The Bacterial Endotoxins Test:A Practical Approach

Karen Zink McCullough

PDABethesda, MD, USADHI Publishing, LLCRiver Grove, IL, USA

Front Matter:Front Matter copy 9/3/11 20:18 Page 1

www.pda.org/bookstore

10 9 8 7 6 5 4 3 2 1

ISBN: 1-933722-49-5Copyright © 2011 Karen Zink McCulloughAll rights reserved.

All rights reserved. This book is protected by copyright. No part of it may bereproduced, stored in a retrieval system or transmitted in any means, electronic,mechanical, photocopying, recording, or otherwise, without written permissionfrom the publisher. Printed in the United States of America.

Where a product trademark, registration mark, or other protected mark ismade in the text, ownership of the mark remains with the lawful owner of themark. No claim, intentional or otherwise, is made by reference to any such marksin the book.

While every effort has been made by the publisher and the author to ensurethe accuracy of the information expressed in this book, the organization accepts noresponsibility for errors or omissions. The views expressed in this book are thoseof the editors and authors and may not represent those of either Davis HealthcareInternational or the PDA, its officers, or directors.

This book is printed on sustainable resource paper approved by the Forest Stewardship Council. Theprinter, Gasch Printing, is a member of the Green Press Initiative and all paper used is from SFI(Sustainable Forest Initiative) certified mills.

PDA Davis Healthcare International Publishing, LLC4350 East West Highway 2636West StreetSuite 200 River GroveBethesda, MD 20814 IL 60171United States United Stateswww.pda.org/bookstore www.DHIBooks.com301-986-0293

Front Matter:Front Matter copy 9/3/11 20:18 Page 2

www.pda.org/bookstore

CONTENTS

Foreword xiiiAuthor’s Preface xvGlossary xix

1 Discovery and Acceptance of the BacterialEndotoxinsTest 1

James F. CooperDiscovery 1

Preparation of Limulus Amebocyte Lysate (LAL) 3Origin of LAL methods 3Comparison of LAL and pyrogen tests 4Commercialization of LAL reagent 5Symposia atWoods Hole 6

Acceptance 6Pyrogen and LAL tests at the FDA 7Industry acceptance of LAL methods 7The LAL-Test Guideline 8Revolutionizing the validation of depyrogenation processes 9LAL Reactive Glucan (LRG) challenges specificity 9Evolution of the compendial Bacterial Endotoxins Test (BET) 10

iii

Contents:Layout 1 9/3/11 20:17 Page iii (Black plate)

www.pda.org/bookstore

Summary 10References 11About the Author 13

2 Understanding Reaction Basics 15Michael E. Dawson

Introduction 15The LAL Clotting Reaction 15

The recombinant Factor C assay 22Endotoxin 24Glucans 25Conclusion 26References 26About the Author 28Appendix 29

3 Constructing and Interpreting Standard Curvesfor Quantitative BET Assays 31

Karen Zink McCulloughThe Endpoint Assay 33The Kinetic Assay 36Accuracy 39Anatomy and Attributes of a Standard Curve 40

Slope 40y-intercept 41Correlation coefficient 41Coefficient of variation 42

Analysis: Effects of Standard Curves on the Accuracy 43of DataScenario 1 — effect of changes in slope 45Scenario 2 — effect of changes in y-intercept 48Scenario 3 — effect of changes in %CV 50Scenario 4 — effect of non-linearity 52Alternate regression analysis 56

Discussion 57Conclusion 60References 61About the Author 62

The Bacterial Endotoxins Test: A Practical Approachiv

Contents:Layout 1 9/3/11 20:17 Page iv (Black plate)

www.pda.org/bookstore

4 Qualifying the Laboratory 63Ronald N. Berzofsky

Equipment 64Qualifying Incubators 65Qualifying Timers 70Qualifying Ancillary Supplies 71Qualifying Procedures 74

Inhibition/enhancement 74Storage of samples and standards 75

Qualifying Reagents 76The LAL reagents 76Endotoxins 77Buffers and solutions 78Qualifying the analyst 80

Dilutions 80Conclusions 82References 83About the Author 83

5 Calculating Endotoxin Limits, MaximumValidDilutions and MinimumValid Concentrations 85

Karen Zink McCulloughEndotoxin Limits 85

Endotoxin limits for small volume parenteral drugsand biologicals 86

Endotoxin limits for large volume parenterals 92Endotoxin limits for radiopharmaceuticals 93Endotoxin limit for drugs administered per square meter

of body surface 94Setting endotoxin limits for a product 95Endotoxin limits in test solutions 96

MaximumValid Dilution (MVD) 97MinimumValid Concentration (MVC) 99Summary 101Problem Set 102References 110About the Author 113

Contents v

Contents:Layout 1 9/3/11 20:17 Page v (Black plate)

www.pda.org/bookstore

6 Assigning Endotoxin Limits to NoncompenialArticles 115

Michael E. DawsonConclusion 129References 129About the Author 130

7 Applying USP Test Requirements: Medical Devices 131Marilyn J. Gould

The Challenge 131USP and Other References Pertaining to Testing Devices 132Historical Perspectives — How Much Endotoxin is Pyrogenic? 133A Reference Standard Endotoxin and Threshold

Pyrogenic Dose 134Comparing the 1987 FDA Guideline Device Limits with

Drug or Biologics Limits 135Intraethecal Endotoxin Limit 135USP <161> Specifies LAL ReagentWater (LRW) as the

Extraction Medium 136SituationsWhen a Solvent Other thanWater

May Be Required 137Qualify the Extraction Medium 138Sample Sizes MayVary with Lot Sizes 138USP Equation to Calculate Limits for the BET 139ExtractionVolumes Less than 40 mL/Device 140ExtractionVolumes Greater than 40mL/Device 141Find a Convenient Volume Other than 40 mL/Device 141MaximumValid Dilution (MVD) 142What is a Device for Purposes of Calculating Limits? 143Critical Surfaces 143Before Testing a Device for the First Time 144What ExtractionVolume Should be Used? 149What if the Ideal ExtractionVolume Exceeds the

Calculated MaximumVolume? 150Multiple extractions in the same volume 150Concentrate the extract 150

Extraction Containers 152Extractions — Physical Considerations 152Challenge and Recovery 153Handling Devices to Set Up Extractions 154Steps to Perform a USP Test on a Medical Device 155One Final Note 157

The Bacterial Endotoxins Test: A Practical Approachvi

Contents:Layout 1 9/3/11 20:17 Page vi (Black plate)

www.pda.org/bookstore

References 158About the Author 161

8 Verifying USPTest Methodology 163Karen Zink McCullough

Spiking 167Double-double method 168Hot spike 169

Test Method Development 170Calculation of the endotoxin limit 171Calculation of the MVD and/or MVC 173Preliminary testing 174

Inhibition/Enhancement, or Verification of the Suitabilityof the Test Method 181Suitability testing for gel clot 182Suitability testing for quantitative tests 185

Some Questions Regarding Suitability Testing 186Chapter Questions 187Appendix. Example Template for a Gel Clot Suitability Study 188References 192About the Author 193

9 ResolvingTest Interferences 195John Dubczak

Inhibition 196Inhibition due to pH 197Inhibition due to high osmolarity 200Inhibition due to chelating agents 200Inhibition due to Ca++ containing formulations 201Inhibition due to protease inhibitors 202Inhibition due to heavy metals 202Inhibition due to detergents 204Inhibition due to proteins 204Inhibition due to liposomes 205

Enhancement 206Enhancement due to beta 1-3 glucans 206Enhancement due to serine proteases 210Enhancement due to detergents 210

Conclusion 212References 212About the Author 214

Contents vii

Contents:Layout 1 9/3/11 20:17 Page vii (Black plate)

www.pda.org/bookstore

10 Performing RoutineTests 215Alan Baines

Introduction 215Equipment and materials 217

Sampling 218Sample containers and accessories 218SamplingWater for Injection (WFI) 219Sampling incoming raw materials 221In-process samples 222Drug product sampling 225Pooling 225Storing endotoxin standards and test samples 227

Establishing a Testing Routine 228Sources of error 228Assay prerequisites 229Controls 233

Negative control 233Positive Product Control (PPC) 233Method 1:“Double-double” method 234Method 2:“Hot spike” method 234Standard series (gel clot) or standard curves(quantitative tests) 236

Sample preparation 237Assay set-up — gel clot 238

Gel clot limits test 238Gel clot assay 240

Assay set-up: quantitative tests 241Standard curves in routine testing 242Archived standard curves 243Product standard curves 244Positive product controls and routine testing 245

Data Analysis and Interpretation 248Calculating and reporting results for the gel clot

limits test 249Gel clot assay 250Quantitative tests 252Converting EU/ml to EU/unit of drug product 252

Robotics and Routine Testing 253Reporting Results 253

Header section 254Standards 255Sample results 257Masking test results 259

The Bacterial Endotoxins Test: A Practical Approachviii

Contents:Layout 1 9/3/11 20:17 Page viii (Black plate)

www.pda.org/bookstore

Reviewing results 259Alert and action limits 260OOS and re-testing 261Trending 262

Appendix 1 Effect of high background on PPC Recovery 264Appendix 2 Robotics 266References 269About the Author 270

11 Investigating Out-of-Specification (OOS)BET Results 271

Karen Zink McCullough1987 FDA Guideline 272Current Thinking: FDA’s 2006 OOS Guidance 274

Principle 1.The laboratory may not test a productinto compliance 274

Principle 2.Test results may not be averaged into compliance 275Principle 3. Investigations into OOS results must be thorough,unbiased, scientific, timely and well documented 276Principle 4. Investigations, where appropriate, must include

associated batches of product 278SOPs for Investigating BET OOS Results 280Phase I Laboratory Investigations: Identifying Critical

Points in the Lab 283Creating the OOS Checklist: Fault Tree Analysis 285

Retesting 292Beyond the Laboratory: Phase II Investigation 292Thoughts on the Pretest from the Beginning of this Chapter 294Summary 295References 295About the Author 295Appendix. Basic checklist for Investigating BET OOS Results 296

12 Structuring a Deyprogenation Study 299John Dubczak

Elements to a Depyrogenation Process Qualification 300Endotoxin Indicators and Their Use 303Dry Heat Pyrogenation 305Dry Heat Process Qualification Studies 306Cleaning/Rinsing Depyrogenation Process Qualification 306

Equipment cleaning/depyrogenation 307

Contents ix

Contents:Layout 1 9/3/11 20:17 Page ix (Black plate)

www.pda.org/bookstore

Container/closure rinsing depyrogenation 308Depyrogenation in Biotechnology 310

Ultrafiltration 311Electrostatic attraction via charge modified media 312Affinity chromatography 313

Other Means of Destroying Endotoxin 314Acid and base hydrolysis 314Oxidation 314

Conclusion 315References 315About the Author 317

13 Setting Alert and Action Limits 319Ronald N. Berzofsky

Process Capabilities vs. Product Specifications 319Standard Deviation vs. Percentiles 321Probability and an Unexpected Event 326Control Charts 328Steps in Calculting Alert and Action Events 329About the Author 332

14 Identifying Critical Control Points in Manufacturing 333Karen Zink McCullough

The Manufacturing Process 334The Product Formulation 337HACCP 338

HACCP Step 1. Conduct a hazard analysis 339HACCP Step 2. Identify ctritical control points (CCP) 339

Process step: receipt and testing of raw materials 341Process step: sterile filtration 342Process step: depyrogenation 343Process step: QC testing 343

HACCP Step 3. Establish control limits 344Process step: testing of raw materials 344Process step: depyrogenation 345Process step: QC testing 345

HACCP Step 4. Establish monitoring procedures 346Process step: raw material testing 346Process step: deyprogenation 347Process step: QC testing 347

HACCP Step 5. Establish corrective actions 347

The Bacterial Endotoxins Test: A Practical Approachx

Contents:Layout 1 9/3/11 20:17 Page x (Black plate)

www.pda.org/bookstore

FMEA 348HACCP Step 6. Establish verification procedures 356HACCP Step 7. Establish record-keeping and documentationprocedures 356

Summary 357References 357About the Author 359

15 Auditing the BET Laboratory 361Karen Zink McCullough

The Audit Cycle 364Planning 365

Preparing an audit checklist 367Preparation 369Execution 373Reporting 374Monitoring 375Summary 378Appendix. Sample audit plan/checklist 379References 382About the Author 383

16 Regulatory Summary 385Karen Zink McCullough

Index 399

Contents xi

Contents:Layout 1 9/3/11 20:17 Page xi (Black plate)

www.pda.org/bookstore

Contents:Layout 1 9/3/11 20:17 Page xii (Black plate)

www.pda.org/bookstore

FOREWORD

Scott Sutton

It is an honor to have been asked to write the foreword to thisimportant book on the Bacterial Endotoxins Test. This volume issignificant not only because of the timing of the work, coming asthe field is in a state of flux with new technologies and newregulations, but also because of its practical, bench-level focus.

There are several books available on the Bacterial EndotoxinsTest. Some approach it from an ecological perspective, focusing onthe horseshoe crab and the impact that farming is having on itspopulation. Others address the topic from a clinical and chemicalperspective, heavy on theory and academic considerations. Theseare valuable contributions to the literature and their review isrecommended for anyone who wishes a broad grounding in thedevelopment and science of the test. However, they are not directedspecifically at the needs of the lab worker in regulated industries. Infact, this book is one of a very limited number of technicallycompetent works directed to the QC lab worker in any topic and forthat reason alone warrants a place on the serious qualityprofessional’s bookshelf.

xiii

Foreword:Layout 1 9/3/11 20:18 Page xiii (Black plate)

www.pda.org/bookstore

Even beyond this consideration, it has to be noted that it isdifficult to bring a diverse group of workers together and producea book that is readable and technically challenging at the same time.The top-rank authors assembled herein have produced such a book,and done it without yielding to the temptation of promoting theirown products. This is especially noteworthy in that most of theauthors work for companies that are in direct competition with eachother, and are generally not shy about disagreeing with each other.The discussions in this book are remarkable in the absence ofcommercial promotion — science and technical methodology takecenter stage throughout.

Enjoy the time you spend with this book. It won’t really matterif you are looking for a historical perspective on the development ofthe test or an explanation of exactly why the y-intercept is animportant consideration of the standard curve, you will almostcertainly learn something new about the bacterial endotoxin testand the role it plays in QC operations in pharma, biopharma,medical devices and all healthcare-related regulated industries

Scott Sutton, Ph.D.Microbiology Network, Inc.

April, 2011

The Bacterial Endotoxins Test: A Practical Approachxiv

Foreword:Layout 1 9/3/11 20:18 Page xiv (Black plate)

www.pda.org/bookstore

AUTHOR’S PREFACE

Welcome.

The goal of this book is to provide sensible and usefulinformation for those who are responsible for the performance andinterpretation of the Bacterial Endotoxins Test (BET). The conceptwas to create a user-friendly reference tool for BET analysts,laboratory managers and regulatory/compliance specialists that ispart lab manual, part textbook, part tutorial, and part non-commercial consultant.

For those laboratories new to BET, this book lays a solidfoundation by providing information and tips for qualifying thelaboratory, calculating endotoxin limits, verifying USP testmethodology, resolving test interferences and performing routinetests for parenteral drugs, biologics and medical devices.

For those laboratories that are experienced in BET, this bookbuilds on basic testing requirements by discussing topics such as

xv

Authors Preface:Layout 1 9/3/11 19:54 Page xv (Black plate)

www.pda.org/bookstore

setting endotoxin limits for noncompendial articles, structuring adepyrogenation study, setting action and alert limits andidentifying endotoxin-specific critical control points inmanufacturing.

The content of this book represents an amazing 200+ author-years of experience in the field. With many thanks to Alan Baines,Ron Berzofsky, Jim Cooper, Mick Dawson, John Dubczak andMarilyn Gould for their extraordinary contributions to this effort,their remarkable perspective and their expert sage advice, I hopethat the information in this book will help everyone to take apractical approach to the Bacterial Endotoxins Test.

Karen Zink McCulloughWhitehouse Station, NJ

April, 2011

The Bacterial Endotoxins Test: A Practical Approachxvi

Authors Preface:Layout 1 9/3/11 19:54 Page xvi (Black plate)

www.pda.org/bookstore