cell protein impurities there is a third immunoassay nov^ available to the bioanalyst for this purpose: the immunoligand assay (ILA). Like the ELISA, it is configured as a double-antibody sandv^ich. The anti-host-cell protein antibodies are separately conjugated to biotin and to fluorescein. A tripartite immune complex is formed betvvreen host-cell protein impurities and these two anti-

PROTEIN IMPURITY ANALYSIS

53

bodies. These immune complexes are then trapped on a biotinylated nitrocellulose membrane by the action of streptavidin. Subsequently, the complex is incubated with an anti-fluorescein-urease conjugate. The urease hydrolyzes urea to ammonia in a volume of 0.5 fiL and the resulting pH change alters the surface potential, which is then measured by a silicon sensor.^^^'^^^ Reportedly, this technology can detect as little as 0.05 ppm of host protein impurities in recombinant biopharmaceuticals.^^'^ This procedure has been used to measure host protein impurities both in recombinant human erythropoietin derived from mammalian cells^^^ and recombinant bovine somatotropin synthesized in £. coli}^^ iv. Detection in Immunoassays

The secondary antibody in immunoassays is conjugated to a moiety capable of eliciting a detectable signal. There are three different types of signals that can be elicited: isotopic, colorimetric, and chemiluminescent. Isotopic signals are derived from labeling the antibody with radioactive iodine. The use of this method is on the decline because of the difficulty working with radioactive hazards and the short shelf life of the reagent due to radioactive decay. Nevertheless, there may be instances where isotopic detection is desirable. The interested reader is directed to references at the end of the chapter describing the preparation of radioactive reagents.^^^"^^^ Chemiluminescence is the production of light from a chemical reaction. The emitted light is detected with either a luminometer or on photographic film. There are several substrates capable of producing these light emissions and luminol (5-amino-2, 3-dihydro-l, 4-phthalazinedione) and adamantyl 1,2-dioxetane aryl phosphate are two of the most popular.^^"^'^^^ The former is used with horseradish peroxidase (HRP) coupled antibodies and the latter is used with alkaline phosphatase (AP)-coupled antibodies. Using an AP-coupled antibody and adamantyl 1, 2-dioxetane aryl phosphate, Bronstein et al}^^ were able to detect as little as 125 pg of protein in a Western blot. Colorimetric detection is the most widely used method of detection in immunoassays. It is based on the action of an antibody-coupled enzyme on substrates to produce a colored product. The two most common enzymes used are HRP and AP. Of these two, HRP has more serious disadvantages. HRP activity is inhibited by azide, certain substrates are suspect carcinogens (e.g., 3,3'-diaminobenzidine), and the results with some substrates on blots can fade with time. HRP activity and sensitivity are also strongly dependent both on the concentration of hydrogen peroxide, which itself is quite unstable, and the pH. AP, on the other hand, is not inhibited by azide but it is inhibited by free phosphate ions. AP-antibody conjugates display excellent stability over time. The color development of the AP substrate p-nitrophenyl phosphate is linear over time. It is stable, nonhazardous, and obtainable in a tablet form to minimize handling. The AP substrate BCIP produces a colored precipitate on blots that does not fade appreciably with time. Many AP-antibody conjugates are commercially available for Western blots. However for the host-cell protein ELISA described in Protocol 10, the analyst has to prepare their own AP conjugate. Two methods are generally employed to create these conjugates. One method utilizes glutaraldehyde and can lead to a

54

DONALD O. O'KEEFE

high molecular weight crosslinked product.^^^ A second method utilizes a heterobifunctional maleimide that produces a better-defined product lacking undesirable crosslinking. A procedure for this method adapted from Ishikawa et al}^^ is described in Protocol 13. Protoco/ / 3 : Conjugation of Alkaline Phosphatase to Antibodies

• Dissolve 8 mg of calf intestinal alkaline phosphatase (CIP) in 1.0 mL of 50 m M Na-borate, pH 7.6, containing 1 m M MgCl2 and 1 m M ZnCl2. • Prepare 80 m M N-succinimidyl 4-(N-maleimidomethyl) cyclohexane1-carboxylate (Pierce Chemical Company) in N,N'-dimethylformamide. • Add 50 IJLL of the maleimide reagent to 1.0 mL of the CIP and

incubate for 1 hr at 30°C with constant moderate agitation. • Separate the derivatized CIP from the reagent by desalting on a Sephadex G-25 column equiUbrated in 100 m M Tris-HCl, pH 7.0, containing 1 m M MgCl2, and 0.1 m M ZnCl2. • Concentrate the maleimide-CIP to 2 mg/mL. • Add 0.5 mL (1 mg) of the maleimide-CIP to 0.5 mL (9.2 mg/mL) of purified antibody Fab' fragments (see reference 117 for the preparation of Fab' fragments) in 50 m M Na-acetate, pH 5.0, and incubate for 20 hr at 4°C. • Stop the reaction by adding 2-mercaptoethylamine to 1 m M and incubating for 20 min at room temperature. • Separate the conjugate from the uncoupled proteins by chromatography on Sephadex G-200 equilibrated in 10 m M Tris-HCl, pH 7.0, 100 m M NaCl, 1 m M MgCl2, 0.1 m M ZnCl2, and 0.02% NaN^, Pool the fractions containing the conjugate and add bovine serum albumin to 1 m g / m L and store at 4°C. • The conjugated CIP-antibody can also be chromatographed on the affinity column of the host-cell protein reference standard to eliminate antibody conjugates that may have lost their binding activity in the coupling procedure.

E. Identification of Host-Cell Protein Impurities

Host-cell protein ELISA have the advantage of quantitating host protein impurities. The disadvantage, hov^ever, is that the quantitation is of a group of impurities. Western blot analysis, on the other hand, provides the analyst w^ith a relative level of an individual impurity compared to other impurities. If the level of one or more host protein impurities appears to be excessive based on the intended use of the drug product then it may be necessary to identify those impurities. This can provide assurance that the impurity is innocuous and it can also define the physicochemical properties of the impurity such that the process can be modified to reduce its presence in future production lots. The identification can also lead to the development of a quantitative assay for monitoring the individual impurity in every lot. For the majority of host-cell protein impurities, the most direct w^ay of identification is through N-terminal sequence analysis. The automated

PROTEIN IMPURITY ANALYSIS

55

gas-liquid protein sequencer can routinely determine the N-terminal sequence of as little as 10 pmole of protein. For a 50 kDa protein this is equivalent to 50 ng, which is 50 ppm for 1 mg of drug product. The isolated impurity is first immobilized on a glass fiber disk followed by repetitive rounds of Edman degradation. The released PTH amino acids are separated by RP-HPLC and identified through the use of an amino acid standard.^^^ Two criteria determine the steps required prior to N-terminal sequence analysis: the level of the impurity and the ability to separate it from the drug product. A high-level impurity that is separable from the drug product by SDS-PAGE can be sequenced directly from a membrane after electroblotting. The procedure is similar to that used for preparing membranes for Western blot analysis but there are critical differences. The electrotransfer buffer is instead 10 m M 3-[cyclohexylamino]-l-propanesulfonic acid (CAPS), pH 11.0, with 10% methanol. The glycine and Tris in the Western transfer buffer would cause high background readings during sequencing. SDS can be added to this buffer as before to facilitate protein transfer. The membrane used is PVDF instead of nitrocellulose because of higher protein binding capacity and superior chemical resistance.^^^ Other types of membranes have also been used; notable is carboxymethylcellulose membranes for more basic proteins.^^^ After SDS-PAGE, the migration of the drug product is determined so that the recombinant protein can be excised from the gel. Since the drug product will be in great excess over any impurity, it is likely that the recombinant protein will overwhelm the membrane after transfer and its sequence will be detected everywhere.^^ The recombinant protein is most easily removed by cutting off the extreme ends of the gel and staining the gel slices with Coomassie Blue to identify its location, and then excising the unstained protein from the gel. Protocol 14: Isolation of Host-Cell Protein Impurities by Electroblotting SDS-PAGE

• Samples are prepared and electrophoresed as described in Protocol 11. Electrotransfer

• The ends of the electrophoresed gel are removed with a scalpel and stained with Coomassie Brilliant Blue R-250 (1 m g / m L in 50% methanol-10% acetic acid). Staining for 10-20 min should be sufficient to see the recombinant protein. If necessary, the gel strips are destained briefly with 50% methanol-10% acetic acid. • The identified region containing the recombinant protein is excised from the gel using a scalpel and discarded. • The electrophoresed gel is then washed in transfer buffer (10 m M CAPS, pH 11.0, 10% methanol) for 10 min to remove protein not within the gel and to wash away Tris and glycine. • A piece of PVDF membrane (ProBlott, Applied Biosystems) is cut to the size of the gel and equilibrated first in methanol and then in transfer buffer for 5-10 min each time. Always wear gloves when handling the PVDF membrane to reduce background sequences.

56

DONALD O. O'KEEFE

• The gel is overlaid with the PVDF membrane and any trapped air bubbles are removed. • The gel and the membrane are then sandwiched between sheets of Whatman 3MM paper and the entire assembly is placed in a Genie Electrophoretic Blotter according to the manufacturer's instructions. • The Genie is filled with transfer buffer and connected to the power supply. Transfer is for 1 hr at 24 A with a Schauer battery charger. Detection and N-terminal Sequencing Preparation

• The PVDF membrane is washed in deionized water and then stained briefly with Coomassie Brilliant Blue R-250 (1 m g / m L in 50% methanol-10% acetic acid) to the point where the protein impurity is visualized but not more than 5-10 min. Alternatively, the membrane is stained with Amido Black or Ponceau S. Destain the membrane with 50% methanol-10% acetic acid as much as possible and then let the membrane air dry. If the impurity is not visualized, then end slices of the PVDF membrane are immunodetected as in Protocol 11 to localize it. • The stained impurity is cut from the membrane on as small a portion as possible. • The membrane strip is cut into small fragments and placed directly into the sequenator for analysis. If no sequence is revealed by the preceding procedure, then it may be because the sequence is blocked at the N terminus or the protein level is too low. Proteins that are blocked at the N terminus can be enzymatically digested on the membrane, the peptides isolated, and an internal sequence can be determined.^^^ If the protein level is too low, then the impurity might not be amenable to the blotting approach for sequence analysis discussed earlier. In these instances, the protein must to be isolated from upstream process intermediates to obtain a fraction enriched in the impurity. This is done by first identifying these fractions by Western blot analysis and then chromatographically purifying the impurity from these fractions. The identification of an £. coli protein impurity in preparations of recombinant acidic fibroblast growth factor was accomplished in this way (see Section IV).^^ Determination of the 10 N-terminal amino acids is generally sufficient to identify the protein. The sequence obtained is compared to published databases using homology search engines.* However, determination of the N-terminal sequence does not ensure that the protein will be identified. With the recent deciphering of the entire genome of £. coli^^^ any protein impurity sequence found in biopharmaceuticals derived from this recombinant bacterium should be identified. The entire protein sequence can then be used to develop peptide antigens for antibodies useful in future assays of the impurity. Unfortunately, protein databases and genome analyses are less complete for other recombinant hosts such as yeast and mammalian cells, so alternative strategies need to be developed. One alternative method is to perform • An Internet link to protein sequence databases and search engines is found at www.sdsc.edu/ResTools/biotools/biotoolsl9.html and a Hnk to Internet resources for sequence analysis is www.sdsc.edu/ResTools/biotools/biotoolsl.html.

57

PROTEIN IMPURITY ANALYSIS

two-dimensional gel electrophoresis. After staining with an appropriate method, the pi and mass coordinates of the impurity are compared to those of two-dimensional gel databases from the host organism.* If the corresponding protein in the database is known, then specific antibodies can be obtained to identify and quantitate that impurity.

III. EXPERIMENTAL SUMMARY Numerous methods exist for determining the protein purity and impurity profile of biopharmaceuticals derived from recombinant DNA. These methods are both qualitative and quantitative in nature and span four different types of analytical technologies. The reasonable detection limits for protein impurities via these technologies are presented in Table 2. No single method or technology is sufficient to give a complete purity assessment or impurity profile for any particular recombinant therapeutic. Instead, several methodologies must be utilized orthogonally, i.e., those that separate and detect protein impurities based on different physicochemical properties. A prudent approach would be to utilize RP-HPLC, CZE, and an immunoassay. This tripartite attack would resolve protein impurities based on hydrophobicity, charge-to-mass ratio, and antibody recognition, respectively. With validated assays in each of these disciplines, it is somewhat unlikely that any detectable impurity would not be resolved from the protein drug product or other impurities. However, impurities below the sensitivity limits will remain undetected. The tripartite strategy proposed is not in complete alignment with that currently recommended by regulatory agencies, many of which still advocate silver staining of polyacrylamide gels as an indispensable and sensitive impurity test. Nevertheless, it is the author's opinion that this will eventually change. As the proposed technologies are developed further, as their usage becomes more widespread, and as their sensitivities become greater and differ * Two-dimensional gel databases are located on the Internet. One for 5. cervisiae is found at www.proteome.com and one for both S. cervisiae and £. coli is found at http://expasy.hcuge.ch/ch2d/.

T A B L E 2 Approximate Protein Impurity Detection Limits for C o m m o n Bioanaiytical Techniques Impurity level Technique SDS-PAGE RP-HPLC CE Immunoassay

%(w/w)

ppm

1 0.04 0.04 < 0.001

10,000 400 400
(15)

where V^ ^ is the volume of the mobile phase m, adsorbed to the sorbent, j8 is the activity coefficient of the sorbent, i^^abs,/ i^ the ratio (mole P-/g sorbent)/(mole P-/mL mobile phase), e^ is the solvent strength parameter of the mobile phase, SQ is the adsorption energy of P^ for a standard sorbent where j8 = 1 from a standard mobile phase where e^ = 0, and A- is the molecular surface area of V-. The variables V^ • and ^ represent characteristic properties of sorbents that predominantly function via hydrogen bond effects. These properties will vary markedly with the amount of adsorbed water or the effective "hydroxylated" or "hydrophilated" content by bonding the hydrophilic ligates to the sorbent. If these hydrophilic ligates have no interaction with the biosolutes per se, but only with the water in the mobile phase, then these chromatographic materials fulfill some of the desirable properties of an ideal HP-SEC sorbent.

92

MILTON T. W. HEARN

In size-exclusion (gel permeation) HPLC separations, the retention can be described in terms of volume units (V^ ^) such that V.,, = Vo + K,,,V,

(16)

where VQ is the void volume and Vp is the total pore volume of the column, and Kp^i is the permeation coefficient of P^, which is assumed in ideal cases to be 0 < Kp^^ < 1. However, when hydrogen bonding effects occur in HP-SEC, then displaced dependencies arise, with K^ • values > 1. Because V^ ^ is related empirically to the logarithm of the molecular weight of a polypeptide or protein [i.e., to In(M^)] over a suitable range of molecular weights, the selectivity in HP-SEC between two proteins or polypeptides P^ and P^ will be controlled by the slope w^ y, expressed by A log MW

Alternative expressions linking V^ ^ to M^ have been developed,"^^ based on the relationship between the radius of gyration R and the molecular mass M^, i.e., Rg cc M", where a = 1 for prolate or rodlike proteins, a = 1/2 for flexible coiled coils, and a = 1/3 for spherical proteins. For a compact globular protein, the hydrodynamic volume, Vf^ can be calculated from

where 17 is the intrinsic viscosity, expressed as the volume of molecules per unit mass; N is Avogadro's number, and v is the Simhas factor. For spherical globular proteins, v = 2.5, while for ellipsoidal proteins v > 2.5. Moreover, according to Tanford,^^ V^ = 1.5444M^, while the Stokes radius of compact globular protein is given by R — 0.81 X My^. Thus, application of the modified Himmel-Squire dependency''^ and related relationships'"^ enables a useful alternative expression to be employed for the characterization of HP-SEC selectivities, namely, -b =

— = ^^^— dlnR dlnM'-''

(19) ^ ^

where Kp^^^ a-

felnM,

(20)

and a/b is related to the pore size distribution of the HP-SEC sorbent, while the slope of the selectivity curve b depends on the pore size distribution. The molecular dimensions of most globular and structural proteins can be readily described in terms of their major elliptical or major-minor prolate axes.'^^''^^ Thus, the major elliptical axis of many globular proteins falls in the range of 50-500 A, while for prolate proteins, e.g., [Q:I]2a!2 collagen, the major and minor axes fall in the ranges 2200-3000 and 120-140 A, respectively. This

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

93

means that most globular proteins with molecular masses up to 7 X 10^ Da can be successfully separated by HP-SEC sorbents with pore sizes greater than 500 A but below '^ 2000 A. Because hydrogen bonding and hydrophobic interaction effects can give rise to anomalous retention behavior for some polypeptides or proteins with HP-SEC sorbents, a common practice is to add a small amount of a suitable organic solvent to the mobile phase in these cases. C. Electrostatic Interactions Coulombic or electrostatic interactions are, like hydrogen bonds, of polar origin. The interactions occur between charged or ionizable groups, which form part of the immobilized ligate, or alternatively are an inherent component of the stationary phase and the complementary charge groups accessible at the surface of the biosolute. Many different types of electrostatic ligates are available^"^ for use as HP-IEX systems, ranging from the familiar (CH3CH2)2N-, (CH3)3N+-, -CH2SO3H, and - C H 2 C O O H moieties to the more exotic malachite green carbinol ligates, such as ((Me)2N-C^H4)2 (NH2-C5H4)COH, which in general are very strong anion exchangers as the corresponding immobilized carbonium species^^ at neutral or slightly acidic pH values. Electrostatic interactions between oppositely charged species are o

effective over distances of the order of 100 A. In the simplest case, the magnitude of the electrostatic force F-^^^ between two point charges Q^ and Q25 separated by a distance r, in a medium of dielectric constant e^ is given by ^iex = — - T

(21)

The amphoteric nature of polypeptides and proteins significantly complicates their treatment as single-charge-state species. The presence of multiple charge states can nevertheless be addressed through the use of average distribution coefficients and weighted mole fractions of individual charge species. Empirically, the dependency of the equiHbrium association constant ^assoc, / o^ ^^^ concentration of the displacing ion can be described in terms of nonmechanistic, stoichiometric models, whereby the mass distribution of the protein, P, of charge state + x is given by

( P ± ^ ) ^ + {x/y)iD^y),

^ ( P ± - ) , + (x/y)iD^y)„

(22)

where D - ^ is the displacing salt counter ion, and the subscripts m and s refer to the mobile and the stationary phases, respectively. The equilibrium association constant i^assoc, i ^^^ ^he ion-exchange process can thus be represented as

94

MILTON T. W. HEARN

where 7.^ represents the ratio ( x / y ) of the effective charge on the polypeptide or protein to that on the displacer counter ion. When near-equihbrium conditions prevail for the chromatographic distribution process, i.e., v^hen T, P, V, O, and the flow^ rate V are constant, and the adsorption process approximates a linear (Langmuirean) isotherm, then this dependency of ^assoc, i ^^d hence the logarithm of the corresponding capacity factor In ¥.\^^on [ D - ^ ] ^ can be v^ritten in terms of a Taylor series, similar to that employed for RP-HPLC or HP-HIC separations, as follow^s:

In^L,,, = a + j8(ln[l/C]) + y ( l n [ l / C ] ) ' + 5 ( l n [ l / C ] ) ' + -

(24)

v^here C is the concentration of the displacing ion, i.e., [ D - ^ ] ^ , and a , j8, y, 6 , . . . , are coefficients dependent on the solubility parameter of the solute, 6-; the zeta potential ^ of the stationary phase: the mobile phase buffer composition, pH, polarizability, and dielectric properties. Over a narrow range of mobile phase compositions, this relationship has often be approximated to a linear dependency, i.e., given in the familiar form lnfe;,,, = lnKai,,, + Z , ( l n [ l / C ] )

(25)

where i^aist,/ i^ ^^e distribution coefficient, a term that includes the Kassoc o the phase ratio O, and the stationary phase electrostatic ligate concentration [ L - ^] in the following manner: jjr _ ^dist,/ -

^assoc,/^L^ -—

J

{'\r\ \^^)

where the constants z^ and z^ adjust for the valency of the solute and the salt species and ZQ is the theoretical maximum number of charges on the protein surface associated with the adsorption process. Thus under linear elution conditions, the slope coefficient Z^ and In K^^^^^ can be determined from the plot of In k[^^i versus l n [ l / c ] . Note, however, that Z^ reflects the apparent number of ionic charges associated with the adsorption of the polypeptide or protein at the Coulombic ligate surface, and is not formally equivalent in mathematical or physicochemical terms to ZQ or Z^. Moreover, curvilinear, rather than linear, plots are more likely to occur for these Infe-^^^ versus l n [ l / C ] dependencies due to the anisotropic nature of the charge distribution on the polypeptides or proteins and the involvement of secondary binding processes, mediated for example by hydrophobic effects. Figure 5 illustrates'^^'^'^ such a case with the plots of In k[^^j versus l n [ l / C ] for the seven proteins using the strong anion exchanger, MonoQ, with the buffer system of 20 m M piperazine, pH 9.6, containing different concentrations of the displacing salt, NaCl. As is apparent for proteins 1 to 5 in Fig. 5, the expected ion exchange adsorption-desorption behavior is clearly evident, while for proteins 6 and 7 ion exclusion effects prevail, consistent with their charge and pi characteristics. Because polypeptides and proteins are amphoteric, Z^ is also expected to show nonlinear dependencies on pH. This behavior has been observed^^'^^

95

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

ln[l/[NaCl]] F I G U R E 5 Plots of Ink,' versus In [I / C ] for seven proteins using the strong anion exchanger, Mono-Q, with the buffer system 20 m M piperazine containing the displacing salt, NaCI. These plots were derived from isocratic measurements using various concentrations of NaCI at pH 9.6 and a flow rate of 1.0 m L / m i n . The protein key is as follows: /, human albumin, pi = 5.85, hA^ — 69,000; 2, hen e^ white ovalbumin, pi 4.70, M,. = 43,500; 3, bovine hemoglobin, pi = 6.80, M,. = 64,500; 4, bovine erythrocyte carbonic anhydrase, pi = 5.89, M,. = 30,000; 5, sperm whale muscle myoglobin, pi = 7.68, M,. = 17,500; 6, hen Q^ white lysozyme, pi = 11.0, M,. = 14,300; 7, horse heart cytochrome c, pi = 9.4, hA^ = 12,400. Data selected from Ref. 76.

for various globular proteins with both strong anion and strong cation HP-IEX sorbents. As a consequence, the minima in the In k[^^ versus pH plots at a defined concentration of displacing salt will not usually occur at the pi value of the polypeptide or protein, but rather at a pH value above or below the pi value. This behavior is a further reflection of the distribution differences of charged groups accessible on the surface of the polypeptide and protein. Thus, the ion-exchange processes of polypeptides and proteins are dependent on the microlocality and extent of ionization of the surface accessible amino acid side chains, or the N- and C-terminal amino and carboxyl groups, respectively, with the retention behavior in the electrostatic modes of HPLC are dependent on the pH of the buffer. This effect can be evaluated for a polypeptide or protein of charge Zi separated under normal HP-IEX conditions, from the dependency

k'

= $

c ([^^' [H:.])1 [[£'..] ^+ ^ a v ii

\

(27)

96

MILTON T. W. HEARN

where [El^^] is the ionic strength of the displacing counterion in the mobile phase, K^^^- is the average distribution coefficient for the various ionized species of the polypeptide or protein, and C is a system constant. Since small changes in pH v^ill result in large changes in k-^^^j, this property can be easily employed in HP-IEX as part of a buffer optimization routine. Unlike other types of interactions, Coulombic forces can be attractive in the case of oppositely charged groups or repulsive in case of identically charged groups at the surface of the interacting molecules. This property can be exploited in charge exclusion effects in some modes of HP-IEX. In various HP-IEX systems, attraction betv^een negatively charged biomacromolecules can also be affected through the use of chelate development with divalent cations like Ca^^, Mg^"^, Cu^"^, Zn^"^, etc., where the interactions can lead to ionic crosslinking processes between the participating biosolutes and the ligate that change the overall selectivity of the system. Thus, the slopes of the In ^'iex,^ versus l n [ l / C ] plots for different salts will not be parallel but be conditional on the polypeptide or protein examined as well as the position in the Hofmeister series where the anion and the cation reside. As noted, the retention of a polypeptide or protein with HP-IEX sorbents primarily arises from electrostatic interactions between the ionized surface of the polypeptide or protein and the charged surface of the HPLC sorbent. Various theoretical models based on empirical relationships or thermodynamic considerations have been used to describe polypeptide and protein retention, and the involvement of the different ions, in HP-IEC under isocratic and gradient elution conditions (cf. Refs.^'^^'^^''*^'"'^"^^). Over a limited range of ionic strength conditions, the following empirical dependencies derived from the stoichiometric retention model can be used to describe the isocratic and gradient elution relationships between the capacity factor In k[^^ I and the corresponding salt concentration [CJ or the median capacity factor In k[^^^, and the median salt concentration [ Q ] of a polypeptide or protein solute, namely, 1 In feU = In i^iex + Z , In - ^

(28)

1 In k,,, = In K,,, + Z , In - ^

(29)

where In K-^^^ is the intercept value of the In k[^^ (or In ~k^^^) versus In ( l / [ CJ) or l n ( l / [ Q ] plots, and is related to the association constant Xassoc ^^^ ^^^ protein-ligand interaction when [CJ -^ 10"^ mol/L (or [ Q ] -> 10"^ mol/L), and Z^ or Z^ represents the slope of the plots derived from the isocratic or gradient data at a defined salt concentration. As expected from molecular surface area arguments, small molecules such as dansyl amino acid derivatives exhibit relatively small Z^ (or Z^) and In Kjgx values, and these values do not change significantly with increasing temperature. Polypeptides and proteins, on the other hand, exhibit much larger Z^ (or Z^) and In K-^^^ values, and these values have a profound temperature sensitivity. Moreover, it is well known that different salts can

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

97

influence the ion-exchange chromatographic behavior of polypeptides or proteins due to the chaotropic or cosmotropic nature of the component cations and anions.^^'^^'^^"^^ The ionic radius and electronegativity of the monovalent and divalent ions can significantly influence the retention behavior of polypeptide or protein w^ith HP-IEX sorbents. Illustrative of these effects are the plots of ^n k[^^ versus [CJ, shown in Fig. 6 for several polypeptides and globular proteins eluted^^ from the cation-exchange HPLC sorbent Lichrospher 1000 SO^ using as the displacing salts CaCl2 and NaCl in the concentrations range 0-1.0 M.

ln[l/ C] F I G U R E 6 Plots of lnk[ versus [I / C ] for a selection of proteins separated on the strong cation exchanger LiChrospher SO3" at 25°C with different concentrations of (a) CaCi2 and (b) NaCI as the displacer salt. Inthese studies, the experinnent data were obtained utilizing linear gradients from 20 to 100 min duration at a flow rate of I m L / min with the buffer system 50 MAI sodium acetate to 50 M M sodium acetate containing I M CaClj or I M NaCI. The protein key is as follows: - • - , angiotensin I; - • - , angiotensin II; - A - , angiotensin III; -w-, arglnlne; - • - , horse heart cytochrome c; - • - , bovine insulin; - ® - , hen egg white lysozyme; - @ - , hen egg white ovalbumin; - A - , bovine pancreatic ribonuclease; - • - , soyabean trypsin inhibitor. Data adapted from Ref. 96

98

MILTON T. W. HEARN

This influence of the valence and activity coefficients of the displacer salt on the retention behavior of polypeptides and proteins can be anticipated from theoretical treatments of the ion-exchange chromatographic separation of proteins. According to the nonmechanistic stoichiometric model of protein retention behavior in HP-IEX^^'^^~^^ the influence of a divalent cation salt such as CaCl2 on the retention behavior of a protein in HP-IEC can be evaluated in terms of the following relationships: As

k'

= K —^

D hi hibiPoCi

(1 - f)

(30)

where K^, A^, and V^ are the equilibrium constant for the interaction of the polypeptide or protein with the ion-exchange sorbent, the accessible surface area of the adsorbent in square meters per gram and the volume of the mobile phase, while z is the number of charge groups on the polypeptide or protein associated with the adsorption and desorption processes, respectively. The term D^- relates to the initial ligand concentration, DQ is the displacing ion concentration in moles per liter and Q is the concentration of counterions associated with the polypeptide or protein, i.e., in the case of cation exchange binding events the concentration of H"^ ions involved with the polypeptide or protein that are substituted by other cations. The relative elutropic strength and activity coefficients of the displacing ions and counterions for the ion-protein interactions and the ion-ligand interactions are represented by the terms a^^ andfc-y,while the fraction of the adsorbent surface covered by the protein following the adsorption interaction is given by the term f. If it is assumed that "near-equilibrium" conditions apply and the amount of the polypeptide or protein loaded onto the HP-IEX sorbent is small, i.e., if only a small percentage of the adsorption capacity ^* is involved in the binding and the polypeptide-ligate or protein-ligate interaction occurs within a linear region of the adsorption isotherm, then the term f ^ 0 and D^^ will remain essentially constant. Under such conditions, the dependence of k[^^ on the concentration of the participating ions can be represented by K. = K^^DI,[a,^b,^D,C.y'

(31)

For a displacing salt with a divalent cation but a monovalent anion, each cation (e.g., Ca^^) will cause the neutralization of two charge group interactions between the polypeptide or protein and the electrostatic ligate, with the concentration of the M^"^ cation DQ (in moles per liter) required to maintain electroneutrality exactly equal to half of the concentration of the accompanying monovalent (e.g., Cl~) counterions, C^ (in moles per liter), associated with the positively charged polypeptide or protein when it is desorbed from the cation exchange HP-IEX sorbent, and hence K.-K,^Di\a,^b,^Dl']~'

(32)

99

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

The logarithmic form of Eq. (32) can be expressed, for the specific illustrative case of CaCl2 as the displacing salt, in the following terms, although analogous expressions can be derived for anion or cation exchange HP-IEX systems with polyvalent ions: In kl^ = In

As

1

+ zln

^,,&,,[Ca2+]

1.5

(33)

or, alternatively. In k[^^ = In K, + zln

«,;^-;

+ 1.52 In

[Ca^-]

(34)

where

K^ =

Ka^DI,

(35)

According to Eqs. (31)-(35), the magnitudes of the slope terms, i.e., the Z^ values, of the isocratically derived In k^^^ versus l n ( l / [ Q ] ) , or the case of gradient derived data In ~k[^^ versus l n ( l / [ Q ] ) , are predicted to be dependent on the valency of the displacing salt. It can also be seen from Eqs. (33)-(35) that the relationship betv^een the slope term Z^ and the effective charge term z can be represented in the form (n X z = Z^) provided the valencies of the displacing ion and counterion are unity, the activity coefficient terms a^^ and bii are independent of the concentrations of the polypeptide or protein, the ligate groups, the displacing ion, the counterion, or the temperature, and the magnitude of the product of a^j and b^^ is close to 1. When the valency n of the displacing ion is > 1, but the counterion remains monovalent, then the relationship depicted by Eqs. (30) and (35) follows the dependency [(w + 1) X z]/n = Z^, again assuming that the value of a^^ Xfc-y-> 1 and the other "ideal" criteria already listed prevail. D. van der Waals Interactions and Weak Polar Interactions van der Waals interactions arise betw^een all atoms that are brought into very close proximity. In these cases, electrodynamic attractions arise betw^een fluctuating dipoles in one atom and other dipoles induced in a neighboring atom. In addition, attractive contributions are generated from permanent dipole-dipole and permanent dipole-induced dipole interactions. Analogous attractive forces are generated betv^een any two macroscopic bodies whose surfaces are separated by very small distances.^^'^^ With weakly polar interactions, segregation of the electronic charges within an aromatic ring gives rise to electron-poor aromatic hydrogens and electron rich 7r-orbitals. This process tends to favor aromatic residues packing edge to face, with a distance dependence of 1/r^. Oxygen and sulphur atoms can also interact with aromatic ring structures via electron-poor hydrogen atoms, with distance dependence oi\/r^ for the uncharged atoms and 1/r^ for the thiolate (S")

I 00

MILTON T. W. HEARN

and carboxylate oxygens. These weakly polar interactions clearly will not represent a significant contribution to a ligand-ligate interaction, or influence the folded status of a polypeptide or protein significantly, although as a secondary contributor, their effects amplified through dipolar interactions with the solvent then become more evident. In the liquid state, the three types of electrodynamic interactions (London, Debye and Keesom dipole interactions) can be treated completely differently from purely macroscopic points of view, in which the interacting bodies are considered as continuous media. The dispersive London forces involve interactions of induced dipoles with a distance dependence of 1/r^. The strength of these forces depends on the polarizabilities of the interacting molecules, with nonpolar atoms such as aliphatic C or H having stronger interactions than polar atoms such as N or O atoms. Moreover, these dispersive forces will favor like groups coming into contact, such as aromatic side chains adopting where possible preferred contacts. These contributions are associated with long-range effects, often called Lifshitz-van der Waals forces or bonds. The energy of Lifshitz-van der Waals interactions decrease monotonically to the distance separating the interacting species (in the configuration of the two parallel slabs). These effects are operative up to 1000 A, in contrast to hydrogen bonds that are effective over only 1.5-5.0 A with an exponential decay of the interaction energy of ^ 1 0 0 A in the case of electrostatic 99

mteractions. E. Metal Ion Coordination Interactions Metal ions can be considered as Lewis acids, with the formation of complexes rationalized in terms of Lewis acid-base interactions. This interaction can be visualized in terms of the ability of a metal atom or ion M to accept a pair of electrons (and thus act as a Lewis acid) from a ligand : X, which is an electron donating base with an accessible lone pair of electrons, i.e., M+ :X^M:X

(36)

The distribution of the electrons as a coordination bond in this Lewis acid-base complex largely dictates the character of the complex, thus the nature of the atoms involved, their ability to form coordination complexes of specific geometry, and their hydration-hydroxylation state in water systems, enable these processes to be categorized^^^ as "hard" or "soft." Typically, a metal ion that acts as a "soft" Lewis acid has outer shell electrons that are easily polarized, low electronegativity characteristics, is easy to oxidize, and contains unshared pairs of electrons in the valence p or d shells. Thus Cu^^ would be considered a "soft" metal ion. In contrast, a metal ion that acts as a "hard" metal ion has a high electronegativity, is difficult to polarize, is hard to oxidize, and contains only high-energy empty orbitals. In this context, Al^"^, Ca^^, and Fe^"^ would all be considered as hard metal ions. The TT-bonding theory of Chatt^^^ adequately rationalizes many of the characteristic features of the coordination interactions of metal ions with polypeptides and proteins. Thus, "soft" metal ions, such as Hg^"^, have a preference for

101

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

"soft" electron donor groups on a polypeptide or protein, such as SH or SMe of Cys or Met residues; borderline metal ions, e.g., Cu^"^, Zn^"^, or Ni^^, have preferences for borderline donor groups associated with Tyr, Trp, or His residues; and "hard" metal ions a preference for "hard" donor groups within Asp, Glu, Lys, and Arg residues, as well as phospho, sulfonato, and sulfate groups. By immobilizing a suitable chelating compound to a support material, immobilized bi- to pentadentate metal ion complexes (IMICs) can be generated as chromatographic sorbents of the type M"'^ CheUH20)^(0U~)j, where m i- j with 0 < m < 4 and 0 < / < 2. The equihbrium association constant, ^u""^ ^cheu often represented as the overall metal ion stability constant j8„, associated with the binding of a metal ion M""^ of valence n"^ to an immobilized monovalent chelating compound CheL can be represented as [M"+ ^he buffer species or added counterion will cause the metal ion to leach from the IMAC sorbent, as commonly observed with many structurally unconstrained bi- and tridentating systems. Metal ion leakage has in fact been one of the major limitations of IMAC procedures with recombinant proteins when structurally unconstrained chelating compounds have been employed to form the metal-ion complexes. This technical facet appears to have now been solved with the development of novel macrocyclic chelating ligand systems with large log j8„ values (see later). The concept of using low molecular weight chelating compounds covalently bound onto chromatographic supports was suggested over 50 years ago by Meinhardt^^^ and subsequently adapted as ligand exchange chromatography to separate metal ions and low molecular weight compounds, predominantly amines, amino acids, mono- and dinucleosides, and nucleotides.^^^'^^"^ The ability of metal ions to selectively bind through coordinative interactions to proteins was turned into practical chromatographic procedure in 1974, when 8-hydroxyquinoline (8-HQ), covalently immobilized to agarose and chelated with Zn^"^ ions, was used to isolate metalloproteins.^^^ Subsequently, Porath and co-workers^^^ recognized the potential of the metal ionbinding properties inherent to immobilized iminodiacetic acid (IDA) and adapted this mode of metal ion interactions with several proteins under the rubic of "immobiHzed metal affinity chromatography (IMAC)." Since then, IDA-based sorbents have been widely employed by many investigators, resulting in this mode of biomimetic chromatography becoming a viable experimental approach for the purification of globular, structural, and membrane proteins at the laboratory scale. In common with many tridentating ligands, significant leakage of border line metal ions occurs under mild elution conditions in HPLC processes with im-M"^-lDA complexes due to their relatively low metal ion stability constants (i.e., 7 < log j8n < 10.5^^^"^^^). As noted earlier, a large number of proteins are, in addition, able to strip metal ions from /m-M"'^-IDA complexes.^^ ^'^^^ To circumvent metal ion leakage, other chelating ligands have been investigated, including ^ns(carboxymethyl)-ethylenediamine (TED),^^^'^^^ a pentadentate ligand that coordinates metal ions via two nitrogen atoms of the secondary amino groups and three oxygen atoms from the three car boxy 1 groups. When Ni^"^ and other M^"^ ions are chelated to the /m-TED ligand, only one vacant coordination site remains available within the coordination sphere of the metal ion for interaction with donor groups on a polypeptide or protein, while three coordination site are available with the corresponding im-Ni^^-IDA complex. The /m-Ni^^-TED complex, therefore, has weaker affinity for proteins, i.e., lower Kp^ value, than the corresponding im-Ni^^-lDA sorbent, although the propensity for metal ion leakage is reduced under comparable elution conditions because of the higher Kj^.^ « chei value. Other chelating ligands have been identified with more optimal association constants for protein binding as well as lower metal ion leakage per se. The combination of hard and soft Lewis acid-base interactions^^^'^^"^ between the acceptor metal ion and donor N - or O-groups of the protein or

103

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

pseudo-cation exchange properties has been of particular interest. Thus, bidentate chelators, e.g., aminohydroxymates (AHM)^^^ or 8-HQs^^^'^^^; tridentate ligands, e.g., O-phospho-serine (OPS), ^^'^ cis-/trans-C2irhoxymethylproline (CMP),^^^ diaminomethyl-pyridine (DAMP)^^^ or dipicolylamine (DPA)^^^; tetradentate ligands, e.g., nitrilotriacetic acid (NTA)^^^'^^^ or carboxy-methylated aspartic acid (CM-ASP)^^^; and pentadentate ligands, e.g., TED or tetraethylene-pentamine (TEPA),^^"^ have all been examined for their potential use in IMAC applications with proteins. Figure 7 illustrates several of the structures of several of these HP-IMAC ligates. Typically, these additional classes of immobilized metal ion-chelating complexes (IMICs) have shown only modest increases in metal ion stability constants (i.e., in the range of 9 < log j8„ < 14), although nevertheless they offer alternative separation options. Recently, a totally different class of IMAC sorbents have been derived from 1,4,7-triazacyclononane (tacn), a macrocyclic chelating ligand, and used in various applications for the purification of human serum proteins and other wild-type proteins.^^^"^^^ Compared to other tri- or tetradentating chelating ligands, these new IMAC systems have much larger stability constants for many borderline metal ions, with values typically higher by at least 4 to 10 orders of magnitude, i.e., in the range of 16 < log j8„ < 30.^^^"^^^ The homologues, fc/s(l,4,7-triazacyclononyl)ethane (dtne) and fc/s(l,4,7-triaza-cyclononyl)-propane (dtnp), involve two tacn macrocyclic rings linked via either an ethyl or a propyl bridge, respectively. Consequently, dtne and dtnp can form two metal ion-binding centers of the type MN3 separated by

OH,

CH^"CO i OH,

(I) CO

CH,-CO

:.^-"^ g ^'--'-

CH

9^2

B t - - -.-.•iOH2

-i^

F I G U R E 7 Representative examples of the structures of different H P - I M A C ligate systems containing a borderline or soft metal metal ion M, such as Ni^"*" or Cu^"*": (I), iminodiacetic acid (IDA); (II), tr/s(carboxymethyl)ethylene-diamine (TED); (III), nitrilotriacetic acid (NTA); and (IV) c/s-trons-carboxymethylproline (CMP).

104

MILTON T. W. HEARN

between 5.6 and 6.8A or alternatively a sandwich-like structure of the type MN6.^^^'^^^ Because of their very high log P„ values, metal ion dissociation does not complicate the solution chemistry of these im-M^^-[tacn]2 species in the presence of proteins. In addition, the spatial orientation of these ligands potentially enables the formation with proteins of either monomeric coordination complexes of the type im-M^^ [ tacn]2-AA [where AA represents a suitable electron donor group of participating amino acid residue(s) of the incoming protein] or alternatively dimeric coordination complexes of the type im-M^^-[tacn]2-[AA]2. Depending on the electronic and steric properties of the functional groups of the participating amino acid residue(s) of the protein and the J-orbital characteristics of the M"^ ion, the protein-ligate interaction with IMAC sorbents derived from dtne and dtnp can involve one or both of the im-M^^-[tacn]2 moieties. Figure 8 illustrates the structures of

[CR,RJ

F I G U R E 8 Representative structures for the im-M"'^-bis-[tacn] ligate in the sandwich MN6 coordination stereochemistry (A) (where M represents a first row or subsequently row transition metal ion) and in the extended MN3 core stereochemistry (D) in the presence of a N - or O - donor group, i.e., an electron-donating nitrogen, oxygen, or sulfur atom within a side chain of a participating amino acid residue of a protein. The relative equilibrium association constant of the protein — im-M"'^-bis-[tacn] complex will depend on the properties of the metal ion, the participating donor solvent, and the size, shape, and the nature of the electron donor group(s) or other surface characteristics of the participating protein. For the Cu^"*" ion, a more extended structure (C) is favored'^'''^° compared to the case of the Ni^"*" ion, where the more compact, sandwich structure (B)I29.I30

105

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

1.5

H 15

in

o^ o

18

300 H • •«—GST-5ATPase-His,

12

1.0

9

o

6
i.o» o^®"" ^^^ rc\o\Q fraction range of 0 to 1.0 (cf. Eqs. 6 - I I ) . As the contact area and the retention factor k\ of the polypeptide or protein increase, the slope of the plot of In k\ versus ^/=o-> i.o increases, resulting in a narrowing of the elution window over which the biosolute can be desorbed from the HPLC sorbent. Cases (a) and (b) are typically observed in RP-HPLC, H P - H I C , H P - I E X , and the other interactive modes of HPLC with small peptides and globular proteins, while cases (c) and (d) are more representative of the behavior evident with more hydrophobic polypeptides, membrane proteins or other nonglobular proteins.

I 30

MILTON T. W. HEARN

commonly observed situation with small peptides separated by RP-HPLC or polar polypeptides and some small proteins in HP-IEX, where relatively small free-energy changes arise over the range of elution strength employed/^'^^'14^'^^4-^^^ Shallow dependencies in terms of the In fe^ versus ^ (or l o g l / [ C J ) dependencies can also be observed under some conditions with more retentive biosolutes, but in this case much larger values of In k'^ are the norm. Such a situation is illustrated by case c, which is representative of the behavior of middle molecular weight proteins and very hydrophobic polypeptides under some RP-HPLC conditions; in HP-IMAC with histidine-rich polypeptides or proteins^^^'^^^; in substrate-analogue affinity displacement HP-IEX of proteins^^^"^^^ where the displacing ionic species or substrate-analogue is typically of low molecular weight. Some examples of polypeptide or protein displacement RP-HPLC or HP-HIC^^^'^^^ also fall within the boundary of case c. Because the ^Q, /? ^'z P • • • ? terms are large with case c biosolutes, small changes in a secondary mobile phase component or condition, e.g., pH or salt type, can lead to significant secondary retention effects. When such behavior is evident, the limiting chromatographic conditions are frequently chosen from practical considerations so that the minimum of the plot of In k] versus ^ (or l n [ l / C j ) corresponds to k'^ values equal to unity. Typically, this criterion is easier to achieve in HP-IEX than RP-HPLC or HP-HIC separations, although sufficient empirical information is now at hand to enable sensible choices to be made for these latter HPLC modes. At this stage of development, insufficient data have been accumulated to enable similar a priori estimations of suitable ranges of ^ to achieve a particular In k] value in the other modes of HPLC, such as HP-IMAC, HP-BMC, etc. In situations associated with the purification of large globular multisubunit proteins or hydrophobic membrane proteins, retention behavior typified by cases a-c with smaller globular proteins and polypeptides in the R P - , IEX-, or BAC-HPLC modes are rarely seen. With the more hydrophobic protein classes retention dependencies approaching case d are often observed, resulting in a narrow desorption window, with evidence of significant secondary high-affinity sorption effects and associated poor mass balance and recovery of bioactivity. Illustrative examples of these different types of retention dependencies are shown in Figs. 2 and 15 as plots of the experimentally determined In fe^ (proportional to AGfsso^ ^) versus the volume fraction of organic solvent if/ for the several hormonal polypeptides and lower molecular weight amino acid derivatives in the presence of an «-octylsilica sorbent, which clearly demonstrate this behavior. From the perspective of a generalized analytical or preparative purification strategy, it is obviously desirable to select chromatographic conditions in which the In k'^ at ^ (or ln(l/[C-])) retention dependencies approximate case a or case b, rather than cases c and d. For the latter two cases the i^assoc, i ^^ the biosolute for the stationary phase is obviously too high, the desorption window suitable for elution is too narrow, and the mass (or bioactivity) recovery potentially is at risk. However, with crude feed stocks implementation of the case a or d scenario should not necessarily be excluded out of hand from a selectivity point of view. For example, situations have been

131

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

0.5 0.0 -0.5

-

-1.0

p-Endorphin 0.26

0.28

0.30

+

0.26

Glucagon 0.28

+

0.30

+

0.32

Volume Fraction, y/ F I G U R E 15 Illustrative examples of the retention dependencies for the polypeptide hormones, j8-endorphin and glucagon, as plots of the experimentally determined In k\ (proportional to AG^jj^^ ,) versus the volume fraction of organic solvent i// measured with a n-butylsilica reversed-phase sorbent at temperatures from 5° to 85°C under isocratic elution conditions at a flow rate of I mL/mIn, encompassing the range of acetonitrile concentrations from 0.25 < \p < 0.30. Data from Ref. 237.

identified where the potential offered by the case a and d scenarios can be exploited, such as the removal of undesirable contaminants during the purification of therapeutic proteins by taking advantage of the so-called "negative-adsorption", "roll-over" adsorption and "feedstock" displacement strategies.^^^'^^"^'^^"^"^^^ For example, in this laboratory, useful adaptations of these approaches have been developed for the removal of trace components of Hageman factor and associated plasminogen activator-prekallikreinrelated proteins from therapeutic grade human immunoglobulins based on a tandem dye-affinity and anion-exchange HPLC method, and similar procedures based on these concepts have been employed^^^ in the fractionation of human a^-plasminogen inhibitor. Gradient or step elution procedures represent the two most commonly adopted methods for the elution of biopolymers from adsorptive HPLC sorbents. These choices are often dictated because of the pronounced dependencies of retention and zone broadening phenomena on the chromatographic conditions. In gradient elution procedures, advantage can be taken of the severity of the In ^^ at ^ (or I n ( l / [ C J ) ) retention dependencies. However, in common with elution carried out under isocratic conditions, these procedures do not necessarily address the important requirements mandated by the conformational dynamics or the desorption kinetics of the biosolute. Significant progress has been made over the past 15 y^^^^i,6,17,32,168,191,208,210,237,238 j ^ ^^^ application and interpretation of the gradient elution data for the simulation of the retention behaviour of polypeptides and proteins in R P - and lEX-HPLC systems. Furthermore, it is often feasible in circumstances of so-called "regular" elution behavior, e.g., with polypeptides that satisfy the case b scenario above, or small globular proteins (case a) to apply experimental data derived^^^'^^^'-^^^'^^^ from analytical, small-scale experiments as normalized integrals of the elution volume.

132

MILTON T. W. HEARN

with the column performance characteristic optimized using computer-assisted techniques, algorithmically based on the use of BioCAD, ProSys, instrumentation, etc., to scale-up the chromatographic bed configuration and to make informed choices of the physical characteristics of the separation media, to achieve appropriate process-scale purification levels. For packed-bed systems, the extent of chromatographic zone broadening of a biosolute can be discussed in terms of the column efficiency, v^hich usually is expressed as the number of theoretical plates N^ or the height equivalent of a theoretical plate H^ • (where H^ ^ value = L/N- and L is the column length). The N- value is thus dependent on a variety of solute and chromatographic parameters including the diffusivities D^f ^, I^sp,/5 ^int,/? D^ ^, and D^ • of the solute within the stagnant film layer, the pore environment, the surface interactive regions of the stationary phase, or the bulk mobile phase, respectively; the column length L; particle diameter dp\ and linear flow velocity u- (equivalent to L/t^J)^ respectively. The theoretical plate number N-, of a solute P^ in a packed bed column can thus be defined in terms of the retention time t^ ^; the peak variance cr/^ of the eluted zone in time units; the peak width at baseline response ^^ ^; the capacity factor k\\ and the column dead time ^Q? according to

N.

= 16

16

(1 + k\)

(62)

When the eluted peak zones assume a Gaussian distribution, the peak width at baseline for a particular biosolute ^^ ^ approximately corresponds to 4cr- (4 X standard deviation), although in reality only ~ 95% of the true peak area of a Gaussian peak will be integrated using this approximation. A major task of current practice with HPLC techniques is to select experimental conditions that maximize the N^ value or minimize the H^ ^ value. To achieve this outcome, it is essential that control is maintained over the different mass transport processes that characterize the zone broadening of a particular biosolute in a packed bed of defined column configuration, flow rate, temperature, sorbent type, or eluent composition. The interplay of these different mass transport processes, which include (i) eddy diffusion, (ii) mobile phase mass transfer, (iii) longitudinal molecular diffusion, (iv) stagnant mobile phase mass transfer, and (v) stationary phase mass transfer (see Fig. 16) have posed some of the more significant challenges to the separation scientists over the past two decades. Nevertheless, many of these challenges have been addressed, initially with the development of well-packed columns containing particles of more uniform size and pore diameter distributions, and subsequently with the development of improved procedures for the chemical modification of the surfaces of the particles. However, with biomacromolecules, some but not all of these band-broadening processes can be controlled from a practical standpoint by the quality of the column bed-packing procedures or the use of small particles of narrow particle size and pore size distributions. Compared to low molecular weight organic analytes, most biopolymers have relatively small effective molecular

133

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Inject Band J^ %%%% Width

@0 ©®

Brownian Motion

• %••• •

Final Band Width

^^ ^^ ^^

Mobile Phase Mass Transfer

i^^^fp Diffusion

t^.

Stagnant Mobile Phase Mass Transfer Stationary Phase Mass Transfer

F I G U R E 16 Schematic representation of the origins of zone-broadening behavior and mass transfer effects of a polypeptide or protein due to Brownian motion, eddy diffusion, mobile phase mass transfer, stagnant fluid mass transfer, and stationary-phase interaction transfer as the polypeptide or protein migrated through a column packed with porous particles of an interactive HPLC sorbent.

diffusivities in HPLC systems. As a consequence, the major problems experienced in the separation of biomacromolecules in the adsorption modes of HPLC as low efficiencies, i.e., small N- or large H^ ^ values, can invariably be traced to inadequate control over three mass transfer aspects, namely, the stagnant mobile phase mass transfer, the film diffusion component, and the stationary phase mass transfer kinetics. Since these kinetics are associated with the diffusive, convective, and perfusive components of the mass transport events, improvements in the physical characteristics of the sorbent and better control over the extent of heterogeneity of the ligates on the surface of the sorbent are essential requirements. In the extreme case of sorbents with very large pores (i.e., with 2000 A < p^ < 5000 A) convective flow conditions can in principle be employed with very high linear flow velocities without sacrificing column capacity and biosolute retention. The development of sorbents with interconnecting macro- or even gigaporous structures^"*^"^"*^ (i.e., with p^ up to 10,000 A) as part of the physical architecture of the sorbent has provided one possible solution to these limitations. Such large-pore materials loosely fall into the category of perfusive sorbents. Similarly, the surface modified so-called "tentacular" sorbents have provided^^'^^^'^^"^'^"^^ an another avenue to address the issue of slow adsorption kinetics or reduced capacity due to restricted diffusion in pores of inappropriately small size. Comparison of peak efficiencies between columns of identical bed dimensions but packed with sorbent particles of different physical or chemical

I 34

MILTON T. W. HEARN

characteristics, or alternatively comparison of columns of different dimensions packed with the same sorbent under different procedures, can be achieved by redefining the height equivalent H^ • in terms of the reduced plate height h^j , while the linear flow velocity u-(= L/IQ) can also be expressed in terms of a reduced velocity such that f^e,^-H,^i/dp

and

v.^u-dp/D^

(63)

These contributions from the various mass transport effects have been formalized in terms of the dependency of h^^^ on u^ for a particular biosolute Pj through the well-known van Deemter-Knox relationships, which take the form h^^. = Avy^ + B/Vi + Cv,

(64)

where the A term expresses the eddy diffusion and mobile phase mass transfer effects and is a measure of the packing quality of the chromatographic bed, the B term encompasses the longitudinal molecular diffusion effects, while the C term incorporates mass transfer resistances within the microenvironment of the stationary phase. Thus, if the diffusional processes prevail, the plots oi h ^ • versus v- will reach a minimum value at a unique v^ value. With well-packed columns, operating at optimal flow rates under carefully selected elution conditions, h^^ values approaching two to five times the particle diameter dp can be achieved. Similarly, at high values of the reduced velocity i;^, the diffusional model predicts that h^ • will become linearly dependent on v^ with slope proportional to C, because under these conditions the Av]^^ and B/v^ terms both are small in comparison to Cv^. The major challenge today for very high efficiency separations of peptides, proteins, and other biomacromolecules with available HPLC packing technology thus remains proper control of the C term effects. The challenge here is to decrease the impact of the C term on Z?^ -, either through the use of advanced nonporous sorbents or by using sorbents that exhibit very shallow h^ • versus v^ dependencies, such as the advanced perfusive or high-flux sorbents.^^^'^46-250 In this regard, sorbents capable of exhibiting little change in h over a wide range of v^ values would in principle enable very high superficial velocities to be employed within a practical separation process. The availability of various nonporous silica-based sorbents in the particle size range of 0.7 to 2.5 jxm. has enabled such separations to be achieved routinely at the analytical scale, while macroporous HP-IEX and RP-HPLC sorbents, i.e., HyperD or Poros RPIO, fulfill similar roles at more preparative scales. As expected, nonporous sorbents of small particle diameters exhibit very high efficiencies and very short analysis times but have unfavorable column backpressure characteristics. Porous materials of similar particle diameters, or alternatively the monolithic rod materials,^^^"^^^ are also now becoming available for researchers to investigate, and similar efficiency criteria have been established with these newer classes of interactive HPLC sorbents. At the process level, the demand for very high efficiency is not as compelling. Selectivity, rather than the number of theoretical plates is often perceived as a

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

135

more important issue here, although clearly particles of better size uniformity or narrower pore size distributions will result in packed columns that are usually more robust, with higher resolution factors and greater productivities. The zone broadening of a biosolutes on elution from a chromatographic system and visualized with an appropriate detector arises from two contributions, the intracolumn contribution that has been discussed and an extracolumn contribution that is due to the characteristics of the instrumentation, tubing, and detector cell. For this reason, in a practical context, the theoretical minimum h^^ value anticipated solely on the basis of the chromatographic bed and flow characteristics can rarely be achieve. Rather, system effects due to the interconnecting tubing, the consistency of the flow rate of the buffer delivery pumps, the characteristics of the detector cell and related instrumental design features contribute to the band-broadening according to the relationship: ^total ^

^column + ^system

V^^^)

where o-^oiumn ^^^ ^system ^^^ ^^^ peak variances arising from zone broadening induced by column effects and by the extracolumn system effects, respectively. With careful attention, the extracolumn system effects can be kept small, i.e., by the appropriate design of the flow through detector cell, with tubing suitable for the chosen separation task, optimal type of injector, and the maintenance of the system at constant temperature, free of flow irregularities or pressure changes, etc. When these precautions are achieved, the impact of o-^ystem o^ ^he overall h^j value can be minimized. Such considerations are particularly important when high-sensitivity analytical microbore HPLC systems are employed, where it is essential that o-^ystcm ^^ ^column? otherwise the opportunity to achieve high-speed, sensitive microbore HPLC analyses will become a futile exercise. Even with preparative HPLC systems, attention to extracolumn system effects is required, since peak overlap may be the consequence due to poor control over fluid mixing in the effluent stream or during elution development. Yll. THE EFFECT OF TEMPERATURE AND THE THERMODYNAMICS OF POLYPEPTIDE- OR PROTEIN-LIGATE INTERACTIONS For the HPLC separation of low molecular weight organic compounds and various biomacromolecules, the "near-equihbrium" criterion has generally been assumed for the binding and desorption behaviour. Changes in thermodynamic parameters due to polypeptide- or protein-ligate interaction can thus be depicted in terms of the Gibbs-Helmholtz relationship, namely, A G ° _ , , = AHl„,,, - TAS«_,,

(66)

Under such binding conditions, the properties of the polypeptide or protein, the surrounding bulk and structured solvent, and the interactive ligate surface have also been usually assumed to be invariant with regard to

I 36

MILTON T. W. HEARN

the temperature T. Hence, the contributions from the corresponding changes in enthalpy AH^^^^^ ^ or entropy AS^^^^^^ to the overall Gibbs free-energy change AG^gg^^, • associated with the solute-sorbent interaction, as well as the phase ratio of the system ^ will also be independent of temperature. When such conditions prevail, then the dependency oiln k'j on 1 / T takes the form of the well-known linear van't Hoff plot. Although this linear van't Hoff plot behavior has been observed experimentally with some small peptides,^'^^'^^'^^^'^^^"^^^ in an increasing number of investigations with polypeptides and proteins studied under such conditions significant divergences from this ideal behavior have been observed.^'^^'^^'^^'^^'^^^'^^^'^^^"^^^ As noted earlier, the interaction of a polypeptide P^ with a chemically defined ligate(s) on a chromatographic sorbent can be described in terms of the logarithm of the capacity factor In fe^, which can be related from fundamental thermodynamic considerations to the temperature T through the expression: In k', = - A H , ° _ , , / R T + A S « _ , , / R + In