Aggregation of Therapeutic Proteins E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 919

Veröffentlichungsjahr: 2010

Beschreibung

This book gives pharmaceutical scientists an up-to-date resource on protein aggregation and its consequences, and available methods to control or slow down the aggregation process. While significant progress has been made in the past decade, the current understanding of protein aggregation and its consequences is still immature. Prevention or even moderate inhibition of protein aggregation has been mostly experimental. The knowledge in this book can greatly help pharmaceutical scientists in the development of therapeutic proteins, and also instigate further scientific investigations in this area. This book fills such a need by providing an overview on the causes, consequences, characterization, and control of the aggregation of therapeutic proteins.

Leseprobe

Table of Contents

Cover

Table of Contents

Half title page

Title page

Copyright page

Dedication

PREFACE

CONTRIBUTORS

CHAPTER 1 Fundamental Structures and Behaviors of Proteins

1.1 THE PROBLEM OF PROTEIN AGGREGATION

1.2 PARALLELS TO PROTEIN FOLDING

1.3 VIEWS OF PROTEIN STABILITY AND AGGREGATION

1.4 MODELS OF AGGREGATION

1.5 MODELS OF PROTEIN FOLDING

1.6 INFLUENCES OF CHEMICAL ALTERATION ON AGGREGATION

1.7 APPROACHES TO PREDICTING AGGREGATION

1.8 CONCLUSIONS

CHAPTER 2 Protein Aggregation Pathways, Kinetics, and Thermodynamics

2.1 INTRODUCTION

2.2 NATIVE AND NONNATIVE AGGREGATION PATHWAYS

2.3 THERMODYNAMICS OF REVERSIBLE SELF-ASSOCIATION

2.4 AGGREGATION KINETICS AND DISTINGUISHING KINETIC PATHWAYS

2.5 CHEMICAL MODIFICATIONS

2.6 EFFECTS OF COSOLVENTS OR COSOLUTES

APPENDIX—DERIVATION OF Γ32 FOR VAN DER WAALS (vdW) MIXTURE

ACKNOWLEDGMENTS

CHAPTER 3 Identification and Impact of Aggregation-Prone Regions in Proteins and Therapeutic Monoclonal Antibodies

3.1 INTRODUCTION

3.2 ENERGY LANDSCAPES, PROTEIN FOLDING, AND AGGREGATION

3.3 PREDICTION OF APRs IN PROTEINS AND BIOTHERAPEUTICS

3.4 CONCLUSIONS AND FUTURE DIRECTIONS

ACKNOWLEDGMENTS

CHAPTER 4 External Factors Affecting Protein Aggregation

4.1 INTRODUCTION

4.2 PROTEIN AGGREGATION PATHWAYS

4.3 EFFECTS OF TEMPERATURE

4.4 EFFECTS OF SOLUTION CONDITIONS AND COMPOSITION ON PROTEIN AGGREGATION

4.5 EFFECTS OF PROCESSING STEPS ON PROTEIN AGGREGATION

4.6 EFFECTS OF SOLID-STATE CONDITION AND COMPOSITION ON PROTEIN AGGREGATION

4.7 SUMMARY

ACKNOWLEDGMENT

CHAPTER 5 Experimental Detection and Characterization of Protein Aggregates

5.1 INTRODUCTION

5.2 AGGREGATE CLASSIFICATION

5.3 ANALYTICAL TOOLS FOR THE CHARACTERIZATION OF AGGREGATES

5.4 SUMMARY

CHAPTER 6 Approaches to Control Protein Aggregation during Bulk Production

6.1 INTRODUCTION

6.2 CANDIDATE SELECTION

6.3 PROTEIN AGGREGATION AND CELL CULTURE

6.4 PROTEIN AGGREGATION AND PURIFICATION

6.5 SUMMARY

CHAPTER 7 Protein Aggregation and Particle Formation: Effects of Formulation, Interfaces, and Drug Product Manufacturing Operations

7.1 INTRODUCTION

7.2 ROLES OF CONFORMATIONAL AND COLLOIDAL STABILITY IN REDUCING RATES OF AGGREGATION

7.3 EFFECTS OF INTERFACES ON PROTEIN AGGREGATION

7.4 CRITICAL PROCESSING STEPS DURING DRUG PRODUCT MANUFACTURING OF BIOPHARMACEUTICALS

7.5 PARTICLES IN PARENTERAL PRODUCTS AND VISIBLE INSPECTION

7.6 SUMMARY AND OUTLOOK

CHAPTER 8 Approaches to Managing Protein Aggregation in Product Development

8.1 INTRODUCTION

8.2 APPROACHES IN FORMULATION DEVELOPMENT

8.3 PROTECTION OF PROTEINS IN VARIOUS PROCESSING STEPS

8.4 AGGREGATION CONTROL BY STRUCTURAL MODIFICATIONS

8.5 SUMMARY

CHAPTER 9 Case Studies Involving Protein Aggregation

9.1 INTRODUCTION

9.2 CASE STUDY 1: AGGREGATION IN THE LIQUID STATE: THE ROLE OF OSMOLYTES IN STABILIZING KGF TOWARD AGGREGATION

9.3 CASE STUDY 2: AGGREGATION IN THE LIQUID STATE: HETEROGENEITY AND NON-LINEARITY IN IgG2 AGGREGATION DURING LONG-TERM STORAGE35

9.4 CASE STUDY 3: AGGREGATION IN THE FROZEN STATE: THE ROLE OF EXCIPIENT CRYSTALLIZATION42

9.5 CASE STUDIES 4 AND 5: AGGREGATION IN THE LYOPHILIZED STATE: ROLE OF RESIDUAL MOISTURE AND MECHANISMS OF EXCIPIENT STABILIZATION47,48

9.6 CASE STUDY 6: PROTEIN PARTICULATION DUE TO NUCLEATION BY FOREIGN MATERIAL IN FILL/FINISH MANUFACTURING OPERATIONS72

9.7 OVERALL DISCUSSION

ACKNOWLEDGMENTS

CHAPTER 10 Aggregation and Immunogenicity of Therapeutic Proteins

10.1 INTRODUCTION

10.2 IMMUNOGENICITY OF THERAPEUTIC PROTEINS

10.3 IMMUNE MECHANISMS RELATED TO PROTEIN IMMUNOGENICITY

10.4 AGGREGATES AND IMMUNOGENICITY

10.5 CONCLUSIONS

CHAPTER 11 Regulatory Perspective on Aggregates as a Product Quality Attribute

11.1 INTRODUCTION

11.2 AN OVERVIEW OF THE REGULATORY PROCESS

11.3 PRODUCT AGGREGATES AND SAFETY CONCERNS

11.4 THE ASSESSMENT OF AGGREGATES: REGULATORY APPROACHES TO CONTROLLING PRODUCT AGGREGATION

11.5 FUTURE CHALLENGES

11.6 SUMMARY

ACKNOWLEDGMENTS

DISCLAIMER

Index

Color Plates

AGGREGATION OF THERAPEUTIC PROTEINS

Copyright © 2010 by John Wiley & Sons, Inc. All rights reserved

Published by John Wiley & Sons, Inc., Hoboken, New Jersey

Published simultaneously in Canada

No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, [phone], fax [phone], or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, [phone], fax [phone], or online at http://www.wiley.com/go/permission.

Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.

For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at [phone], outside the United States at [phone] or fax [phone].

Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at www.wiley.com.

Library of Congress Cataloging-in-Publication Data

Aggregation of therapeutic proteins / edited by Wei Wang, Christopher J. Roberts.

p. ; cm.

Includes bibliographical references.

ISBN 978-0-470-41196-4 (hardback)

ISBN 978-1-118-04358-5 (ebk)

 1. Protein drugs. 2. Aggregation (Chemistry) I. Wang, Wei, 1957 Mar. 10– II. Roberts, Christopher John, 1972–

 [DNLM: 1. Recombinant Proteins—therapeutic use. 2. Cell Aggregation. 3. Protein Conformation. 4. Protein Folding. 5. Recombinant Proteins—metabolism. QU 55 A266 2010]

RS431.P75A34 2010

615.5′8—dc22

2010008428

To my parents, Jiamei Wang and Yuzhen Ma, for their unlimited love and great appreciation of higher education.

WW

To my wife, for her unending patience, support, and love.

CJR

PREFACE

Protein therapeutics have played an indispensable and increasing role in human health for the past three decades. Protein drugs, or biopharmaceuticals, are used in the treatment of a number of debilitating or even deadly human diseases, including diabetes and a number of forms of cancer. The growth of therapeutic proteins as candidates in drug development pipelines is at a record speed, outpacing significantly that for small molecule drugs. Such a pace is fueled by the record investment in biotechnology. According to a recent survey, the global biotech industry raised a total of $53 billion in 2007, a 13% growth compared to the previous year. It is estimated that the high annual growth rate may continue at levels above 15% over the next 10 years.

However, development of commercial protein drugs has not been straightforward in most cases. In comparison to small molecule drugs, production of recombinant protein drugs requires sophisticated and careful control of long fermentation/cell culture and purification processes, characterization with a much more complex suite of analytical chemistry tools, and protection of proteins against a variety of sources for instability and degradation. One such instability is the high tendency of protein molecules to aggregate under a wide range of processing and storage conditions. This propensity for aggregation is arguably the most common and troubling manifestation of protein instability during development of protein biotherapeutics. This is because protein aggregates usually exhibit either reduced or no biological activity and, more importantly, might have stronger immunogenicity and/or cellular toxicities. As such, protein aggregation has to be controlled to a satisfactory level before such a product can be commercialized. Although significant progress has been made in recent years, control or even moderate inhibition of protein aggregation has been largely semiempirical in practice.

Much of the published fundamental work in protein and polypeptide aggregation over the past three decades has focused on two main areas: aggregation as an off-pathway reaction during recombinant in vivo expression and aggregates as potentially causative agents in so-called protein deposition diseases such as Alzheimer’s disease, Parkinson’s disease, prion diseases (bovine spongiform encephalopathy and Creutzfeldt–Jakob disease), Huntington’s disease, Down’s syndrome, as well as a variety of other inherited or contracted amyloidoses. At least some of the basic physics and chemistry of many of the well-studied systems are expected to hold for aggregation of therapeutic proteins, although the latter are often larger and more structurally complex than many of their model counterparts. In addition, therapeutic proteins often encounter a much more diverse set of environments than those typically explored in biological contexts, for example, extremes of pH, solvent composition, temperature, and exposure to air–water and other bulk interfaces.

Therefore, understanding and overcoming protein aggregation remains a key area of intensive research, both in academia and biopharmaceutical companies. It is inherently interdisciplinary, spanning fields such as structural biology, chemistry and biochemistry, biophysics, pharmaceutics, and biochemical engineering. Although it is impossible to be exhaustive in any text covering so broad a field as protein aggregation, the intention of this text is to summarize current understanding and recent progress regarding protein aggregation in the context of biopharmaceutical products. This will hopefully facilitate and stimulate new and continued investigations into the principles of protein aggregation, and the application of those principles to more rapid and predictable commercialization of protein drugs and effective protection of human lives in the future.

This book is composed of 11 chapters. The first two chapters offer the audience overviews of the principles and importance of protein structure, folding and misfolding, native versus non-native interactions, self-association, competing aggregation pathways, how these manifest thermodynamically and kinetically, and how they change as a function of canonical experimental parameters such as solvent composition and temperature.

Chapters 3 and 4, respectively, discuss the internal or intrinsic (protein sequence-related) and external or extrinsic (environmental) factors that affect/control protein aggregation. Chapter 5 discusses and compares analytical methodologies available for monitoring and/or characterizing protein aggregation in research and in commercial development settings, and proposes a systematic nomenclature to help alleviate confusion and ambiguities that often arise when comparing reports from different laboratories.

Chapters 6–8 focus on control, inhibition, and monitoring of aggregation: during expression and purification (Chapter 6), during downstream processing (Chapters 6 and 7), from the context of product formulation (Chapters 6–8), as it relates to bulk interfaces (Chapter 7), when trying to optimize the final drug product (Chapter 8), and how this is coupled to current and historical questions of protein and non-protein particulates in biopharmaceutical products (Chapter 7).

Illustrative case studies on protein aggregation are offered in Chapter 9, providing more detailed discussions to complement the more general presentations in previous chapters. Chapter 10 presents the current understanding as well as key outstanding questions regarding immunogenicity issues for therapeutic proteins and protein aggregates. The last chapter provides an overview of the regulatory process and considerations in developing protein drugs, with a focus on protein aggregation and stability issues.

We are indebted to all of the authors who contributed to this text and offer our sincere thanks for their efforts and dedication, as well as their patience with us during the long process that building a text such as this becomes. We also thank Dr. Erinc Sahin for critical reading of the full manuscript and for composing the cover art.

We hope the resulting volume serves as a valuable and comprehensive resource for scientists and engineers in the industry and academia and for both experienced and new researchers in the field.

Wei Wang Chris Roberts

February 2010

CONTRIBUTORS

Tsutomu Arakawa, Alliance Protein Laboratories, Thousand Oaks, CA

John F. Carpenter, Center for Pharmaceutical Biotechnology, Department of Pharmaceutical Sciences, University of Colorado Denver, Aurora, CO

Rohini Deshpande, Amgen, Inc., Thousand Oaks, CA

Vasco Filipe, Leiden/Amsterdam Center for Drug Research (LACDR), Division of Drug Delivery Technology, Biologics Formulation Group, Gorlaeus Laboratories, Leiden, The Netherlands

Stefan Fischer, Formulation R&D Biologics and Parenterals, Pharmaceutical and Analytical R&D, F. Hoffmann-La Roche Ltd., Basel, Switzerland

Linan Ha, Division of Monoclonal Antibodies, Office of Biotechnology Products, Office of Pharmaceutical Science, Center for Drug Evaluation and Research, US Food and Drug Administration, Bethesda, MD

Andrea Hawe, Leiden/Amsterdam Center for Drug Research (LACDR), Division of Drug Delivery Technology, Biologics Formulation Group, Gorlaeus Laboratories, Leiden, The Netherlands

Yijia Jiang, Amgen, Inc., Thousand Oaks, CA

Wim Jiskoot, Leiden/Amsterdam Center for Drug Research (LACDR), Division of Drug Delivery Technology, Biologics Formulation Group, Gorlaeus Laboratories, Leiden, The Netherlands

Devendra S. Kalonia, Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT

Sohye Kang, Amgen, Inc., Thousand Oaks, CA

Susan L. Kirshner, Division of Therapeutic Proteins, Office of Biotechnology Products, Office of Pharmaceutical Science, Center for Drug Evaluation and Research, US Food and Drug Administration, Bethesda, MD

Sandeep Kumar, BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., Ches­terfield, MO

Jennifer S. Laurence, Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS

Ning Li, BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., Chesterfield, MO

Tiansheng Li, HTL Biosolutions Inc., Newbury Park, CA

Yi Li, Merck Research Laboratories, West Point, PA

Hanns-Christian Mahler, Pharmaceutical and Device Development, Pharma Technical Development Biologics Europe, F. Hoffmann-La Roche Ltd., Basel, Switzerland

C. Russell Middaugh, Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS

Linda O. Narhi, Amgen, Inc., Thousand Oaks, CA

Rahul S. Rajan, Process and Product Development, Amgen Inc., Thousand Oaks, CA

Theodore W. Randolph, Center for Pharmaceutical Biotechnology, Depart­ment of Chemical and Biological Engineering, University of Colorado, Boulder, CO

Christopher J. Roberts, Department of Chemical Engineering and Center for Molecular and Engineering Thermodynamics, University of Delaware, Newark, DE

Huub Schellekens, Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Utrecht, The Netherlands

Vikas K. Sharma, Early Stage Pharmaceutical Development, Genentech, Inc., South San Francisco, CA

Joseph Shultz, Amgen, Inc., Thousand Oaks, CA

Satish K. Singh, BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., Ches­terfield, MO

Stan Speaker, BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., Chester­field, MO

Daniela I. Verthelyi, Division of Therapeutic Proteins, Office of Biotech­nology Products, Office of Pharmaceutical Science, Center for Drug Evalu­ation and Research, US Food and Drug Administration, Bethesda, MD

Wei Wang, BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., Chester­field, MO

Xiaoling Wang, BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., Ches­terfield, MO

Nicholas W. Warne, BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., Andover, MA

Wendy C. Weinberg, Division of Monoclonal Antibodies, Office of Biotech­nology Products, Office of Pharmaceutical Science, Center for Drug Evalu­ation and Research, US Food and Drug Administration, Bethesda, MD

CHAPTER 1

Fundamental Structures and Behaviors of Proteins

JENNIFER S. LAURENCE and C. RUSSELL MIDDAUGH

Protein aggregation has been increasingly recognized as a problem limiting the efficacy and shelf life of protein therapeutics and as an indicator and cause of numerous disease states. Elucidating the molecular mechanisms behind aggregation has become a central focus of investigation in order to improve therapeutics and to understand the relationship between aggregate formation and cellular toxicity in protein misfolding diseases. Innovations in analysis techniques, particularly of solid-state materials, and computational molecular modeling approaches have provided higher resolution information about the structure of aggregates as well as key insights into the mechanisms of aggregate formation. These breakthroughs, coupled with understanding gained from solution experiments and biological systems, have just begun to enable strategies to combat aggregation, including the design and evaluation of peptides and small molecules that inhibit the growth or that facilitate the dissociation of aggregates. This chapter describes the fundamental properties of proteins and the current understanding of underlying mechanisms that influence native folding and the formation of aggregates.

1.1 THE PROBLEM OF PROTEIN AGGREGATION

Protein aggregation has significant influence in the pathology, onset, and progression of most, if not all, misfolding diseases. Over 40 human diseases have been linked to aggregation of a specific protein, including hemoglobin in sickle cell anemia, the widely recognized Aβ peptides in Alzheimer’s disease, the PrP prion protein in Creutzfeldt–Jakob’s and related diseases, expanded polyglutamine tracts in Huntington’s disease, amylin-induced β-cell death in diabetes, and α-synuclein in Parkinson’s disease.1 Moreover, studies of non-disease-associated proteins in vitro show that aggregates and amyloid fibers can be induced to occur from almost any protein, suggesting it is a ubiquitous phenomenon reflecting a common mechanism.2 Therapeutic proteins used to treat various diseases can also produce ill effects when aggregates are present, in some cases contributing to amyloid plaque formation in vivo.3,4 Aggregates have been observed to form in therapeutic proteins during purification and storage, and the administration of proteins containing aggregates has been shown to stimulate immune responses, causing effects ranging from mild skin irritation to anaphylaxis.5,6 As such, major efforts are underway to stabilize therapeutic proteins against aggregation. Thus, the goal of understanding the fundamental properties of proteins that contribute to aggregation and the mechanisms by which they aggregate is of critical importance for determining how to prevent and treat numerous diseases.

In vivo protein aggregation appears to be an ever-present problem caused by thermal fluctuations and chemical changes that disrupt the physical structure of these delicate molecules. Consequently, cells have evolved several mechanisms by which they prevent aggregates from interfering with normal function.7 Improperly folded proteins are removed from cells before they can initiate aggregation by being degraded into smaller peptides via the proteosome or lysosomal enzymes. Alternatively, intracellular proteins can be refolded to their native conformation by interaction with chaperone proteins, which are often expressed at elevated levels in response to thermal (heat shock) or chemical stress. Chaperones bind to hydrophobic patches on misfolded proteins and use an energy-dependent process to alter their conformation, therein providing the protein with additional attempts to find its native fold.8 When the capacity of the aforementioned machinery is exceeded, aggregates may form,9 as is often observed in recombinant expression systems. As one might expect, coexpression with chaperones can reduce the formation of aggregates in some cases. Chaperones have been demonstrated to affect aggregation not only by improving recovery of soluble protein but also conversely to promote aggregation when present at high levels. When aggregates form in vivo, sequestration mechanisms exist that recognize aggregated species and shuttle them to designated storage locations within the cell, such as the bacterial inclusion body and aggresome or newly discovered IPOD and JUNQ sites in eukaryotic cells.10,11 When the cellular machinery is overwhelmed by excessive damage to normal proteins or by mutations that generate a less stable form of a protein that accelerates aggregation, disease or death may result. Evidence for this is found in that increased amounts of proteosomal and chaperone proteins are found colocalized with aggregates in these inclusions.

Recombinant expression has become an increasingly important method for producing large amounts of protein for therapeutic and biotechnology applications. Production of recombinant protein is often frustrated by aggregation in the host. Yield can sometimes be improved by decreasing the temperature at which the protein is made or by coexpression with chaperone proteins (e.g., GroEL) to aid folding in vivo and to reduce sequestration to inclusion bodies.8 Nonetheless, proteins are often shuttled to inclusion bodies. Aggregated proteins, however, may be folded in vitro from the insoluble state. Single-domain proteins less than 150 residues, which are directed to inclusion bodies, can sometimes be extracted from the solid aggregate and refolded. Denaturing conditions are used to disrupt associations between chains, and the denatured material is diluted into a non-denaturing solution so that it may refold into its native form. When the native form of the protein contains disulfide bonds, folding is carried out under defined redox conditions to facilitate proper disulfide formation. This approach is not very efficient, typically resulting in a substantial fraction of the protein returning to an insoluble state. This observation suggests that proteins may follow different pathways during the course of folding, of which only some are productive. Very limited success has been had using this approach with large, multidomain proteins or those with numerous or more complex posttranslational modifications. The difficulty in refolding these proteins probably derives from increased competition between alternative interactions with those of the native state. These incorrect associations may lead to misfolding when the rate of protein production or the context in which the protein is produced is altered. Addition of chaperones at the dilution step has been used to enhance refolding of proteins that otherwise aggregate. The strategy is also being applied to stabilize purified proteins during storage. Once the active form is purified, proteins are commonly maintained at cold temperatures to restrict their conformational flexibility and to preserve their structural integrity.