FORMULATION ANDPROCESS

DEVELOPMENTSTRATEGIES FOR

MANUFACTURINGBIOPHARMACEUTICALS

Edited by

Feroz JameelSusan Hershenson

A JOHN WILEY & SONS, INC., PUBLICATION

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FORMULATION ANDPROCESS

DEVELOPMENTSTRATEGIES FOR

MANUFACTURINGBIOPHARMACEUTICALS

FORMULATION ANDPROCESS

DEVELOPMENTSTRATEGIES FOR

MANUFACTURINGBIOPHARMACEUTICALS

Edited by

Feroz JameelSusan Hershenson

A JOHN WILEY & SONS, INC., PUBLICATION

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

Published by John Wiley & Sons, Inc., Hoboken, New JerseyPublished simultaneously in Canada

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CONTENTS

FOREWORD ix

PREFACE xiii

CONTRIBUTORS xvii

PART I PREFORMULATION AND DEVELOPMENTOF STABILITY-INDICATING ASSAYS: BIOPHYSICALCHARACTERIZATION TECHNIQUES 1

1 THE STRUCTURE OF BIOLOGICAL THERAPEUTICS 3Sheryl Martin-Moe, Tim Osslund, Y. John Wang, Tahir Mahmood,Rohini Deshpande, and Susan Hershenson

2 CHEMICAL INSTABILITY IN PEPTIDE AND PROTEINPHARMACEUTICALS 41Elizabeth M. Topp, Lei Zhang, Hong Zhao, Robert W. Payne, GabrielJ. Evans, and Mark Cornell Manning

3 PHYSICAL STABILITY OF PROTEIN PHARMACEUTICALS 69Byeong S. Chang and Bernice Yeung

4 IMMUNOGENICITY OF THERAPEUTIC PROTEINS 105Steven J. Swanson

5 PREFORMULATION RESEARCH: ASSESSING PROTEINSOLUTION BEHAVIOR DURING EARLY DEVELOPMENT 119Bernardo Perez-Ramirez, Nicholas Guziewicz, and Robert Simler

6 FORMULATION DEVELOPMENT OF PHASE 12BIOPHARMACEUTICALS: AN EFFICIENT AND TIMELYAPPROACH 147Nicholas W. Warne

vi CONTENTS

7 LATE-STAGE FORMULATION DEVELOPMENT ANDCHARACTERIZATION OF BIOPHARMACEUTICALS 161Adeola O. Grillo

8 AN EMPIRICAL PHASE DIAGRAMHIGH-THROUGHPUTSCREENING APPROACH TO THE CHARACTERIZATIONAND FORMULATION OF BIOPHARMACEUTICALS 173Sangeeta B. Joshi, Akhilesh Bhambhani, Yuhong Zeng,and C. Russell Middaugh

9 FLUORESCENCE AND PHOSPHORESCENCE METHODS TOPROBE PROTEIN STRUCTURE AND STABILITY IN ICE: THE CASEOF AZURIN 207Giovanni B. Strambini

10 APPLICATIONS OF SEDIMENTATION VELOCITY ANALYTICALULTRACENTRIFUGATION 231Tom Laue

11 FIELD FLOW FRACTIONATION WITH MULTIANGLE LIGHTSCATTERING FOR MEASURING PARTICLE SIZE DISTRIBUTIONSOF VIRUS-LIKE PARTICLES 253Joyce A. Sweeney and Christopher Hamm

12 LIGHT-SCATTERING TECHNIQUES AND THEIR APPLICATIONTO FORMULATION AND AGGREGATION CONCERNS 269Michael Larkin and Philip Wyatt

PART II DEVELOPMENT OF A FORMULATIONFOR LIQUID DOSAGE FORM 307

13 EFFECTIVE APPROACHES TO FORMULATION DEVELOPMENTOF BIOPHARMACEUTICALS 309Rajiv Nayar and Mitra Mosharraf

14 PREDICTION OF AGGREGATION PROPENSITY FROM PRIMARYSEQUENCE INFORMATION 329Mark Cornell Manning, Gabriel J. Evans, Cody M. Van Pelt,and Robert W. Payne

15 HIGH-CONCENTRATION ANTIBODY FORMULATIONS 349Steven J. Shire, Jun Liu, Wolfgang Friess, Susanne Jorg,and Hanns-Christian Mahler

CONTENTS vii

16 DEVELOPMENT OF FORMULATIONS FOR THERAPEUTICMONOCLONAL ANTIBODIES AND Fc FUSION PROTEINS 383Sampathkumar Krishnan, Monica M. Pallitto, and Margaret S. Ricci

17 REVERSIBLE SELF-ASSOCIATION OF PHARMACEUTICALPROTEINS: CHARACTERIZATION AND CASE STUDIES 429Vikas K. Sharma, Harminder Bajaj, and Devendra S. Kalonia

PART III DEVELOPMENT OF A FORMULATIONFOR LYOPHILIZED DOSAGE FORM 457

18 DESIGN OF A FORMULATION FOR FREEZE DRYING 459Feroz Jameel and Mike J. Pikal

19 PROTEIN CONFORMATION AND REACTIVITYIN AMORPHOUS SOLIDS 493Lei Zhang, Sandipan Sinha, and Elizabeth M. Topp

20 THE IMPACT OF BUFFER ON SOLID-STATE PROPERTIESAND STABILITY OF FREEZE-DRIED DOSAGE FORMS 507Evgenyi Y. Shalaev and Larry A. Gatlin

21 STABILIZATION OF LYOPHILIZED PHARMACEUTICALSBY CONTROL OF MOLECULAR MOBILITY:IMPACT OF THERMAL HISTORY 521Suman Luthra and Michael J. Pikal

22 STRUCTURAL ANALYSIS OF PROTEINS IN DRIED MATRICES 549Andrea Hawe, Sandipan Sinha, Wolfgang Friess, and Wim Jiskoot

23 THE IMPACT OF FORMULATION AND DRYING PROCESSESON THE CHARACTERISTICS AND PERFORMANCEOF BIOPHARMACEUTICAL POWDERS 565Vu L. Truong and Ahmad M. Abdul-Fattah

PART IV MANUFACTURING SCIENCES 58724 MANUFACTURING FUNDAMENTALS FOR

BIOPHARMACEUTICALS 589Maninder Hora

25 PROTEIN STABILITY DURING BIOPROCESSING 605Mark Cornell Manning, Gabriel J. Evans, and Robert W. Payne

viii CONTENTS

26 FREEZING AND THAWING OF PROTEIN SOLUTIONS 625Satish K. Singh and Sandeep Nema

27 STRATEGIES FOR BULK STORAGE AND SHIPMENT OFPROTEINS 677Feroz Jameel, Chakradhar Padala, and Theodore W. Randolph

28 DRYING PROCESS METHODS FOR BIOPHARMACEUTICALPRODUCTS: AN OVERVIEW 705Ahmad M. Abdul-Fattah and Vu L. Truong

29 SPRAY DRYING OF BIOPHARMACEUTICALS AND VACCINES 739Jim Searles and Govindan Mohan

30 DEVELOPMENT AND OPTIMIZATION OF THEFREEZE-DRYING PROCESSES 763Feroz Jameel and Jim Searles

31 CONSIDERATIONS FOR SUCCESSFUL LYOPHILIZATIONPROCESS SCALE-UP, TECHNOLOGY TRANSFER,AND ROUTINE PRODUCTION 797Samir U. Sane and Chung C. Hsu

32 PROCESS ROBUSTNESS IN FREEZE DRYING OFBIOPHARMACEUTICALS 827D. Q. Wang, D. MacLean, and X. Ma

33 FILLING PROCESSES AND TECHNOLOGIES FOR LIQUIDBIOPHARMACEUTICALS 839Ananth Sethuraman, Xiaogang Pan, Bhavya Mehta,and Vinay Radhakrishnan

34 LEACHABLES AND EXTRACTABLES 857Jim Castner, Pedro Benites, and Michael Bresnick

35 PRIMARY CONTAINER AND CLOSURE SELECTIONFOR BIOPHARMACEUTICALS 881Olivia Henderson

36 PREFILLED SYRINGES FOR BIOPHARMACEUTICALS 897Robert Swift

37 IMPACT OF MANUFACTURING PROCESSES ON DRUGPRODUCT STABILITY AND QUALITY 917Nitin Rathore, Rahul S. Rajan, and Erwin Freund

INDEX 941

FOREWORD

Since the introduction of recombinant therapeutic proteins in the 1980s, dozens ofproducts have been successfully commercialized, and hundreds of new ones are cur-rently in clinical trials. These products provide uniquely effective treatments fornumerous human diseases and disorders. They have revolutionized the practice ofmedicine, saving and improving countless lives. But the most promising protein-baseddrug will not be of benefit to patients unless it can be manufactured, shipped, stored,and delivered to the patient, while minimizing degradation of the protein. This is adaunting challenge because proteins can readily aggregate, even under solution con-ditions that greatly favor the native state. Also, proteins are susceptible to numerouspathways of chemical degradation. Adding to the challenge is the potential that evenif a small fraction of the protein molecules in a dose is degraded, an immunogenicresponse may be triggered with the potential to cause adverse effects in patients.Furthermore, the therapeutic protein must be produced at commercial scale using acomplicated process that has been developed and documented to consistently resultin a high-quality product. Also, the appropriate analytical methods must be devel-oped and validated to ensure that degradation products can be quantified accuratelyand precisely. Clearly, the successful development of a commercialized therapeuticprotein product requires multidisciplinary efforts of experienced, skilled scientists,engineers, and managers, and tremendous expenditure of capital. It is critically impor-tant for management to be cognizant of these challenges and to provide the appropriateresources to the development efforts for therapeutic proteins, as well as to establishreasonable timelines for this work, which is so vital to ensuring product quality andprotecting patients safety.

Over the years, as recombinant therapeutic proteins have been developed, thefield as a whole has had to learn how to do this properly, and many of the importantguiding principles and practical strategies had to be learned on the job as productswere being developed. There were no established academic or industrial foundationsfor these efforts, because never before had recombinant proteins been used to treathuman diseases and disorders. Fortunately, during this time, many of the leaders inthe key disciplines published papers and books describing the continually improving,state-of-the-art approaches to stabilizing proteins, analyzing degradation products, anddeveloping successful formulations.

An example of this type of on-the-job training was the research focusing on devel-oping stable lyophilized formulations of proteins. In the early to mid-1980s, expertisefrom parenteral sciences and process engineering were applied to formulation and

x FOREWORD

process development for freeze-dried therapeutic proteins. The results of early effortswere often commercially viable freeze-drying cycles and formulations that providedgood cake structure but did not stabilize the protein very well. At the same time,researchers from materials sciences, food sciences, and even zoology were working tounderstand the mechanisms by which various excipients succeeded or failed to stabi-lize proteins during freezing, drying, and storage in the dried solid. Combined effortsfrom all of these disciplines gradually led to determination of these mechanisms aswell as to the discernment of the key physical properties (and associated analyticalmethods) that govern long-term storage stability of dried proteins. As a result, wenow have fairly straightforward, rational approaches to development of stable freeze-dried protein formulations. But many challenges remain, particularly understanding thequantitative linkages between different degradation pathways (e.g., oxidation, aggre-gation) and physical properties of the dried formulation (e.g., glassy-state dynamics,protein structure).

Also, during the years of development of therapeutic proteins the types of degra-dation products that could be studied, and the quality and resolution of analyticalmethods have vastly improved. These improvements allow for better understandingof causes and pathways for degradation. However, they also lead to more stringentcriteria for the definition of a stable protein product. There are still many analyticalchallenges. For example, size exclusion chromatography (SEC) is the key method usedto quantify levels of protein aggregates and monomer. But this method can providemisleading results because aggregates can dissociate or form during SEC and/or adsorbto the column resin. Thus, values obtained from SEC may not actually represent thetrue aggregate levels in the protein drug container, so there is a continued effort toinvestigate methods that can be used to corroborate results from SEC. Currently themost promising approach is analytical ultracentrifugation (AUC). But this method hasits own challenges in proper sample handling, data analysis, and appropriate trainingof personnel. The field must continue to strive to improve SEC and AUC methods foraggregate quantification and to explore new methods (e.g., field flow fractionation).

Today we benefit from the numerous advances in the field that have been madeover the last few decades. But we also face many new challenges to developing safeand effective therapeutic proteins. For example, monoclonal antibody products thathave doses with relatively high protein concentrations (e.g., 100 mg/mL) can bedifficult to manufacture, stabilize sufficiently, and analyze properly. Additionally, theuse of prefilled syringes as product containers has recently led to new issues withprotein stability that had to be resolved. In general, we must conduct research to gainmore fundamental insights into the effects of the various product containers and theircomponent materials on protein stability. Similarly, we must work to understand howvarious key processes steps (e.g., filling vials or syringes with pumps) affect proteinstability, to increase awareness of these issues, and to create effective strategies toinvestigate these potential problems and to mitigate them.

Another challenge facing many companies is the need to develop consistentapproaches for protein formulation studies, characterizing analytical methods, andstudying protein stability during various processing steps. This does not mean that

FOREWORD xi

there should be platform formulations for drug product or platform analytical meth-ods; indeed, any platform approach must be confirmed for each individual molecule,and there are many examples of surprising results. Rather, it is important to incor-porate the scientific knowledge that has been gained across the industry in rationalapproaches that are developed and agreed on by educated and experienced personnel toensure product quality and safety. As more companies develop global operations, suchan approach may have the added benefits of promoting best practices between sitesand individual researchers, minimizing unproductive conflicts, and speeding productdevelopment.

As has been the case throughout the history of working with recombinant thera-peutic proteins, the field will take on current and future challenges and learn how toovercome them. Certainly, with future insights into disease pathologies and creation ofnew therapeutic protein categories, delivery approaches, and analytical methods, evenmore challenges will arise. With the strong foundation of excellence in therapeuticprotein product development and rational approaches to delineate and solve problems,the field will successfully overcome these barriers, and new medicines will be madeavailable for the benefit of patients.

In this book, experts from around the world provide comprehensive overviewsof the many important steps involved inand the critical insights needed forthesuccessful development of therapeutic proteins. The book is a state-of-the-art sum-mary of what we have learned together as a field as we have worked to define thetheory and practice of proper development of safe and effective medicines based onbiotechnology. Moreover, it documents how researchers from numerous companiesand universities contribute to furthering our insights and expertise for developingtherapeutic proteins. The editors and authors are to be congratulated for their leader-ship in these efforts and their willingness to continue to communicate openly aboutwhere we are as a field and where we are going.

John Carpenter

PREFACE

The unraveling of the human genome, the concomitant explosion of proteomics, and anever-increasing interest in proteins to treat an expanding range of medical indicationshave lead to growing interest in the development and production of biomolecules fortherapeutic use. The identification of a new candidate drug compound is preceded bysubstantial scientific efforts and considerable capital investment. In order to realize thevalue to patients and the healthcare industry, the new drug molecule must be formu-lated and manufactured in an appropriate dosage form that can be conveniently usedby the patient. Understanding the underlying challenges at each step of developmentand commercialization of the drug product dosage form is central to the successfullaunch of a biological therapeutic.

In order for proteins to manifest their proper biological and therapeutic effect,their conformational and structural integrity must be maintained at all stages of thedevelopment and commercialization process. Biomolecules are generally very sensitiveto their microenvironment due to their complex and fragile structures. Once a newbiologic has been identified for therapeutic use and product development, the first stepsin the development process are determination of the physical and chemical propertiesof the molecule, identification of the major degradation pathways, and developmentof stability-indicating analytical methods as well as other biophysical characterizationtechniques. The information gathered from these early studies is used to identifyexcipients and conditions that will keep the protein therapeutic molecule in the nativeconformation and promote long-term product stability. Several chapters in this bookdiscuss the latest biophysical and biochemical characterization techniques, as well asapproaches to conducting the early physicochemical characterization studies.

The protein or peptide drug active must then be formulated for preclinical andclinical testing in conditions that preserve the chemical and physical integrity of themolecule, as well as render it in a form suitable for administration to patients. This isgenerally accomplished by screening the protein under a variety of excipients andconditions and monitoring stability as a function of time, temperature, and otherstresses to identify/select the best conditions for further development. Liquid dosageforms may be preferred because of their greater convenience and lower manufactur-ing costs. However, lyophilized formulations may be required in some cases to attainadequate shelf-stability or where enhanced stability at higher temperatures or otherspecial features are desired. At early stages, lyophilization may also offer a fasteror more reliable path to develop an initial clinical formulation. This book containsa number of chapters relating to early formulation development strategies, platform

xiv PREFACE

approaches for initial antibody formulations, high-throughput strategies based on sta-tistical design, and design space considerations. Additional chapters focus on thechallenges associated with stability and analysis in the development of high concen-tration antibody formulations, and the impact of high concentrations on manufacturingand dose delivery. Case examples are provided to illustrate these approaches and offerspecific applications.

Concurrent with preclinical and clinical testing of the candidate drug compound,the process development group will typically evaluate additional options availablefor expression, recovery, purification, and characterization of the drug substance forcommercial production. Alternative formulations of the drug product for commercialuse will also typically be explored. At this stage, the requirements in terms of sta-bility, shelf-life, and ruggedness are typically much greater than for the earlier stagesof development. In addition the focus on minimizing cost of goods and increasingthroughput and manufacturing ease and consistency are significantly greater at thisstage. Robust conditions for storage and shipment of the bulk drug substance mustbe identified. During subsequent commercial manufacturing, the purified bulk drugsubstance needs to be processed and prepared for successful fill/finish of final dosageform and, may go through freeze-thawing, formulation, mixing, filtration and fillingoperations prior to finishing as a lyophilized or liquid dosage form. Although theseunit operations have been studied during earlier stages, the stresses generated and themechanisms of denaturation in a manufacturing setting may be different, depending onscale, equipment and facility. Chapters dedicated to drug product process developmentdiscuss in detail, illustrated with case studies, methodology to develop, characterizeand optimize for scalability all the manufacturing processes relating to drug productprior to their transfer to manufacturing sites. Additionally, these chapters provide guid-ance on formulation design considerations to stabilize the drug against the stresses thattypically arise during large-scale manufacturing and commercialization in the cGMPenvironment.

There is growing interest in devices to simplify injection, particularly for productsthat will be sent home with patients for self-administration. This has led to increasedinterest in more complex container closures, such as prefilled syringes, either as stand-alone injection devices or as a component of a more complex injection device suchas an auto-injector. The more complex primary containers may introduce additionalstresses for the protein drug, as well as increased manufacturing challenges. Severalchapters address considerations common to all container closures, as well as specificissues related to the more complex primary containers such as prefilled syringes.

Once the commercial formulation and configuration have been recommended andall the process parameters are locked into, the process is transferred to manufacturing.In simple terms technology transfer is referred to as transfer of a new product designfrom development (internal or external) into an operational environment for validationand robust sustained production. It can be between sites at a single company or fromcompany to company and may involve a scale change or adaptation to a differentequipment train. It is very complex operation that demands in-depth understanding ofmanufacturing challenges associated with the design of the facility, equipment train,

PREFACE xv

scale, and operational procedures, besides development of robust processes and analyt-ical methods. Chapters relating to technology transfer will discuss the manufacturingchallenges and requirements and provide guidance to the reader as to when in thedevelopment phase these requirements need to be incorporated to mitigate the risk offailures and delays in getting the product to the market.

In recent years the field has evolved rapidly in many dimensions. The dramaticexpansion in number and diversity of protein therapeutics, new scientific and technicalapproaches, the evolving regulatory landscape, and changes in marketing requirementsand expectations for patient compliance make it imperative to update the availableinformation. This book provides a comprehensive overview and guide to formulationand process development as well as manufacturing of biopharmaceutical drug product,covering both fundamentals and specialized considerations. Case histories are includedto illustrate challenges and successful approaches for each phase as well as variousclasses of protein therapeutics, along with thoughtful analysis of lessons learned.Contributors have been selected from both industry and academia and have a widerange of experience and expertise in this area. The book will benefit scientists andengineers involved at various stages of product development, commercial production,project management, clinical, regulatory affairs, and quality assurance, and can serveas an introduction and reference for students who are contemplating a career in thebiopharmaceutical industry.

Color versions of some of the text illustrations can be found at the following ftpsite address:ftp://ftp.wiley.com/public/sci_tech_med/formulation_biopharmaceutical

Feroz JameelSusan Hershenson

Thousand Oaks, CaliforniaLa Jolla, CaliforniaMay 2010

CONTRIBUTORS

Ahmad M. Abdul-Fattah, Biogen Idec, San Jose, CaliforniaHarminder Bajaj, Process Development Sciences, Maxygen, Inc., Redwood City,

California

Pedro Benites, Lanthens Medical Imaging, North Billerica, MassachusettsAkhilesh Bhambhani, Macromolecule and Vaccine Stabilization Laboratory, Depart-

ment of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas

Jim Castner, Ph.D., Senior Principal Scientist, Lanthens Medical Imaging, NorthBillerica, Massachusetts

Byeong Chang, Ph.D, Symyx Technologies, Inc., Camarillo, CaliforniaRohini Deshpande, Scientific Director, Division of Translational Sciences, Amgen

Inc., Thousand Oaks, California

Gabriel J. Evans, Legacy BioDesign LLC, Loveland, ColoradoWolfgang Friess, Professor, Department of Pharmacy, Pharmaceutical Technology

and Biopharmacy, Ludwig MaximiliansUniversity Munich, Munich, Germany

Erwin Freund, Scientific Director, Drug Product and Device Development, AmgenInc., Thousand Oaks, California

Larry A. Gatlin, Global Head Technical Development, Parenteral Center of Emphasis,Pfizer Global Research & Development, Groton/New London Laboratories, Pfizer,Groton, Connecticut and Product Novartis Consultant

Adeolla O. Grillo, Human Genome Sciences, Inc., Rockville, Maryland.Nicholas Guziewicz, BioFormulations Development, Genzyme Corporation, Fram-

ingham, Massachusetts

Christopher Hamm, Research Chemist, Merck & Co., Inc., Rahway, New JerseyAndrea Hawe, Division of Drug Delivery Technology, Leiden/Amsterdam Center for

Drug Research, Leiden University, Leiden, The Netherlands

Olivia Henderson, Principal Scientist I, Protein Pharmaceutical Development, BiogenIdec,

Susan Hershenson, Vice President, Pharmaceutical and Device Development, Genen-tech Inc., South San Francisco, California

Chung C. Hsu, Director, Genentech Inc., San Francisco, CaliforniaManinder Hora, Vice President, Product Operations & Quality, Facet Biotech, Red-

wood City, California

xviii CONTRIBUTORS

Feroz Jameel, Ph.D., Drug Product and Device Development, Amgen Inc., ThousandOaks, California

Wim Jiskoot, Professor, Division of Drug Delivery Technology, Leiden/AmsterdamCenter for Drug Research, Leiden University, Leiden, The Netherlands

Sangeeta B. Joshi, Associate Director, Macromolecule and Vaccine StabilizationLaboratory, Department of Pharmaceutical Chemistry, University of Kansas,Lawrence, Kansas

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

Sampath Kumar Krishnan, Principal Scientist, Process and Product Development,Amgen Inc., Thousand Oaks, California

Michael Larkin, Research and Development, Wyatt Technology Corporation, SanFrancisco, California

Tom Laue, Professor, Department of Biochemisty, University of New Hampshire,Durham, New Hampshire

Jun Liu, Late Stage Pharmaceutical and Processing Development Department andPharmaceutical and Device Development Department, Genentech Inc., San Fran-cisco, California

Suman Luthra, Pfizer Global Research and Development, Pfizer Inc., Groton, Con-necticut

X. Ma, Department of Formulation, Freeze-Drying, and Drug Delivery, Global Bio-logical Development, Bayer HealthCare, Berkeley, California

D. MacLean, Department of Formulation, Freeze-Drying, and Drug Delivery, GlobalBiological Development, Bayer HealthCare, Berkeley, California

Hanns-Christian Mahler, Director, Formulation R&D Biologics, Pharmaceutical andAnalytical R&D, F. Hoffmann-La Roche, Switzerland

Tahir Mahmood, Department of Chemistry and Worm Institute of Research Medicine,The Scripps Institute, La Jolla, California

Mark Cornell Manning, Legacy BioDesign LLC, Loveland, ColoradoSheryl Martin-Moe, Director, Late Stage Pharmaceutical and Processing Develop-

ment Department, Genentech Inc., San Francisco, California

Susanne Jorg, Pharmaceutical and Analytical Development, Novartis Pharma AG,Basel, Switzerland

Bhavya Mehta, Drug Product and Device Development, Amgen Inc., Thousand Oaks,California

C. Russell Middaugh, Professor, Macromolecule and Vaccine Stabilization Labora-tory, Department of Pharmaceutical Chemistry, University of Kansas, Lawrence,Kansas

Govindan (Dan) Mohan, Ph.D., President, Applied Prime Technologies, Cupertino,California

Mitra Mosharraf, HTD Biosystems Inc., Hercules, California

CONTRIBUTORS xix

Rajiv Nayar, President, Product Development, HTD Biosystems Inc., Hercules, Cal-ifornia

Sandeep Neema, Senior Director, Pharmaceutical Sciences, Global Biologics, PfizerInc., Chesterfield, Missouri

Tim Osslund, Principal Scientist, Division of Translational Sciences, Amgen, Inc.,Thousand Oaks, California

Chakradhar Padala, Ph.D., Senior Scientist, Drug Product and Device Development,Amgen Inc., Thousand Oaks, California

Monica M. Pallitto, Principal Scientist, Process and Product Development, AmgenInc., Thousand Oaks, California

Xiaogang Pan, Bayer Technology Services (Asia), Shanghai, ChinaRobert W. Payne, Legacy BioDesign LLC, Loveland, ColoradoBernardo Perez-Ramirez, BioFormulations Development, Genzyme Corporation,

Framingham, Massachusetts

Micheal J. Pikal, Professor, Department of Pharmaceutical Sciences, School of Phar-macy, University of Connecticut, Storrs, Connecticut

Vinay Radhakrishnan, Group Leader, Pharmaceutical R&D, Global Biologics, PfizerInc., Chesterfield, Missouri

Rahul S. Rajan, Principal Scientist, Process and Product Development, Amgen Inc.,Thousand Oaks, California

Theodore W. Randolph, Gillespie Professor of Bioengineering, Department of Chem-ical and Biological Engineering, University of Colorado, Boulder, Colorado

Nitin Rathore, Senior Scientist, Drug Product and Device Development, Amgen Inc.,Thousand Oaks, California

Margaret S. Ricci, Director, Process and Product Development, Amgen Inc., Thou-sand Oaks, California

Samir U. Sane, Group Leader, Genentech Inc., San Francisco, CaliforniaJim Searles, Ph.D., Director of Development, Aktiv-Dry LLC, Boulder, ColoradoAnanth Sethuraman, Senior Scientist, Drug Product and Device Development,

Amgen Inc., Thousand Oaks, California

Evgenyi Y. Shalaev, Ph.D., FAAPS, Associate Research Fellow, Parenteral Cen-ter of Emphasis, Pfizer Global Research & Development, Groton/New LondonLaboratories, Pfizer, Groton, Connecticut

Vikas K. Sharma, Early Stage Pharmaceutical Development, Genentech Inc., SanFrancisco, California

Steven J. Shire, Group Leader, Late Stage Pharmaceutical and Pharmaceutical andDevice Development Department, Genentech Inc., San Francisco, California

Robert Simler, BioFormulations Development, Genzyme Corporation, Framingham,Massachusetts

Satish Singh Pharmaceutical Sciences, Global Biologics, Pfizer Inc., Chesterfield,Missouri

xx CONTRIBUTORS

Sandipan Sinha, Research Fellow, Department of Pharmaceutical Chemistry, Uni-versity of Kansas, Lawrence, Kansas

Giovanni B. Strambini, Professor, CNR, Institute of Biophysics, Pisa, ItalySteven J. Swanson, Ph.D., Executive Director, Clinical Immunology, Amgen, Inc.,

Thousand Oaks, California

Joyce A. Sweeney, Senior Investigator, Merck & Co., Inc., Rahway, New JerseyRobert Swift, Senior Principal Engineer, Drug Product and Device Development,

Amgen, Inc., Thousand Oaks, California

Elizabeth M. Topp, Professor, Department of Pharmaceutical Chemistry, Universityof Kansas, Lawrence, Kansas

Vu L. Truong, Vice President, Aridis Pharmaceuticals, San Jose, CaliforniaCody M. Van Pelt, Legacy BioDesign LLC, Loveland, ColoradoD. Q. Wang, Department of Formulation, Freeze-Drying, and Drug Delivery, Global

Biological Development, Bayer HealthCare, Berkeley, California

Y. John Wang, Late Stage Pharmaceutical and Processing Development Department,Genentech Inc., San Francisco California

Nicholas W. Warne, Ph.D., Director, Formulations Group, Wyeth BioPharma,Andover, Massachusetts

Philip Wyatt, Wyatt Technology Corporation, Santa Barbara, CaliforniaBernice Yeung, Ph.D, Symyx Technologies, Inc., Camarillo, CaliforniaYuhong Zeng, Macromolecule and Vaccine Stabilization Laboratory, Department of

Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas

Lei Zhang, Department of Pharmaceutical Chemistry, University of Kansas,Lawrence, Kansas

Hong Zhao, Department of Pharmaceutical Chemistry, University of Kansas,Lawrence, Kansas

PART IPREFORMULATION

AND DEVELOPMENTOF STABILITY-INDICATING

ASSAYS: BIOPHYSICALCHARACTERIZATION

TECHNIQUES

1

THE STRUCTURE OFBIOLOGICAL THERAPEUTICSSheryl Martin-Moe, Tim Osslund, Y. John Wang, TahirMahmood, Rohini Deshpande, and Susan Hershenson

1.1. INTRODUCTION

The first synthetic drug, acetylsalicylate, produced in 1895 and patented as aspirin[1] (Bayer in 1900), marks the beginning of the modern pharmaceutical industry.Throughout the early to mid-1900s there was significant emphasis on developmentof synthetic antibiotics for infectious diseases and small organic molecules, whichcontinues to this day as the mainstay of the traditional (small-molecule) pharmaceuti-cal industry. Advances in understanding the mechanism of reproductive functions andmetabolic diseases, such as diabetes and short stature, led to the discovery of polypep-tide hormones. The early polypeptide hormone drugs were purified from organs, suchas insulin from animal pancreas or growth hormone from cadaver pituitary. Thefirst animal-derived insulin preparation to become commercially available was Iletin,derived from bovine or porcine sources (Eli Lilly in 1923) [2]. Growth hormone,which had to be derived from a human source, was originally produced by specialorder in hospital laboratories and only commercialized much later in the United Statesin 1976 [3]. Although these products were breakthroughs in the treatment of diabetesand dwarfism, there were serious limitations with this type of production, includingavailability of organs and issues with transmission of infectious diseases [4].

Formulation and Process Development Strategies for Manufacturing Biopharmaceuticals,edited by Jameel and HershensonCopyright 2010 John Wiley & Sons, Inc.

4 THE STRUCTURE OF BIOLOGICAL THERAPEUTICS

During World War II, Edwin Cohn developed the plasma fractionation processwhereby blood components such as human serum albumin were produced to sup-plement blood loss in wounded soldiers [5]. Polyclonal antibody therapeutics derivedfrom human plasma, such as immunoglobulins, were available as treatment as early asthe 1940s [6]. In 1968 the first of the blood enzymes, antihemophilic factor VIII wascommercialized by Baxters Hyland Division [7,8]. Although these products repre-sented life-saving breakthroughs for the treatment of patients with chronic conditionssuch as hemophilia, the production of plasma proteins also suffered from safety andproduction-scale limitations since they, too, were isolated from natural sources [9,10].

Advances in the synthesis of peptides in solution and by solid phase made itpossible to produce peptides on the industrial scale [11]. However, until the adventof recombinant DNA technology, purification from natural or semisynthetic sources,with the attendant limitations of scale and/or concerns about impurities and infectiousagents, remained the only means of producing the larger polypeptide therapeutics,such as insulin and human growth hormone.

The biotechnology industry began formally in 1976 with the founding of thefirst biotechnology company, Genentech. Recognizing the significance of being ableto manipulate genes using the newly discovered tools of restriction endonucleasesand DNA ligases, Stanley Cohen and Herbert Boyer (a co-founder of Genentech)outlined in a series of papers the foundation of modern biotechnology [12]. Recom-binant DNA production systems are integrally related to the structure and functionof the protein products. The initial phase of recombinant production started withexpression and purification in bacterial systems such as Escherichia coil . Althoughsuccessful for many products, there can be serious challenges with refolding someproteins purified from E. coli . In addition, if posttranslational modifications, such asglycosylation, are required for activity, then E. coli is not viable as a host since themachinery for this is not present. Subsequently, methods were developed for cloningand expression of DNA sequences in yeast where refolding was not an issue but post-translation modifications were limited, and in insect cell systems using Baculoviruswhere modifications were closer (but still not identical) to those produced by humancells but where scalability was an issue [13]. It was not until mammalian cell systemssuch as Chinese hamster ovary (CHO) cells were established that recombinant DNAtechnology could produce products closely resembling the full range of human pro-tein structures. Mammalian systems enabled production of larger and more complexprotein therapeutics because of the ability of these cell systems to fold proteins cor-rectly and add posttranslational modifications such as N -linked and O-linked glycansessential for the biological activity and stability of many proteins. In addition, effortswere initiated to improve pharmaceutical or pharmacokinetic properties of proteins.Approaches included modification of the sequence, addition of glycosylation sites,and fusions with domains such as the Fc portion of an antibody or chemical modifi-cations such as addition of poly(ethylene glycol) or lipid. Today there are well overa hundred protein therapeutics on the market in the United States alone, representinga wide variety of structural classes; and the natural human proteins and chemicallymodified derivatives continue to be a fruitful source of new therapeutic products [14].

NATIVE HUMAN PROTEINS AND ANALOGS 5

The first monoclonal antibody therapeutics to be introduced were murine anti-bodies, based on the work of Kohler and Milstein on the continuous expression ofmonoclonal antibodies in mouse hybridoma systems [15]. The first monoclonal anti-body product produced by this technology was muromonab-CD3 (Orthoclone OKT-3by Ortho in 1986), for reversal of kidney transplant rejection [16]. Over the followingdecade methods for increasing the human content of therapeutic antibodies throughproduction of humanmurine chimeras, followed by humanized and fully human MAbconstructions were developed. Today fully human antibodies are now commerciallyproduced [17]. Currently monoclonal antibodies constitute the most rapidly growingclass of human therapeutics and the second largest class of drugs after vaccines. Thereare 26 approved monoclonal antibody-based biopharmaceutical products, mainly forthe treatment of cancer and autoimmune diseases, and there are well over a 100 drugcandidates currently under clinical development [18].

Antibodies have served as a natural biomolecular scaffold for various applica-tions. The variable region serves as the antigen-binding site and provides an effectivehumoral response against foreign substances and invading pathogens. A key advan-tage of antibody therapeutics is their high level of specificity for the relevant diseasetargets, which minimizes cross-reactivity and off-target toxicity and can thereby serveto reduce adverse effects compared to other therapeutic approaches. New technologiesfor generating humanized and fully human monoclonal antibody therapeutics are beingdeveloped with the aims of extending the range of targets, increasing the efficiency ofproduction. In addition, the field continues to experiment with modified forms of thebasic monoclonal antibody platform to extend the already impressive performance ofthis dominant class of biological therapeutics [19,20].

The following section introduces some of the major structural classes of recom-binant therapeutic proteins. Consideration has been limited to this class and doesnot include related topics such as synthetically derived peptides, protein vaccines, ordiagnostic reagents. Even for therapeutic proteins, the chapter is by no means com-prehensive, but is rather intended to provide illustrative examples of each major class.In addition some of the recent trends in the development of new classes of therapeuticproteins are briefly considered. Figure 1.1 illustrates a few examples of the structuralrange of current therapeutic protein structures, in comparison to small-molecule drugssuch as aspirin or an antibiotic.

1.2. NATIVE HUMAN PROTEINS AND ANALOGS

1.2.1. Polypeptide Hormones, the First Recombinant Therapeutics

The first wave of the biotechnology industry in the 1980s targeted replacement ofexisting, nonrecombinant therapeutic proteins with high-purity, fully human proteinsproduced using the new recombinant DNA technology. The first human protein to becloned and expressed was a polypeptide hormone, somatostatin, in 1977, based onthe work by Herbert Boyer et al. in laboratories at the University of California, SanFrancisco and the City of Hope [21]. Soon after, the human polypeptide hormonesinsulin [22] and growth hormone, or somatotrophin [23], were cloned and expressed

6 THE STRUCTURE OF BIOLOGICAL THERAPEUTICS

Aspirin

Penicillin

MAB

Erythropoietin

Insulin

Figure 1.1. Molecular representations comparing the structural complexity of types ofparenteral drugs: aspirin (180 Da), penicillin (334 Da), insulin (5808 Da), erythropoietin

(36,000 Da), and a monoclonal antibody (MAB) (150,000 Da).

by recombinant DNA methods at Genentech for commercial purposes. To understandthe pressing medical need for recombinant sources, consider the cases of insulin andgrowth hormone.

Prior to the production of recombinant insulin, insulin-dependent diabetics weretreated with insulin preparations from either bovine or porcine sources. Human insulinwas made from conversion of porcine insulin using a combination of enzymatic andchemical treatment of the porcine product [24]. The three-dimensional conformationis similar for insulin analoges from human, bovine, or porcine sources, in that theA chain forms two antiparallel helices, and the B chain forms a single helixfollowed by a turn and a strand. The arrangement of the chains buries the disulfidebonds and the aliphatic side chains in the nonpolar core. Insulin has the tendencyto self-associate forming dimer, hexamer, and multimers but is complexed with zincto form the hexamer [25,26]. Although the basic structure of the human, porcine,and bovine insulins is similar, the preparations derived from animal tissues containedmany impurities (proinsulin, arginine insulin, and desamidoinsulin), some of whichelicited immune responses exacerbated by the dosing frequency. Additionally, seem-ingly minor sequence differences between human and bovine insulin, in particular, mayhave elicited antibody responses that reduced biological activity over time, resultingin insulin resistance or altered pharmacokinetic profiles [27].

Recombinant human insulin was licensed to Eli Lilly by Genentech for develop-ment, and was the first recombinant human protein therapeutic to be approved by theUS Food and Drug Administration (FDA) in 1982. The original Genentech productionprocess involved insertion of the nucleotide sequence coding for the insulin A and B

NATIVE HUMAN PROTEINS AND ANALOGS 7

chains into two different E. coli cells that were cultured separately at large scale. Afterpurification, the A and B chains were incubated together under appropriate conditionsto promote interchain disulfide bonds. Eli Lilly improved on this method by insertionof a proinsulin nucleotide sequence into E. coli , allowing a single fermentation andpurification process. After purification there is proteolytic excision of the C peptideto produce human insulin [24,28]. Today there are a number of insulin and insulinanalog products on the market produced by a variety of host cells and processes; forexample, in addition to a number of E. coli -derived products, at least one recombinanthuman insulin is produced in yeast (Novolin by Novo Nordisk, approved in 2005).

Insulin variants have also been designed with specific pharmacokinetic proper-ties. In 1987 the first group to design fast-acting insulin was at Novo Nordisk [29].The FDA eventually approved three analogs of insulin for fast onset: insulin lispro(Humalog by Lilly, in 1996), insulin aspart (NovoLog by Novo Nordisk in 2000), andinsulin glulisine (Apidra by Aventis in 2000). They differ from insulin in Lys-Pro atB28 and B29, Asp at B28, and Lys and Glu at B3 and B29, respectively [16]. Thesemodifications generally disrupt the hexamer association, shifting the equilibrium infavor of the monomer that is absorbed more rapidly [26].

For long-acting insulin, FDA granted approval of insulin glargin (Lantus by Aven-tis in 2000) and insulin detemir (Levemir by Novo Nordick in 2005) [16]. Insulinglargin contains an extra Gly on the A chain and two Arg residues on the B chain.Insulin detemir is novel as B30 is omitted and a fatty acid (myristate) is attached atB29 Thr [30,31] (see Table 1.1 for current insulin products).

In the case of growth hormone, patients (mainly children) had been treatedwith drug derived from the pituitary gland of human cadavers. These patients oftenexperienced loss of response to the therapy, which was also linked to induction ofneutralizing antibodies attributed to the quality of the preparations [32]. These obser-vations created a drive to introduce high-quality, native human protein therapeuticsthat would avoid or minimize these adverse reactions and exposure to infectiousagents [23]. Genentechs somatrem (Protropin, met-hGH) was approved for treatinggrowth hormone deficiency in children in 1985. The approval process was expeditedby the FDA after a number of deaths caused by virus contamination in pituitarysomatropin [33]. The plasmid used at that time was designed for E. coli cytoplasmicexpression, resulting in a protein, somatrem, with an extra methionine at the N ter-minus of human growth hormone. By designing a plasmid with a signal sequence,it was shown that E. coli could remove the signal sequence during the secretionprocess to produce growth hormone without N -terminal methionine. On the basis ofthis sequence difference, Lillys recombinant somatropin, Humatrope, also receivedorphan drug exclusivity and received approval in 1987 [16]. By the end of 2008, therewere several recombinant somatropins on the market (Table 1.1).

Some of the smaller peptide hormones may be produced by either recombinantDNA technology or chemical synthesis. Nesiritide (Natrecor, commonly known asbrain or B-type natriuretic peptide) has the same 32 amino acid sequence as theendogenous peptide, which is produced by the ventricular myocardium, a single chainwith a disulfide bond between cysteines at 10 and 26. In the late 1990s, Scios made

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