regulated bioanalytical...
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REGULATEDBIOANALYTICALLABORATORIES
Technical and Regulatory Aspectsfrom Global Perspectives
MICHAEL ZHOU Ph.D.
Synta Pharmaceuticals Corporation
REGULATED BIOANALYTICAL LABORATORIES
REGULATEDBIOANALYTICALLABORATORIES
Technical and Regulatory Aspectsfrom Global Perspectives
MICHAEL ZHOU Ph.D.
Synta Pharmaceuticals Corporation
Copyright � 2011 by John Wiley & Sons, Inc. All rights reserved
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Library of Congress Cataloging-in-Publication Data:
Zhou, Michael.
Regulated bioanalytical laboratories : technical and regulatory aspects from global
perspectives / Michael Zhou.
p. cm.
Includes index.
ISBN 978-0-470-47659-8 (cloth)
1. Medical laboratories–Qualtiy control. 2. Biological laboratories–Qualtiy control.
3. Pharmaceutical technology–Qualtiy control. I. Title.
R860.Z56 2011
610.28’4–dc22
2010022398
Printed in United States of America
10 9 8 7 6 5 4 3 2 1
CONTENTS
Preface xiii
Acknowledgment xvii
Contributors and Advisors xix
1 Introduction, Objectives, and Key Requirements
for GLP Regulations 1
1.1 Introduction 1
1.1.1 Good Laboratory Practices 1
1.1.2 Bioanalytical Laboratories—Bioanalysis 4
1.1.3 Good Laboratory Practices Versus Bioanalytical Labs/
Bioanalysis 7
1.2 Objectives and Key Requirements for GLP Regulations 8
1.3 Fundamental Understanding of GLP Regulations and Principles 10
1.3.1 Elements of Good Laboratory Practices 11
1.4 Key Elements of Bioanalytical Methods Validation 16
1.4.1 Reference Standards 19
1.4.2 Method Development—Chemical/Chromatographic
Assay 20
1.4.3 Calibration/Standard Curve 21
1.4.4 Stability 21
1.4.5 Reproducibility 23
1.4.6 Robustness or Ruggedness 23
v
1.5 Basic Principles of Bioanalytical Method Validation and
Establishment 23
1.5.1 Specific Recommendations for Method Validation 24
1.5.2 Acceptance Criteria for Analytical Run 29
References 33
2 Historic Perspectives of GLP Regulations, Applicability,
and Relation to Other Regulations 35
2.1 Historic Perspectives of GLP Regulations 35
2.1.1 Economic Assessment 39
2.1.2 Environmental Impact 40
2.2 Applicability and Relations to Other Regulations/Principles 42
2.2.1 GLP, GCP, GMP, and Part 11 42
2.2.2 General Terminologies and Definitions of GxPs (GLP,
GCP, and cGMP) 47
2.3 Comparison of FDA GLP, EPA GLP Regulations, and OECD
GLP Principles 47
2.3.1 US and OECD GLP Similarity and Differences 53
2.4 Applications of GLP to Multiple Site Studies 55
2.4.1 Roles and Responsibilities 57
2.4.2 Performance of the Studies 61
2.4.3 Applications of GLP to In Vitro Studies for Regulatory
Submissions 64
2.5 21 CFR Part 11 in Relation to GLP Programs 66
2.5.1 A New Risk-Based Approach 67
2.5.2 Understanding Predicate Rule Requirements 67
2.5.3 21 CFR Part 11 Best Practices 68
2.5.4 Use of Electronic Signatures 71
2.6 GLP, cGMP, and ISOApplicabilities, Similarity, and Differences 74
2.6.1 GLPs, cGMPs, ISO 17025:2005: How Do They Differ? 74
2.6.2 GLPs Versus GMPs 74
2.6.3 GLPs Versus ISO/IEC 17025:2005 75
2.6.4 ISO Versus GLPs 76
2.7 Good Clinical Practices and Good Clinical Laboratory Practices 78
2.8 Gap andCurrent Initiatives onRegulatingLaboratoryAnalysis in
Support of Clinical Trials 80
References 84
3 GLP Quality System and Implementation 87
3.1 GLP Quality System 87
3.1.1 Regulatory Inspection for GLP Quality System 95
3.1.2 Good Laboratory Practice Inspections 99
3.1.3 GLP Quality System Objectives 103
3.2 Global GLP Regulations and Principles 106
3.2.1 General 106
vi CONTENTS
3.2.2 Responsibilities and Compliance 107
3.2.3 Statement of Compliance in the Final Report 107
3.2.4 Protocol Approval 108
3.2.5 Assignment of Study Director 108
3.2.6 Laboratory Qualification/Certification 108
3.2.7 Authority Inspections 108
3.2.8 Archiving Requirements 108
3.3 Implementation of GLP Regulations and OECD Principles 109
3.3.1 Planning (Master Schedule) 114
3.3.2 Personnel Organization 115
3.3.3 Curriculum Vitae 115
3.3.4 Rules of the Conducts of Studies 116
3.3.5 Content of Study Protocol 116
3.3.6 Approval of Study Protocol 118
3.3.7 Distribution of Study Protocol 118
3.3.8 Protocol Amendment 118
3.3.9 Standard Operating Procedures 119
3.3.10 SOP System Overview 119
3.3.11 Characterization 121
3.3.12 Test Item/Article Control before Formulation 121
3.3.13 Preparation of the Dose Formulation 123
3.3.14 Sampling and Quality Control of Dose Formulation 125
3.4 Initiatives and Implementation of Bioanalytical Method
Validation (Guidance for Industry BMV—May 2001) 126
3.4.1 Summary 127
References 128
4 Fundamental Elements and Structures for Regulated
Bioanalytical Laboratories 131
4.1 Introduction 131
4.2 Fundamental Elements for Bioanalytical Laboratories 133
4.2.1 Document Retention and Archiving 136
4.3 Basic Requirements for GLP Infrastructure and Operations 139
4.4 GxP Quality Systems 143
4.4.1 Laboratory Instrument Qualification and Validation 149
4.4.2 Procedural Elements and Function that Maintain
Bioanalytical Data Integrity for GLP Studies 150
References 166
5 Technical and Regulatory Aspects of Bioanalytical Laboratories 167
5.1 Fundamental Roles and Responsibilities of
Bioanalytical Laboratories 167
5.1.1 Technical Functions of Bioanalytical Laboratories 168
5.1.2 Basic Processes in Bioanalytical Method Development,
Validation, and Sample Analysis 173
CONTENTS vii
5.2 Qualification of Personnel, Instrumentation, and
Analytical Procedures 178
5.2.1 FromRegulatory Perspectives: Personnel, Training, and
Qualification 183
5.2.2 Facility Design and Qualifications 186
5.2.3 Equipment Design and Qualification 186
5.2.4 Analytical/Bioanalytical Method Qualification and
Validation along with Related SOPs 197
5.3 Regulatory Compliance with GLP Within
Bioanalytical Laboratories 204
5.4 Joint-Effort from Industries and Regulatory Agencies 206
5.4.1 Ligand-Binding Assays In-Study Acceptance Criteria 213
5.4.2 Determination ofMetabolites during Drug Development 216
5.4.3 Incurred Sample Analysis 216
5.4.4 Documentation Issues 217
5.4.5 Analytical/Validation Reports 218
5.4.6 Source Data Documentation 218
5.4.7 Final Report Documentation 219
5.4.8 Stability Recommendation 219
5.4.9 Matrix Effects for Mass Spectrometric-Based Assays 221
5.4.10 System Suitability 222
5.4.11 Reference Standards 222
5.4.12 Validation Topics with No Consensus 222
5.4.13 Specific Criteria for Cross-Validation 223
5.4.14 Separate Stability Experiments Required at � 70�C if
Stability Shown at � 20�C 223
5.4.15 Stability Criteria for Stock Solution Stability 224
5.4.16 Acceptance Criteria for Internal Standards 224
5.4.17 Summary 224
References 226
6 Competitiveness of Bioanalytical Laboratories—Technical
and Regulatory Perspectives 229
6.1 Technical Aspect of Competitive Bioanalytical Laboratories 229
6.2 Bioanalytical Processes and Techniques 232
6.2.1 Sample Generation, Shipment, and Storage 232
6.2.2 Sample Preparation 233
6.3 Enhancing Throughput and Efficiency in Bioanalysis 243
6.3.1 Chromatographic Separation 244
6.3.2 Selective and Sensitive Detection 251
6.4 Technical Challenges and Issues on Regulated Bioanalysis 254
6.4.1 Matrix Effect 254
6.4.2 Method Validation and Critical Issues during Sample
Analysis 256
6.4.3 Method Transfer 258
viii CONTENTS
6.5 Regulatory Aspects of Competitive Bioanalytical Laboratories 264
6.5.1 General Consideration 264
6.5.2 Historical Perspective 265
6.5.3 Personnel—Training and Qualification 267
6.5.4 Facility—Design and Qualifications 269
6.5.5 Equipment Design and Qualification 270
6.5.6 Standard Operating Procedures 272
6.5.7 Laboratory/Facility Qualification Perspectives 272
6.6 Advanced/Competitive Bioanalytical Laboratories 277
6.6.1 Strategy Versus Tactics 278
6.6.2 Bioanalytical Laboratory Assessment 279
6.6.3 Capacity 279
6.6.4 Experience 280
6.6.5 Quality 281
6.6.6 Performance and Productivity Measures 281
6.6.7 Information Technology and Data Management 282
6.6.8 Communication 282
6.6.9 Financial Stability 283
6.6.10 Ease of Use 283
6.6.11 Contracting Bioanalytical Services 284
6.6.12 The Contracting Process 284
6.7 Applications andAdvances in Biomarker and/or Ligand-Binding
Assays within Bioanalytical Laboratories 286
References 290
7 Sponsor and FDA/Regulatory Agency GLP Inspections and Study
Audits 297
7.1 GLP versus Biomedical Research Monitoring and Mutual
Acceptance of Data for Global Regulations and Inspections 298
7.2 Purposes and Benefits of Regulatory Inspections/Audits 303
7.2.1 Criteria for Selecting Ongoing and Completed Studies 304
7.2.2 Areas of Expertise of the Facility 305
7.2.3 Establishment Inspections 305
7.2.4 Organization and Personnel (21 CFR 58.29, 58.31,
58.33) 305
7.2.5 Quality Assurance Unit (QAU; 21 CFR 58.35) 307
7.2.6 Facilities (21 CFR 58.41–58.51) 308
7.2.7 Equipment (21 CFR 58.61–58.63) 309
7.2.8 Testing Facility Operations (21 CFR 58.81) 310
7.2.9 Reagents and Solutions (21 CFR 58.83) 311
7.2.10 Animal Care (21 CFR 58.90) 311
7.2.11 Test and Control Articles (21 CFR 58.105–58.113) 312
7.2.12 Test and Control Article Handling (21 CFR 58.107) 313
7.2.13 Protocol and Conduct of Nonclinical Laboratory Study
(21 CFR 58.120–58.130) 314
CONTENTS ix
7.2.14 Study Protocol (21 CFR 58.120) 314
7.2.15 Test System Monitoring 314
7.2.16 Records and Reports (21 CFR 58.185–58.195) 314
7.2.17 Data Audit 316
7.2.18 General 316
7.2.19 Final Report Versus Raw Data 317
7.2.20 Specimens Versus Final Report 318
7.2.21 Refusal to Permit Inspection 318
7.2.22 Sealing of Research Records 318
7.2.23 Samples 319
7.3 Typical Inspections/Audits and Their Observations 320
7.4 Regulatory Challenges for Bioanalytical Laboratories 321
7.4.1 Introduction 321
7.4.2 Analysis of Current FDA Inspection Trends 324
7.4.3 Discussion and Analysis of Specific Potential FDA 483
Observation Issues 325
7.4.4 Method Validation Issues 325
7.4.5 Batch Runs Acceptance Criteria Issues 329
7.4.6 Events/Deviations Investigation/Resolution Issues 331
7.4.7 Test Specimen Accountability Issue 333
7.4.8 Recommendations to Support an Effective FDA
Inspection Readiness Preparation 334
7.5 Handling and Facilitating GLP or GxP Audits/
Inspections 334
7.5.1 General Preparation for an Inspection 336
7.5.2 Why Are Audits/Inspections Needed and Conducted? 342
7.5.3 Written Policy in Place 342
7.5.4 Positions on Controversial Issues 343
7.5.5 The Inspection Coordinator 344
7.5.6 Follow-Up Procedures 348
7.5.7 Summary 349
References 351
8 Current Strategies and Future Trends 353
8.1 Strategies from General Laboratory and Regulatory
Perspectives 354
8.2 Strategies from Technical and Operational Perspectives 356
8.3 Biological Sample Collection, Storage, and Preparation 360
8.3.1 Sample Collection and Storage 360
8.3.2 Sample Preparation Techniques 361
8.3.3 Off-Line Sample Extraction 364
8.3.4 On-Line Sample Extraction 364
8.4 Strategies for Enhancing Mass Spectrometric Detection 366
8.4.1 Enhanced Mass Resolution 368
8.4.2 Atmospheric Pressure Photoionization 369
x CONTENTS
8.4.3 High-Field Asymmetric Waveform Ion Mobility
Spectrometry 370
8.4.4 Electron Capture Atmospheric Pressure Chemical
Ionization 370
8.4.5 Mobile Phase Optimization for Improved Detection and
Quantitation 371
8.4.6 Anionic and Cationic Adducts as Analytical Precursor
Ions 372
8.4.7 Derivatization 372
8.5 Strategies for Enhancing Chromatography 374
8.5.1 Ultra-Performance Chromatography 375
8.5.2 Hydrophilic Interaction Chromatography for Polar
Analytes 376
8.5.3 Specialized Reversed-Phase Columns for Polar
Analytes 377
8.5.4 Ion-Pair Reversed-Phase Chromatography for Polar
Analytes 378
8.6 Potential Pitfalls in LC–MS/MS Bioanalysis 378
8.6.1 Interference from Metabolites or Prodrugs due to
In-Source Conversion to Drug 378
8.6.2 Interference from Metabolites or Prodrugs due to
Simultaneous M þ Hþ and M þ NH4þ Formation or
Arising from Isotopic Distribution 379
8.6.3 Pitfall in Analysis of Two Interconverting Analytes due
to Inappropriate Method Design 383
8.6.4 Matrix Effect 383
8.7 Trends in High-Throughput Quantitation 386
8.7.1 System Throughput 386
8.7.2 High-Speed HPLC 386
8.8 Trends in Hybrid Coupling Detection Techniques 388
8.9 Trends in Internal R&D and External Outsourcing 388
8.10 Trends in Ligand-Binding Assays and LC–MS/MS
for Biomarker Assay Applications 397
8.11 Trends in Study Design and Evaluation
Relating to Bioanalysis 399
8.12 Trends in Applying GLP to In Vitro Studies in Support of
Regulatory Submissions 403
8.13 Trends in Global R&D Operations 404
8.14 Trends in Regulatory Implementations 407
8.14.1 Calibration Range and Quality Control Samples 407
8.14.2 Incurred Sample Reproducibility (Duplicate Sample
Analysis) 408
8.14.3 LIMS and Electronic Data Handling, Security,
Archiving, and Submission 409
8.15 Trends in Global Regulations and Quality Standards 412
CONTENTS xi
8.16 Trends in Compliance with 21 CFR Part 11 414
8.16.1 21CFR Part 11 Software Requirements 415
8.16.2 Building a Roadmap for Compliance with
21 CFR Part 11 415
8.16.3 Low Hanging Fruits in the Roadmap for Compliance
with 21 CFR Part 11 416
8.17 Summary 419
References 421
9 General Terminologies of GxP and Bioanalytical Laboratories 431
9.1 General Terminologies for GxP and Bioanalytical Laboratories 431
9.2 GLP Basic Concepts and Implementation 469
9.2.1 The Study Protocol 470
9.2.2 Raw Data 471
9.2.3 The GLP Archive and the Archivist 472
9.2.4 Expansion of GLP Scope 473
9.2.5 OECD GLP 473
9.3 GLP Guidance Documents 474
9.3.1 FDA Guidance for Industry on Bioanalytical Method
Validation 474
9.3.2 OECD GLP Guidance Documents 474
9.3.3 Swiss GLP Guidance Documents 475
References and Sources for Above Terminologies 475
Appendix A Generic Checklist for GLP/GXP Inspections/Audits 479
Appendix B General Template for SOP 489
Appendix C Typical SOPs for GLP/Regulated Bioanalytical
Laboratory 493
Quality Assurance—GLP 493
Bioanalytical—GLP Laboratories 494
Appendix D Basic Equipment/Apparatus for Bioanalytical
Laboratory 497
Appendix E Website Linkages for Regulated Bioanalysis 499
Index 503
xii CONTENTS
PREFACE
TheGood Laboratory Practice (GLP) regulations were established in the 1970s by the
United States Food and Drug Administration (FDA), and published in the Code of
Federal Regulations (21 CFR Part 58). The Organization for Economic Cooperation
and Development (OECD) established the Principles of GLP in 1981. United States
Environmental Protection Agency (EPA) adopted its own set of GLP regulations
shortly thereafter, governing the research surrounding pesticides and toxic chemicals.
Bioanalytical laboratories have increasingly become critically important in data
generation for discovery, preclinical, and clinical development in life science
industries. Bioanalysis, employed for the quantitative determination of drugs and
their metabolites in biological fluids, plays significant roles in the evaluation and
interpretation of bioequivalence (BE), bioavailability (BA), pharmacodynamic (PD),
pharmacokinetic (PK), and toxicokinetic (TK) studies. The quality of these studies,
which are often used to support regulatory filings, is directly related to the quality of
the underlying bioanalytical data. It is therefore important that guiding principles for
the validation of these analytical methods be established and disseminated to the
pharmaceutical and life science communities.
The focus and working groups from American Association of Pharmaceutical
Scientists (AAPS), European Bioanalysis Forum (EBF), Food and Drug Adminis-
tration, European Medicine Agency (EMEA), and other related organizations have
held a series of workshops and seminars focusing on key issues relevant to bioana-
lytical methodology and provided a platform for scientific discussions and delibera-
tions. As bioanalytical tools and techniques have continued to evolve and significant
scientific and regulatory experiences have been gained, the bioanalytical community
has continued its critical review of the scope, applicability, and success of the
presently employed bioanalytical guiding principles. Life science products including
xiii
foods, nutritional supplements, medicine/drug discovery, research, and development
expand to wide scope of life sciences meaning varieties of industries such as
environmental toxicology, food nutritional analysis, biotechnology, biopharmaceu-
ticals, pharmaceuticals, hospitals, diagnostic/medical device industries, and so on.
Bioanalytical sciences basically support above-mentioned areas under FDAGLP and
other related regulations, principles, and guidelines. Below are some example
elements/infrastructure that are required for regulated Bioanalytical Laboratories:
(1) Responsible management team with quality system
(2) Qualified personnel selection, staffing, and training
(3) Standard operating procedures (SOPs)
(4) Installation, operational, and performance qualification (IQ, OQ, and PQ,
respectively) of facilities, instrumentation, and software
(5) Quality control (QC) procedures and staffing
(6) Quality assurance unit (QAU)
(7) Data generation and security assessment
(8) Documentation and archival process
(9) Laboratory information management system (LIMS)
(10) Final gap analysis
This book provides useful information for bioanalytical/analytical scientists,
analysts, quality assurance managers, and all personnel in bioanalytical laboratories
through all aspects of bioanalytical technical and regulatory perspectives within
bioanalytical operations and processes. Readers will learn how to develop and
implement strategies for routine, nonroutine, and standard bioanalytical methods
and on the entire equipment hardware and software qualification process. The book
also gives guidelines on qualification of certified standards and in-house reference
material aswell as on people qualification. Finally, it guides readers through stressless
internal and third party laboratory audits and inspections. Highly comprehensive
content with specific chapter by chapter is elaborated making it easy not only to learn
the subject but also to quickly implement the recommendations.
It takes account to most national and international regulations and quality and
accreditation standards such as GLP, cGMP, GCP, and GCLP from US FDA, ICH,
WHO, and EU, accreditation standards such as ISO17025 and to corresponding
interpretation and inspection guides. The text beginswith an introductory overview of
the roles of bioanalytical laboratories in pharmaceutical and biotechnology drug
development. Regulatory wise, it describes some fundamental understanding of
regulatory aspectswithin bioanalytical laboratories—current and future requirements
as far as GLP and/or GxP quality systems, facility, and personnel infrastructure and
qualification along with continuing improvement on a daily basis. From technical
standpoints, the book also elaborates the strategies for sample preparation, along with
essential concepts in extraction chemistry. Particular strategies for efficient use of
automation within bioanalytical laboratories are also presented. With regards to
xiv PREFACE
instrumental analysis, fundamental approach is presentedwithin the areas of LC–MS/
MS and other hyphenated analytical techniques. Ligand-binding assays are also
discussed to recognize its increasingly crucial applications within bioanalytical
laboratories. Important objectives that can be accomplished when the strategies
presented in this book are followed include: improved efficiency inmoving discovery
compounds to nonclinical and clinical status with robust analytical methods; auto-
mation for sample preparation; modern analytical equipment, and improved knowl-
edge and expertise of laboratory staff. It has been widely accepted that good sciences
are not enough to meet regulatory requirements. In author�s opinion, good laboratorypractices may not improve any “poor” sciences, but indeed make “good sciences”
better as to ensure the quality, integrity, and reconstructability of data. GLP or GxP is
all about documentation. In anotherword, nothing has been properly donewithout any
documentation. GLP principles may also enhance the opportunity of Limiting waste
of resources, Ensuring high quality of data, Acquiring comparability of results, and
Deriving to mutual recognition of scientific findings worldwide and ultimately
Securing the health and well-being of our societies, as being LEADS concept per
author�s perspectives.
PREFACE xv
ACKNOWLEDGMENT
The need of this book has been apparent as noted that Good Laboratory Practice must
be followed while generating data for regulatory consideration. I am grateful to peer-
reviewers for their positive feedback and encouragement since this project started.
The staff at John Wiley and Sons has provided me with tremendous support. In
particular, I would like to thankmy editor, Jonathan Rose, who has been an invaluable
resource during the project as well as other advice. I am truly indebted to all of the
contributors for their willingness of sharing their experiences, knowledge, and
perspectives on bioanalytical technical and regulatory aspects. The contributions as
well as the many discussions and interactions are worth noting! My special gratitudes
extend to Dr. Vinod P. Shah for his expert advice and inputs. The growth of
bioanalytical laboratory operations and contributions in life science industries has
been truly remarkable. I am thankful to have had the opportunity to interact with so
many people (contributors, reviewers, and advisors) who shared a common passion
for the analytical/bioanalytical sciences and regulatory compliance for the betterment
of the world and health.
Finally, I thank my wife, family, and friends for their courage and continued
support for everything I do.
xvii
CONTRIBUTORS AND ADVISORS
Author sincerely appreciates all of the advice, review, suggestions, and edits from
following field experts on respective chapters and sections.
Frank Chow Lachman Consultants
1600 Stewart Avenue, Suite 604
Westbury, NY 11590, USA
Howard M. Hill Huntingdon Life Sciences
Huntingdon, Cambridge, PE28 4HS, UK
Mohammed Jemal Bristol-Myers Squibb
Route 206 and Province Line Road
Princeton, NJ 08543, USA
Marian Kelley MKelley Consulting LLC
1533 Glenmont Lane
West Chester, PA 19380, USA
Jean Lee Amgen Inc
100 Amgen Center Drive
Thousand Oaks, CA 91320, USA
Raymond Naxing Xu Abbott Laboratories
100 Abbott Park Road
Abbott Park, IL 60064, USA
xix
1INTRODUCTION, OBJECTIVES,AND KEY REQUIREMENTSFOR GLP REGULATIONS
1.1 INTRODUCTION
1.1.1 Good Laboratory Practices
Good laboratory practices (GLPs 21 CFR PART 58) is a standard by which laboratory
studies are designed, implemented, and reported to assure the public that the results
are accurate/reliable and the experiment can be reproduced accordingly [1], at any
time in the future. In less technical terms, GLP is the cornerstone of all laboratory-
based activities in any organization that prides itself on the quality of the work it
performs. And, despite its immediate association with the pharmaceutical sector,
GLPs can (and should) be applied to virtually all industries in which laboratory work
is conducted, including companies involved in drug development, manufacturing,
foods, pesticides (agrochemicals), drink production, and engineering testing. In
addition, commercial testing laboratories (for toxicology, metabolism, materials,
and safety, for example), research establishments, and universities—in fact, all
laboratories engaged in product or safety testing or research and development—
should adopt and apply the doctrines of GLP.
GLP is not a luxury. It is a necessity for any professional laboratorywishing to gain
and retain the respect of its employees, clients, regulators, and perhaps most
importantly, its competitors. If a company is seen to be applying and adhering to
the highest standards of laboratory practice, it will gain significant competitive
advantage and will compete successfully for business and recognition within its
Regulated Bioanalytical Laboratories: Technical and Regulatory Aspects from Global Perspectives,
By Michael Zhou
Copyright � 2011 John Wiley & Sons, Inc.
1
operational environment. Conversely, without rigidly enforced GLPs, good clinical
practice (GCP) [2], good manufacturing practices (GMPs) [3], or GxPs—a scientific
organizationwill not achieve the commercial success and respect that its products and
personnel deserve.
Published GLP regulations and guidelines have a significant impact on the daily
operations of analytical and/or bioanalytical laboratories. GLP is a regulation that
enhances good analytical practice. Good analytical/bioanalytical practice is impor-
tant, but it is not enough. For example, the laboratory must have a specific
organizational structure and procedures to perform and document laboratory work.
The objective is not only quality of data but also traceability and integrity of data.
However, the biggest difference between GLP and non-GLP work is the type and
amount of documentation. GLP functions as a regulation, which deals with the
specific organizational structure and documents related to laboratory work in order to
maintain integrity and confidentiality of the data. The entire cost of GLP-based work
is about 40% or more additional (from case to case) when compared to non-GLP
operations. For aGLP inspector, it should be possible to look at the documentation and
to easily find out the following:
. Who has done a study
. How the experiment was carried out
. Which procedures have been used, and
. Whether there has been any problem and if so
. How it has been addressed and solved where applicable
And this should not only be possible during and right after the study has been
finished but also 5–10 or more years later.
From worldwide perspectives, good practice rules govern drug/product develop-
ment activities in many parts of theworld.World Health Organization (WHO), which
has published documents on current good manufacturing practices (cGMPs) and
GCPs, has not previously recommended or endorsed any quality standard governing
the nonclinical phases of drug/product development. GLPs are recognized rules
governing the conduct of nonclinical safety studies, ensuring the quality, integrity, and
reliability of their data. To introduce the concepts of GLP to scientists in developing
countries, workshops onGLP have been organized in these regions. As an outcome of
the workshops (industries and regulatory bodies), it became apparent that some
formal guidance would be needed for the successful implementation of the GLP
regulations.
The first scientific working group on GLP issues was convened on November 25,
1999, in Geneva, to discuss quality issues in general and the necessity for a WHO
guidance document on GLP in particular. The working group concluded that it was
important to avoid the coexistence of two GLP standards, the Principles of good
laboratory practice of the Organization for Economic Cooperation and Development
(OECD) [4] being the internationally recognized and accepted standard, and
recommended that theOECDPrinciples be adopted byWHOforResearch&Training
2 INTRODUCTION, OBJECTIVES, AND KEY REQUIREMENTS FOR GLP REGULATIONS
in Tropical Disease (TDR) as the basis of this guidance document. The experts also
recognized the need to address quality issues in areas other than the strictly regulated
safety studies for regulatory submission, and recommended that some explanation be
included in this guidance document. The working group further recommended that
WHO/TDR should request OECD’s permission to publish the existing OECD GLP
textwith aWHOendorsement, and to supplement it with an explanatory introduction.
Classical drug development (drug life cycle) is characterized by four well-defined
stages as follows:
Stage 1: The first stage, the discovery of potential new drug products, is neither
covered by a regulatory standard, nor are studies demonstrating proof of
concept. This area may well require some international standards or guidance
documents in the future.
Stage 2: The position of GLP studies within the drug development process is
specific to the second stage. These studies are termed “nonclinical” as they are
not performed in human. Their primary purpose is safety testing. Toxicology
and safety pharmacology studies, with a potential extension to pharmacoki-
netics and bioavailability, are those studies where the compliance with GLP is
required, which is the rather restricted scope of GLP.
Stage 3: The third stage, following on from safety studies, encompasses the clinical
studies in human. Here, GCP is the basis for quality standards, ethical conduct,
and regulatory compliance. GCP must be instituted in all clinical trials from
Phase I (to demonstrate tolerance of the test drug and to define human
pharmacokinetics), through Phase II (where the dose–effect relationship is
confirmed), to Phase III (full-scale, often multicenter, clinical efficacy trials in
hundreds and thousands of patients).
Stage 4: The fourth stage is postapproval. Here the drug is registered and available
on the market. However, even after marketing, the use of the drug is monitored
through formalized pharmacovigilance procedures. Any subsequent clinical
trials (Phase IV) must also comply with GCP.
A brief summary of different stages is shown in Table 1.1.
TABLE 1.1 Stages Defined Within Discovery and Development Programs
Stage I Stage II Stage III Stage IV
Establish discovery
assessment of
compounds with
in vitro and/or
in vivo data
(not regulated
under GxP)
Demonstrate
efficacy, identify
side effects
including Tox and
assessment of
pharmacokinetics
(GLP and GCP)
Gain more data on
safety and
effectiveness in
multicenters with
thousands of
patients (GLP,
GCP, cGMP)
Monitor claims or
demonstrate new
indications;
examine special
drug–drug
interactions; assess
pharmacokinetics
(GLP, GCP, cGMP)
INTRODUCTION 3
1.1.2 Bioanalytical Laboratories—Bioanalysis
Bioanalytical laboratories have increasingly become center of excellence and
critically important in data generation for discovery, preclinical and clinical devel-
opment in life science industries. Bioanalysis is a broad term that is derived from
analytical applications to biologicalmaterials (matrices) such as human and/or animal
biological fluids and materials (blood, plasma, serum, urine, feces, tissues, etc.),
biopharmaceutical (peptides, protein, etc.), and biochemistry (DNA, RNA, organo-
nucleotides, etc.). The main focus of this book is within the aspects of liquid
chromatography–tandem mass spectrometry (LC–MS/MS) and to certain extent
of immunochemistry assays—enyzme-linked immunosorbent assays (ELISA) or
ligand-binding assays (LBAs). Bioanalysis is mainly referred to the quantitative
determination of drugs and their metabolites, and other life science products in
various sample matrices. However, it should also apply to qualitative analysis
(identification and elucidations) of drug degradants, metabolites, impurities, and
other analytes of interests. The techniques (chromatographic-based and ligand-
binding-based assays) are used very early in the drug discovery and development
process to provide support to product discovery programs on metabolite fate and
pharmacokinetics of chemicals in living cells and animals. They are referred by FDA
Guidance for Industry Bioanalytical Method Validation for chromatographic-based
and ligand-binding-based assays [5]. Their uses continue throughout the nonclinical
and clinical product development phases into postmarketing support and may
sometimes extend into clinical therapeutic monitoring. Recent developments and
industry trends for rapid sample throughput and data generation are introduced and
discussed in following chapters, together with examples of how these high throughput
needs are met in bioanalysis.
1.1.2.1 High-Throughput Bioanalytical Sample Preparation Methods and
automation strategies are authoritative reference on the current state-of-the-art in
sample preparation techniques for bioanalysis. The following related chapters focus
on high-throughput (rapid productivity) techniques and describe exactly how to
perform and automate these methodologies, including useful strategies for method
development and optimization. A thorough review of the literature is included
describing high-throughput sample preparation techniques: protein removal by
precipitation; equilibrium dialysis and ultrafiltration; liquid–liquid extraction; solid
phase extraction; and various online techniques. A schematic diagram of analytical/
bioanalytical techniques used in automation is shown in Figure 1.1.
Among the sample preparation scheme, protein precipitation (PPT) is the most
commonly used approach for a simple, fast, and unique process of removing
unwanted materials from analyte(s) of interest for analysis or in some case for
further cleanup. High selectivity and sensitivity are also imperative for bioanalytical
laboratories to deal with sample analyses with great demand in method limits of
quantitation (LOQ), wide dynamic range (linearity and range), free of interferences
(specificity and selectivity), and other highly challenging requirements such as
multiple compounds (analytes—parent drugs, prodrugs, and their degradants/
4 INTRODUCTION, OBJECTIVES, AND KEY REQUIREMENTS FOR GLP REGULATIONS
metabolites), various sample types (matrices), and different analytical techniques
including LC–MS/MS,GC–MS/MS, LC–NMR, ICP–MS, and other advanced hybrid
techniques. In addition to above analytical techniques, immunoassays (ELISA and/or
ligand-binding assays—LBAs or alike) are also widely used within bioanalytical
laboratories, especially in biopharmaceutical and biotechnology industries where
relatively large molecules are dealt such as peptides and proteins as part of
drug development compounds and applying to different therapeutic areas. Rapid
advances in chromatographic as well as ligand-binding assay technologies have
been observed to meet the needs in product research and development processes.
More details of description are elaborated on above analytical and bioanalytical
techniques as powerful methodologies in trace level qualitative and quantitative
analyses.
There have been varieties of separation and detection techniques involved in
analytical and bioanalytical methodologies as indicated in Figure 1.2. More recent
years, biomarker analysis in various therapeutic areas has become incredibly
significant in drug/product development and monitoring programs. Without any
doubt, this has increasingly become part of bioanalytical capabilities. Biomarker
Analytical/Bioanalytical Sample Prep. Chemistry and Techniques
Protein Precipitation Liquid–LiquidExtraction
Solid Phase Extraction
Automation: Liquid Handling Workstations and Robots
TomtecQuadra 96 Plus
Packard MultiProbe IIEx
TecanGenesis Freedom
HamiltonSTAR
FIGURE 1.1 General schematic of analytical/bioanalytical techniques used in laboratory
operations/automation.
Analytical/Bioanalytical Separation/Detection Techniques
Spectroscopy/LBAsSpectrophotometryChromatography
GC, HPLC, CE, UV–VIS, FT-IR, NMR, MS/MS, LBAs, etc.
Small and Large Molecules
Ionic and PolarSpecies
Volatile andNonvolatile
Liquid, Gas, and Solids
FIGURE 1.2 Commonly used techniques in analytical/bioanalytical separation and
detection.
INTRODUCTION 5
measurements now support key decisions throughout the drug development process,
from lead optimization to regulatory approvals. They are essential for documenting
exposure–response relationships, specificity and potency toward themolecular target,
untoward effects, and therapeutic applications. In a broader sense, biomarkers
constitute the basis of clinical pathology and laboratory medicine. The utility of
biomarkers is limited by their specificity and sensitivity toward the drug or disease
process and by their overall variability. Understanding and controlling sources of
variability is not only imperative for delivering high-quality assay results, but
ultimately for controlling the size and expense of research studies. Variability in
biomarker measurements is affected by biological and environmental factors (e.g.,
gender, age, posture, diet, and biorhythms), sample collection factors (e.g., preser-
vatives, transport and storage conditions, and collection technique), and analytical
factors (e.g., purity of reference material, pipetting precision, and antibody speci-
ficity). The quality standards for biomarker assays used in support of nonclinical
safety studies fall under GLP (FDA) regulations, whereas, those assays used to
support human diagnostics and healthcare are established by Clinical Laboratory
Improvement Amendments (CLIAs) and Centers for Medicare &Medicaid Services
(CMSs) regulations and accrediting organizations such as the College of American
Pathologists (CAPs). While most research applications of biomarkers are not
regulated, biomarker laboratories in all settings are adopting similar laboratory
practices in order to deliver high-quality data. Because of the escalation in demand
for biomarker measurements, the highly parallel (multiplexed) assay platforms that
have fueled the rise of genomics will likely evolve into the analytical engines that
drive the biomarker laboratories of tomorrow. The role of biomarkers in drug
discovery and development has gained precedence over the years. As biomarkers
become integrated into drug development and clinical trials, quality assurance and, in
particular, assay validation become essential with the need to establish standardized
guidelines for bioanalytical methods used in biomarker measurements. New bio-
markers can revolutionize both the development and use of therapeutics but are
contingent on the establishment of a concrete validation process that addresses
technology integration and method validation as well as regulatory pathways for
efficient biomarker development. Perspective focuses on the general principles of the
biomarker validation process with an emphasis on assay validation and the collab-
orative efforts undertaken by various sectors to promote the standardization of this
procedure for efficient biomarker development. It is important to point out that
biomarker method validation is distinct from pharmacokinetic validation and routine
laboratory validation. The FDA has issued guidance for industry [5] on bioanalytical
method validation for assays that support pharmacokinetic studies that are specific for
chromatographic and ligand-binding assays, and that are not directly related to the
qualification or validation of biomarker assays.Whereas routine laboratory validation
refers to laboratories that do testing on human specimens for diagnosis, prevention, or
treatment of any disease and falls under the jurisdiction of the Clinical Laboratory
Improvement Amendments of 1988, there is little regulatory guidance on biomarker
assay validation. Hence, a “fit-for-purpose” approach for biomarker method devel-
opment and validation is derived with the idea that assay qualification or validation
6 INTRODUCTION, OBJECTIVES, AND KEY REQUIREMENTS FOR GLP REGULATIONS
should be tailored to meet the intended purpose of the biomarker study. Numerous
applications using bioanalytical techniques have generated enormous interests and
some case reveal ultimate solutions in drug efficacies and other indications that are
critical to the success in drug/product development and approval processes.
1.1.3 Good Laboratory Practices Versus Bioanalytical Labs/Bioanalysis
Recently, more and more debate and discussion around the connection between GLP
and Bioanalysis are surfaced. It is noted that there is no direct reference from GLP
regulations to bioanalysis. However, it has become common terminology and
acceptance when people refer to GLP–Bioanalysis. In a regulatory term, it may be
referred as regulated bioanalysis to support programs or studies under GLP compli-
ance. There is a misconception in some quarters that GLP is required for the conduct
of clinical studies. This is not correct. The introduction to the OECD Principles of
GLP (and the introduction to the USFDA GLPs in 21 CFR part 58) makes clear that
they apply only to the portions of nonclinical (preclinical) studies. The relevant
documents for clinical studies are the various codes of GC(R)P (e.g., ICH; TGA). The
USFDA and other registration authorities do require a demonstration of the quality of
test data from clinical studies. In the United States, this may well be by means of
conformance with Clinical Laboratories Improvement Act (CLIA) [6]. In Australia,
this is best demonstrated by the testing laboratory’s NATA accreditation (in Medical
Testing, Chemical Testing, etc.)1. Nevertheless, bioanalytical laboratories generate
data in support of clinical studies and ultimately as part of data submissions to
regulatory agencies. More detailed discussions on above techniques and guidelines
are available in respective chapters of this book. The regulatory environment in which
clinical trials are conducted continues to evolve. The changes are generally focused on
requiring more rigorous control within the organizations performing clinical trials in
order to ensure patient safety and the reliability of data produced. The global
acceptance of the ICH Guideline for GCP and the implementation of the European
Union Clinical Trials Directive (2001/20/EC) are two clear examples of such change.
For some years, it has been internationally recognized that clinical laboratories
processing specimens from clinical trials require an appropriate set of standards to
guide good practices. With that aim in mind, the Good Clinical Laboratory Practice
Guidelines [7] were drafted and published in 2003 by a working party of the Clinical
Committee of the British Association of ResearchQuality Assurance (BARQA). This
guidance identifies systems required and procedures to be followed within an
organization conducting analysis of samples from clinical trials in compliance with
the requirements of GCP. It thus provides sponsors, laboratory management, project
managers, clinical research associates (CRAs), and quality assurance personnel with
the framework for a quality system in analysis of clinical trial samples, ensuring GCP
compliance overall of processes and results.
1 The National Association of Testing Authorities (NATA)—Australia’s national laboratory accreditation
authority. NATA accreditation recognizes and promotes facilities competent in specific types of testing,
measurement, inspection, and calibration.
INTRODUCTION 7
1.2 OBJECTIVES AND KEY REQUIREMENTS FOR GLP
REGULATIONS
The ability to provide timely, accurate, and reliable data is essential to the role of
analytical and bioanalytical chemists and is especially true in the discovery, devel-
opment, and manufacture of pharmaceuticals and life science products. Analytical
and bioanalytical data are used to screen potential drug candidates, aid in the
development of drug syntheses, support formulation studies, animal PK/Tox, clinical
safety and efficacy programs, monitor the stability of bulk pharmaceuticals and
formulated products, and test final products for release. The quality of analytical and
bioanalytical data is a key factor in the success of a drug or product development
program. The process of method development and validation has a direct impact on
the quality of these data.
Although a thorough validation cannot rule out some potential problems, the
process ofmethod development and validation should address themost commonones.
Examples of typical problems that can be minimized or avoided are synthesis
impurities that coelute with the analyte peak in an HPLC assay; a particular type
of column that no longer produces the separation needed because the supplier of the
column has changed themanufacturing process; an assaymethod that is transferred to
a second laboratory where they are unable to achieve the same detection limit; and a
quality assurance audit of a validation report that finds no documentation on how the
method was performed during the validation.
Problems increase as additional people, laboratories, and equipment are used
to perform the method. When the method is used in the developer’s laboratory, a
small adjustment can usually be made to make the method work, but the flexibility
to change it is lost once the method is transferred to other laboratories or used for
official product testing. This is especially true in the pharmaceutical and life
science industries, where methods are submitted to regulatory agencies and changes
may require formal approval before they can be implemented for official testing/
intended use. The best way to minimize method problems is to perform adequate
validation experiments during development and establishment. Analysis of chemi-
cals/drugs in the complex environments/matrices in which they occur are carried out
by a vast range of institutions for a variety of purposes, from pharmaceutical and
agrochemical companies to hospital biochemistry labs and industry laboratories, from
environmental monitoring to safety and toxicity testing of new drugs/products. The
range of compounds for analysis is enormous, from naturally occurring compounds
such as vitamins to man-made chemicals from the pharmaceutical and agrochemical
industries. The following chapters offer an integrated, readable reference text
describing the full range of analytical techniques and regulatory requirements
available for such small molecules (mostly) and large molecules in an up-to-date
manner and should be useful and appeal to all involved in the rapidly growing field of
bioanalytical sciences.
. Responsibilities should be defined for the sponsor management, for the study
management, and for the quality assurance unit.
8 INTRODUCTION, OBJECTIVES, AND KEY REQUIREMENTS FOR GLP REGULATIONS