Copyright © 2005 by Marcel Dekker. Copyright © 2005 by Marcel Dekker. Although great care has been taken to provide accurate and current information, neither the author(s) nor the publisher, nor anyone else associated with this publication, shall be liable for any loss, damage, or liability directly or indirectly caused or alleged to be caused by this book. The material contained herein is not intended to provide specific advice or recommendations for any specific situation. Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Analytical techniques for biopharmaceutical development / Tim Wehr, Roberto Rodriguez- Diaz, Stephen Tuck, editors. p. ; cm. Includes bibliographical references and index. ISBN 0-8247-2667-7 (alk. paper) 1. Protein drugs Analysis Laboratory manuals. [DNLM: 1. Pharmaceutical Preparations analysis Laboratory Manuals. 2. Biopharmaceutics methods Laboratory Manuals. 3. Chromatography methods Laboratory Manuals. 4. Electrophoresis methods Laboratory Manuals. 5. Spectrum Analysis methods Laboratory Manuals. QV 25 A5338 2005] I. Wehr, Tim. II. Rodríguez-Díaz, Roberto. III. Tuck, Stephen (Stephen F.) RS431.P75A536 2005 615'.7 dc22 2004058292 ISBN: 0-8247-0706-0 This book is printed on acid-free paper. Headquarters Marcel Dekker, 270 Madison Avenue, New York, NY 10016, U.S.A. tel: 212-696-9000; fax: 212-685-4540 World Wide Web http://www.dekker.com Copyright © 2005 by Marcel Dekker. All Rights Reserved. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Current printing (last digit): 10987654321 PRINTED IN THE UNITED STATES OF AMERICA Copyright © 2005 by Marcel Dekker. v Contents About the Editors vii Contributors ix 1. Analytical Techniques for Biopharmaceutical Development 1 Stephen Tuck 2. Introduction to the Development of Biopharmaceuticals 5 Roberto Rodriguez-Diaz 3. Protein Assay 13 Stephen Tuck and Rowena Ng 4. Use of Reversed-Phase Liquid Chromatography in Biopharmaceutical Development 27 Tim Wehr 5. Practical Strategies for Protein Contaminant Detection by High-Performance Ion-Exchange Chromatography 67 Pete Gagnon 6. Practical Strategies for Protein Contaminant Detection by High-Performance Hydrophobic Interaction Chromatography 81 Pete Gagnon Copyright © 2005 by Marcel Dekker. vi Contents 7. Use of Size Exclusion Chromatography in Biopharmaceutical Development 95 Tim Wehr and Roberto Rodriguez-Diaz 8. Slab Gel Electrophoresis for Protein Analysis 113 David E. Garfin 9. Capillary Electrophoresis of Biopharmaceutical Proteins 161 Roberto Rodriguez-Diaz, Stephen Tuck, Rowena Ng, Fiona Haycock, Tim Wehr, and Mingde Zhu 10. Mass Spectrometry for Biopharmaceutical Development 227 Alain Balland and Claudia Jochheim 11. Analytical Techniques for Biopharmaceutical Development — ELISA 279 Joanne Rose Layshock 12. Applications of NMR Spectroscopy in Biopharmaceutical Product Development 305 Yung-Hsiang Kao, Ping Wong, and Martin Vanderlaan 13. Microcalorimetric Approaches to Biopharmaceutical Development 327 Richard L. Remmele, Jr. 14. Vibrational Spectroscopy in Bioprocess Monitoring 383 Emil W. Ciurczak Copyright © 2005 by Marcel Dekker. vii About the Editors Roberto Rodriguez-Diaz is Senior Scientist at Dynavax Technologies in Berke- ley, California. He has extensive experience in product development in the bio- pharmaceutical industry, and his research has focused on development of analytical methodology ranging from determination of low molecular weight reactants to analysis of protein-oligonucleotide conjugates. He holds a B.S. degree from the University of Michoacan, Morelia, Mexico. Tim Wehr is Staff Scientist at Bio-Rad Laboratories in Hercules, California. He has more than 20 years of experience in biomolecule separations, including development of HPLC and capillary electrophoresis methods and instrumentation for separation of proteins, peptides, amino acids, and nucleic acids. He has also worked on development and validation of LC-MS methods for small molecules and biopharmaceuticals. He holds a B.S. degree from Whitman College, Walla Walla, Washington, and earned his Ph.D. from Oregon State University in Corvallis. Stephen Tuck is Vice President of Biopharmaceutical Development at Dynavax Technologies in Berkeley, California. He has over 14 years of experience in pharmaceutical chemistry. He was involved in the development of Fluad™ adju- vated flu vaccine as well as various subunit vaccines, adjuvants, vaccine conju- gates, and protein therapeutics. He earned his B.Sc. and Ph.D. degrees from Imperial College, University of London, United Kingdom. Copyright © 2005 by Marcel Dekker. ix Contributors Alain Balland Analytical Sciences, Amgen, Seattle, Washington Emil W. Ciurczak Integrated Technical Solutions, Golden’s Bridge, New York Pete Gagnon Validated Biosystems, Inc., Tucson, Arizona David E. Garfin Garfin Consulting, Kensington, California Fiona Haycock Dynavax Technologies, Berkeley, California Claudia Jochheim Analytical Biochemistry, Corixa, Seattle, Washington Yung-Hsiang Kao Genentech, Inc., South San Francisco, California Joanne Rose Layshock Chiron Corporation, Emeryville, California Rowena Ng Dynavax Technologies, Berkeley, California Richard L. Remmele, Jr. Pharmaceutics, Amgen, Inc., Thousand Oaks, California Roberto Rodriguez-Diaz Dynavax Technologies, Berkeley, California Copyright © 2005 by Marcel Dekker. xContributors Stephen Tuck Dynavax Technologies, Berkeley, California Martin Vanderlaan Genentech, Inc., South San Francisco, California Tim Wehr Bio-Rad Laboratories, Hercules, California Ping Wong Genentech, Inc., South San Francisco, California Mingde Zhu Bio-Rad Laboratories, Hercules, California Copyright © 2005 by Marcel Dekker. 1 1 Analytical Techniques for Biopharmaceutical Development Stephen Tuck INTRODUCTION Before the days of mass literacy, medicine was more art than science, and people recognized a pharmacy by the four traditional colored bottles that represent earth, fire, air, and water. Medicine has come a long way since the days of the apothecary with its impressive collection of powders and bottles; drugs today are highly regulated and must comply with standards set by the U.S., Europe, Japan, and other countries. A drug must be shown to be efficacious and meet rigorous standards of purity, composition, and potency before being approved for use in the patient population. These regulations provide confidence to the patient that a prescribed medicine will achieve its therapeutic goal. Whether in the form of a pill, a capsule, an injection, a tablespoon of syrup, or an inhaler, analytical methods ensure the identity, purity, potency, and ultimately the performance of these drugs. Analytical methods are important not only in the development and manu- facture of commercial biopharmaceutical drugs, they also play a vital role in the whole drug development life cycle. Drug discovery and preclinical research require development and application of analytical methodologies to support iden- tification, quantitation, and characterization of lead molecules. It is difficult to perform a comparative potency assay on lead molecules if one does not know how much of each is going into the assay or how pure the molecule is. Analytical methods are typically developed, qualified, and validated in step with the clinical Copyright © 2005 by Marcel Dekker. 2 Tuck phase of the molecule. Techniques used during discovery and preclinical devel- opment will be qualified for basic performance. When the drug is approaching early human clinical trials, and compliance to regulations becomes the order of the day, the analytical scientist begins developing assays that International Con- ference on Harmonization (ICH) guidelines define as “appropriate for their intended applications.” Analytical methods will be required for characterizing the protein’s physical-chemical and biological properties, developing stable formu- lations, evaluating real-time and accelerated stability, process development, pro- cess validation, manufacturing, and quality control. The objective of this book is to provide both an overview and practical uses of the techniques available to analytical scientists involved in the development and application of methods for protein-based biopharmaceutical drugs. The emphasis is on considering the analytical method in terms of the stage of the development process and its appropriateness for the intended application. The availability of techniques will reveal whether or not the analytical problem has a potential solution. Then will come the question of whether or not the technique is a truly appropriate solution. The theoretical considerations behind choosing the technique may be solid. However, the practicality of the method may not hold up to inspection. Consider this question: “Can one develop a stable 2 to 8°C formulation of a protein that has a propensity to aggregate and lose activity?” Several challenges face the analytical scientist. Activity is obviously a key stability-indicating assay, but is best used as a confirmatory assay because it is usually an expensive in vitro or in vivo assay that is time consuming and may not be sensitive enough to differentiate between formulations. A highly automatable or high-throughput surrogate assay would be more appropriate if it can be demonstrated to be stability indicating and to correlate with activity. If one simply wants to detect a confor- mational change, then there are techniques; one might consider nuclear magnetic resonance (NMR) as a technique that has the potential for detecting such a structural change. On further inspection, NMR is a technique that requires expen- sive equipment, highly trained operators, and significant quantities of protein. In addition, sample throughput time is slow, so all of these factors suggest that it is probably not a good screening assay for sample-intensive formulation-screening studies. However, NMR could be a good assay for characterizing the structure of the molecule and confirming that its conformation has changed. NMR is an assay requiring serious consideration prior to development, whereas gel electro- phoresis is a workhorse method that is used throughout the development process and across many areas. Each chapter of this book describes an analytical technique and discusses its basic theory, applications, weaknesses and strengths, and advantages and disadvantages, and, where possible, compares it to alternate methods. The aim is not to go into significant theoretical considerations of the technique, but rather to provide information on how and when to apply the method with examples. Copyright © 2005 by Marcel Dekker. [...].. .Analytical Techniques for Biopharmaceutical Development 3 The basic theory allows the reader to discern what considerations need to be addressed in order to evaluate the technique for the application at hand The chapters are organized to follow the order in which one might need to employ the methods during the biopharmaceutical development cycle It is difficult to do much of anything analytical. .. and identity of the final formulation The techniques described here should provide the beginning of a palette from which to develop analytical solutions Copyright © 2005 by Marcel Dekker 2 Introduction to the Development of Biopharmaceuticals Roberto Rodriguez-Diaz INTRODUCTION Although the purpose of this book is not to serve as a guideline for all aspects of biopharmaceutical development, and even less... important for development scientists to familiarize themselves with the regulatory process, which defines the development stages of a biopharmaceutical Along this path there are several checkpoints that must be passed before reaching the next plateau These checkpoints (or phases) affect all groups within Copyright © 2005 by Marcel Dekker 8 Rodriguez-Diaz a company For example, for methods development. .. biological products, are fairly unique, and thus require a tailored developmental scheme In other words, what works for the development of one drug does not necessarily work for the development of another one For example, highly specific issues about drugs often necessitate a “case-by-case” approach by regulatory agencies What is most important for a company is to demonstrate the reasoning, safety profile,... laboratories provide support for most of the activities at the biopharmaceutical company They are responsible for characterizing the molecules in development, establishing and performing assays that aid in the optimization and reproducibility of the purification schemes, and optimizing the conditions for fermentation or cell culture, including product yields Some of the characterization techniques will eventually... Marcel Dekker Introduction to the Development of Biopharmaceuticals 7 interface with the contract laboratory to ensure that proper assays are performed, and most likely will participate in the decision-making process derived from the data obtained Because biopharmaceutical development is a lengthy, expensive process, the odds of commercialization for a drug are maximized by developmental groups screening... scientists will provide support for many of the activities in a biopharmaceutical company They are responsible for characterizing the molecules in development, establishing and performing assays that aid in optimization and reproducibility of the purification schemes, and optimizing conditions for fermentation or cell culture to include product yields Some of the characterization techniques will eventually... colorimetric protein assays in biopharmaceutical development, although many alternative assays now exist As described in the following chapter, there are many biopharmaceutical applications of protein assays Assigning the protein concentration for the drug substance, drug product, or in-process sample is often the first task for subsequent analytical procedures because assays for purity, potency, or identity... required for most biopharmaceuticals What follows is not an exhaustive or up-to-the-minute survey of the methods available for protein quantitation, but a practical guide to selecting the appropriate assay for each stage of drug development A case study further illustrates the application of the standard protein methods to the drug development process The reader is referred to reviews on the topic for further... be validated Ideally, the method used here will be the method anticipated for commercial production As the drug goes through development, it is usual for formulations, dosage strengths, and even delivery vehicles to change Hence a major challenge for the protein assay at this juncture is for it to remain suitable through the development life cycle It is worthwhile to develop a drug product protein . Mingde Zhu 10. Mass Spectrometry for Biopharmaceutical Development 227 Alain Balland and Claudia Jochheim 11. Analytical Techniques for Biopharmaceutical Development — ELISA 279 Joanne Rose. Dekker. v Contents About the Editors vii Contributors ix 1. Analytical Techniques for Biopharmaceutical Development 1 Stephen Tuck 2. Introduction to the Development of Biopharmaceuticals 5 Roberto Rodriguez-Diaz 3 Hercules, California Copyright © 2005 by Marcel Dekker. 1 1 Analytical Techniques for Biopharmaceutical Development Stephen Tuck INTRODUCTION Before the days of mass literacy, medicine was more art