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Evaluation, Validation and Implementationof New Microbiological Testing Methods

Technical Report No. 33

PDA

May /June 2000

Vol.54, No. 3, May / June 2000, Supplement TR33 i

Task Force Members

Brian Bauer, Ph.D., Merck & Co., Elkton, VirginiaMark Claerbout, Lilly Research Laboratories, Indianapolis, IndianaWarren Casey, Ph.D., GlaxoWellcome R&D, Research Triangle Park, North CarolinaAnthony M. Cundell, Ph.D., Wyeth-Ayerst Pharmaceuticals, Pearl River, New York (Chair)Martin Easter, Ph.D., Celsis Ltd., Cambridge, EnglandEdward Fitzgerald, Ph.D. Consultant, (USP Microbiology Subcommittee)Carol Gravens, BioMerieux, Inc., Hazelwood, MissouriDavid Hussong, Ph.D., CDER, FDA, Rockville, MarylandMichael Korcynzski, Ph.D., PDA Training Institute, Baltimore, Maryland (USP Microbiology Subcommittee)Robin Lerchen, American Pharmaceutical Partners, Melrose Park, IllinoisFrederic J. Marsik, Ph.D. CDER, FDA, Rockville, MarylandAmy Meszaros, StatProbe Inc, Ann Arbor, MichiganJeanne Moldenhauer, Ph.D., Jordan Pharmaceuticals, Inc., Elk Grove, IllinoisManju Sethi, Qualicon, Wilmington, DelawareScott Sutton, Ph.D., Alcon Laboratories, Fort Worth, Texas (USP Microbiology Subcommitee)Martin Tricarico, Chemunex (USA), Monmouth Junction, New JerseyAmanda Turton, Millipore Corp, Bedford, MassachusettsChristine Vojt, Johnson & Johnson Diagnostics Inc., Rochester, New YorkKirsty Wills, Celsis Ltd., Cambridge, EnglandJon Wuannlund, Becton Dickinson Microbiology Systems, Cockeysville, Maryland

PDA TECHNICAL REPORT NO.33EVALUATION, VALIDATION AND IMPLEMENTATIONOF NEW MICROBIOLOGICAL TESTING METHODS

Table of Contents

Part One: Selection of New Microbiological Methods1.0 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1 Scope of Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Purpose of Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3 Overview of Document Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2.0 Technology Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.1 Generic Description of Types of Microbiological Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2 Technology Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32.3 Growth-based Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.3.1 ATP Bioluminescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32.3.2 Colorimetric Detection of CO

2 Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.3.3 Measurement of Change in Head Space Pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.3.4 Impedance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.3.5 Biochemical Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.4 Viability-based Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62.4.1 Solid Phase Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62.4.2 Flow Fluorescent Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

2.5 Artifact-based Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72.5.1 Fatty Acid Profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72.5.2 Mass Spectometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72.5.3 ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72.5.4 Fluorescent Probe Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72.5.5 Bacterial Endotoxin-Limulus Amebocyte Lysate Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7

2.6 Nucleic Acid-based Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82.6.1 DNA Probe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82.6.2 Ribotyping/Molecular Typing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82.6.3 Polymerase Chain Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

3.0 Regulatory Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103.1 General Classification of Microbiological Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

3.1.1 In-Process Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103.1.2 Product Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103.1.3 Qualitative Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103.1.4 Quantitative Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

3.2 Compendial Microbiological Test Method References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103.2.1 Water for Pharmaceutical Purposes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103.2.2 Antimicrobial Effectiveness Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103.2.3 Microbial Limit Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113.2.4 Sterility Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113.2.5 Environmental Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113.2.6 Microbial Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Vol.54, No. 3, May / June 2000, Supplement TR33 iii

3.3 Changing a Microbiological Test Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113.3.1 The Regulatory Perspective on the Introduction of New Microbiological Test Methods . . . . 113.3.2 The Compendial Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

3.4 Obtaining Compendial Change . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133.5 Regulators Assessment of New Microbiological Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Part Two: How to Validate and Implement a New Microbiological Test Method

4.0 The Validation Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .154.1 The Equipment Qualification Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

4.1.1 Vendor/Specification Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164.1.1.1 Test Method Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174.1.1.2 Vendor Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

4.1.2 Validation Plan Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184.1.3 Installation Qualification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184.1.4 Operation Qualification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204.1.5 Performance Qualification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

4.2 Validation Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214.2.1 Preparation of Test Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234.2.2 Variability of Microbiological Methods: Special Note . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25

4.2.2.1 Sample Distribution Error . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254.2.2.2 Cell Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254.2.2.3 Metabolic Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

4.2.3 Protocol Design Using Recommended Validation Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264.3 Special Considerations for the Validation of Microbiological Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

4.3.1 Use of Multiple Pieces of the Same Equipment within the Laboratory and Company . . . . . . . . . . . . . 294.3.2 Unique Testing Requirements for Microbiological Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

5.0 Glossary of Terms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

6.0 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

iv PDA Journal of Pharmaceutical Science & Technology

PART ONE:Selection of New Microbiological Methods

1.0 INTRODUCTION

1.1 Scope of Document

This document is intended to provide guidance forthe successful evaluation, validation, and implemen-tation of new microbiological methods needed bythe pharmaceutical, biotechnology, and medical de-vice industries to assure product quality. Applica-tions for these methods include but are not limitedto Microbial Limit Testing, Sterility Testing, Anti-microbial Effectiveness Testing, MicrobiologicalMonitoring of Clean Rooms and Other ControlledEnvironments, Water for Pharmaceutical PurposesMonitoring, and Microbial Characterization andIdentification.

The intended audience of this report is microbiolo-gists who are responsible for the validation of themicrobiological test methods used in the routine mi-crobiology testing laboratory. The document shouldbe of interest to suppliers of testing equipment, mi-crobiology managers and supervisors, validationspecialists, quality control personnel responsiblefor the approval of validation protocols and the re-lease of product, and regulatory agencies.

1.2 Purpose of Document

Microbiological testing plays an ever increasing rolein the pharmaceutical laboratory. In response to this,a variety of new methodologies have emerged in re-cent years which automate existing methods, makeuse of surrogate markers for growth, or are based onwholly new technologies. These new methodolo-gies offer significant improvements in terms of thespeed, accuracy, precision, and specificity withwhich testing can be performed.

The majority of testing performed today relies oncentury-old methods, based on the recovery andgrowth of microorganisms, using solid or liquid mi-crobiological growth media. This is true in part be-cause these methods can be very effective and havea long history of application in both industrial andclinical settings. However, they are often limited by

slow microbial growth rates, the unintended selectiv-ity of microbiological culture, and the inherent vari-ability of microorganisms in their response to culturemethods. In spite of the limitations of current culturemethods, acceptance of new and potentially superiormethods is often slow. We believe this is due in partto a lack of clear guidance regarding the demonstra-tion of their equivalence to existing methods accept-able to regulatory agencies and validation of the equip-ment associated with the new methods. This techni-cal report hopes to provide guidance to assist with theevaluation, validation, and implementation of thenewer microbiological methods.

Considerable guidance can be found regarding thevalidation of chemical methods. Examples includeUSP General Informational Chapter <1225> Valida-tion of Compendial Methods (1), and a recent publi-cation by the International Conference on Harmoni-zation (ICH) Validation of Analytical Methods (2).These publications provide very specific instructionregarding the demonstration of new analytical chem-istry methods and their equivalence to existing meth-ods. In contrast, virtually no guidance specific to mi-crobiological testing has been published. Possibleexceptions are the ASM Cumitech publication Verifi-cation and Validation of Procedures in the ClinicalMicrobiology Laboratory (3), that addresses pathogenisolation and identification and antimicrobial suscep-tibility testing, and the new USP General InformationChapter <1227> Validation of Microbial Recoveryfrom Pharmacopeial Articles (4). However, moreguidance is needed. Because microbiological meth-ods are inherently different than chemical assays, thislack of agreed upon demonstration criteria can presentserious obstacles to their implementation.

When instrumentation is developed for existing mi-crobiological methods to automate sample handling,result reading, or data management or to miniaturizethe test procedure, it is not difficult to demonstrate theequivalency of the alternate method using guidelinesdeveloped for chemical assays, because the test re-mains essentially the same. In a similar fashion, whena new technology continues to rely on the measure-ment of microbial growth (e.g., impedance, ATPbioluminescence or other metabolic changes in a

Vol.54, No. 3, May / June 2000, Supplement TR33 1

microbial culture), equivalence can be readily dem-onstrated. However, when a new method is basedon novel technology without direct ties to the ex-isting method (e.g., microbial identification by DNAamplification versus patterns of biochemical reac-tions, or counting fluorescent- labeled bacterial cellsinstead of colony-forming units on an agar plate),demonstration of equivalency may require a newapplication of the validation principles, although themethod provides higher quality results.

The application of these new technologies may becompared to the replacement of the Most ProbableNumber-multiple fermentation tubes with mem-brane filtration methods for counting coliforms inwater, and the change from solely morphologicalfeatures to physiological and biochemical charac-teristics to DNA-based methods for the identifica-tion of bacteria. For example, the ability to countindividual, viable, fluorescent-labeled microbialcells by scanning a membrane with a laser shouldbe superior to counting colony-forming units; thenucleic acid contained in a microorganism is con-servatively unique to that species, i.e., genotypicidentification, hence is superior to patterns of bio-chemical reactions that are currently used to iden-tify bacteria, i.e., phenotypic identification.

This document, was developed as a collaborativeeffort amongst representatives from test manufac-turers, the pharmaceutical and device industry, stan-dards organizations, and regulatory agencies. It isintended to provide a general approach to the intro-duction of new microbiology methods in a govern-ment-regulated environment. It is anticipated thatby providing agreed upon performance standards,the development, demonstration and implementa-tion of superior methods will be greatly accelerated.

1.3 Overview of Document Structure

This document was written to establish industry-wide criteria on what constitutes an acceptable mi-crobiology test and how to prove it to the satisfac-tion of a regulatory agency.

The document is divided into two major sections.In the Method Selection section, a review of micro-biology testing methods is followed by an overviewof compendial applications which make use of thesetests. A discussion of requirements for regulatoryacceptance and economic justification completes thesection. In the Validation section, criteria used forvalidation and demonstration of equivalence are de-fined, and approaches to validation methods anddocumentation are described. Finally, a decision tree,glossary of commonly used terms, and a bibliogra-phy are provided to present an overview of the en-tire process.

2.0 TECHNOLOGY OVERVIEW

2.1 Generic Description of Types of Microbiological Methods

Microbiological test methods can be divided intothree general categories, based on their function:1) detection of the presence or absence of micro-organisms in a test sample, 2) enumeration of mi-croorganisms present in a test sample, and 3) char-acterization and identification of microorganismseither present in test samples or from a pure cul-ture.

Presence/absence tests may be designed to detectdiverse types of microorganisms, as in sterilitytest methods, or may be intended for detection ofspecific microbial species or genera, as in the testsfor Pharmacopeial Indicator Organisms. USP, Ph.Eur. and JP have the same set of indicator organ-isms, i.e., Escherichia coli, Pseudomonasaeruginosa, Staphylococcus aureus, and Salmo-nella species.

The results obtained when testing samples by dif-ferent enumeration methods based on classical mi-crobiological techniques may vary greatly, de-pending on the test conditions, e.g., media typeand incubation temperature, conditions, and du-ration.

An example is the difference seen in plate countsfrom water samples using a low nutrient medium

2 PDA Journal of Pharmaceutical Science & Technology

such as R2A agar with a 5 to 7 day incubationperiod at ambient temperature, versus Soybean –Casein Digest agar incubated for 48 hours at 30to 35°C. Significant differences in counts mayalso be seen when comparing non-growth baseddirect detection methods with classical methods,with higher counts obtained by the former due tosuboptimal culture conditions in microbiologicalmedia.

Many types of microbial characterization andidentification test methods are used, ranging fromclassical Gram’s staining and simple manual testsfor specific enzymes associated with bacterialcells such as catalase and cytochrome oxidase,to compendial methods using the appearance ofmicrobial colonies on selective/differentialgrowth media, to semi-automated biochemicalprofiling systems, to methods based on the de-tection of specific nucleic acid sequences or cellmembrane fatty acid composition.

The test methods vary greatly in their cost, easeof use, duration, and performance as measuredby analytical parameters such as specificity, sen-sitivity, reproducibility, and ruggedness.

2.2 Technology Review

As discussed earlier, the century-old methods de-veloped by the pioneering microbiologists Pasteur,Koch, Lister, and many others is the technology basefor current compendial microbiological test meth-ods. These methods are based on providing condi-tions to allow microbial cells present in test samplesto grow and replicate sufficiently to allow their de-tection, typically by visual examination of a plate orbroth. Specific compendial tests will be discussedlater in the section on Regulatory Review, so thefocus of this section will be on newer technologies.

New technology for microbiology quality assur-ance testing is a rapidly developing area. The TaskForce has attempted to include all technologieswith current application in the pharmaceutical in-dustry. However, exclusion of a technology or

application of a technology is not intentional, anddoes not imply that the technology is not suitable.This review is simply the best available informa-tion at the time. Without doubt, further develop-ments will occur during the lifetime of this docu-ment, which will mean that new technologies or newapplications of existing technologies will be broughtto market. For convenience, the technologies aredivided into 1) growth-based technologies, 2) vi-ability-based technologies, 3) cellular componentor artifact-based technologies, and 4) nucleic acid-based technologies.

Reference to representative suppliers (see Tables 1,2, 3, and 4) is merely given as examples compiled bythe Task Force members and does not infer commer-cial endorsement on the part of the Task Force or PDA.

2.3 Growth-based Technologies

These methods are based on the measurement ofbiochemical or physiological parameters that reflectthe growth of the microorganisms.

2.3.1 ATP Bioluminescence

The presence of Adenosine Triphosphate (ATP) isa well-documented marker for cell viability. Allcells store energy in the form of ATP. An increasein cell numbers results in an increase in ATP lev-els. ATP bioluminescence utilizes the luciferin-lu-ciferase reaction of the firefly to detect the pres-ence of ATP by measuring the light emitted. Thepresence or absence of microbial contamination ina sample can be determined by measuring the in-crease in ATP levels following incubation. ATP bi-oluminescence reduces the test time to approxi-mately one third of that taken by the traditionalmethod. This is because it is a more sensitive end-point detection system, using sensitive chemistryand instrumentation, rather than relying on the hu-man eye. This means that identical principles are inuse, i.e., multiplication of microorganisms, but theamount of replication required for detection is sig-nificantly reduced: 103 cells per mL as opposed to 107

or greater, to determine turbidity or colonies on a plate.


2.3.2 Colorimetric Detection of CO Production

Test samples are placed in culture bottles for moni-toring. The samples are incubated, agitated, andmonitored for the presence of microorganisms.These systems use colorimetric detection of CO2production from the growth of organisms. Somesystems will detect color change, flag a positivesample, and alert the user. These systems are oftenreferred to as non-invasive microbial detection sys-tems and can accommodate a large number ofsamples. Although commonly used clinically forblood cultures, the method could be used for steril-ity testing.

2.3.3 Measurement of Change in Head Space Pressure

These systems are based on non-invasive, continu-ous, automated monitoring of microbial cultures.Electronic transducers are used to sense positive ornegative pressure changes in the head space of eachculture bottle. These changes are caused by micro-bial growth. If significant production and/ or con-sumption of gas is detected, samples are flagged aspositive. Large quantities of samples can be placedinto these instruments for testing with frequentmonitoring of the head space pressure. Althoughcommonly used clinically for blood cultures, themethod could also be used for sterility testing.

2.3.4 Impedance

Impedance measures microbial activity by elec-trical methods. These instruments measure ionicchanges occurring within the growth media asbacteria multiply. The impedance detection timeis inversely proportional to the number of micro-organisms present at initial inoculation. Bacteriametabolize larger, weakly charged molecules andproduce smaller highly charged by-products. Forexample, large weakly charged molecules such asproteins are hydrolyzed to many highly chargedamino acids. Two electrodes are then used tomeasure these ionic changes in the broth or agarculture.

2.3.5 Biochemical Assays

Microbial cell suspensions of pure cultures aretested with a series of biochemical substrates. Mi-croorganisms are known to have particular reac-tions to these biochemicals, e.g., carbohydrate uti-lization. By matching the biochemical resultswith a database of corresponding results, one candetermine the identification of the organism be-ing tested. Many of these methods are performedand recorded manually. High volume, automatedinstruments are also commercially available toread miniature cultures and identify the microor-ganisms from the pattern of reactions in the data-base.

In the following tables, presence/absence, enu-meration and identification methods are designedas P, E, and I respectively.

2

2


SteriScreen and MicroStar RapidMicrobiology and MicroCount DigitalSystems (Millipore Corp, Bedford, MA& RapiScreen (Celsis, Evanston, IL)

Bact/Alert (Organon-Teknika, Durham,NC) & ESP Microbial Detection System(AccuMed International, Detroit, MI)

API Systems (bioMerieux, Hazelwood,MO), BIOLOG Systems (Biolog, SanDiego, CA) & VITEK System(bioMerieux, Hazelwood, MO)

Bactometer (bioMerieux, Hazelwood,MO) & Malthus Microbial DetectionSystem (Malthus Diagnostics NorthRidgeville, OH)

Spiral Plating System (Spiral Biotech,Bethesda, MD)

Iso-Grid (QA LifeScience, San Diego,CA)

Conventional

Conventional

Conventional

Raw material & productscreening, water monitor-ing, pre-sterile filtrationbioburden monitoring (E)Sterility testing (P)

Sterility testing (P)

Microbial identification (I)

Bioburden monitoring,antimicrobial effectivenesstesting (E)

Bioburden monitoring (E)

Bioburden monitoring (E)Pathogen monitoring (E)

Bioburden monitoring,water monitoring,antimicrobial effectivenesstesting (E)

Bioburden monitoring,water monitoring, D-Valueanalysis (E)

Bioburden monitoring (E)Sterility testing (P),Water monitoring (E)

ATPBioluminescence

Colorimetric Detectionof Carbon DioxideProduction HeadspacePressure

Biochemical &PhysiologicalReactions

Impedance/Conductivity

Spiral plating

Hydrophobic GridMembrane FilterMethods

Pour Plate Method

Most ProbableNumber Multiple -Tube Method

Membrane FiltrationMethod

Table 1: Growth-based Microbiological Methods

Technology Representative Commercial Products Principal Applications



2.4 Viability-based Technologies

2.4.1 Solid Phase Cytometry

The method of solid phase cytometry utilizes mem-brane filtration to separate potential microbial con-taminants from filterable samples prior to labelingof the captured cells with a universal viability sub-strate. Once within the cytoplasm of metabolicallyactive microorganisms, the non-fluorescent sub-strate is enzymatically cleaved to release free fluo-rochrome by the hydrolytic enzyme esterase. Onlyviable microorganisms with membrane integrityhave the ability to retain the marker used in the as-say. The filter is then automatically scanned by alaser based detector, and the number of fluorescent-labeled cells immediately reported.

Since the method eliminates the need for cell mul-tiplication, sensitivities to the single cell level in-dependent of volume filtered, are possible for allmicrobial cells including spores, as well asstressed cells and fastidious organisms. Since amuch more sensitive end point detection mecha-nism is employed when compared to growth basedprocedures, near real-time results are available fora full range of viable microorganisms.

2.4.2 Flow Fluorescence Cytometry

In addition to filterable products, this approachhas now been applied to non-filterable productsby employing similar labeling chemistries tohighly sensitive flow cytometers.

Table 2: Viability-based Microbiological Methods

Direct Epifluorescent Filter Technique(DEFT) Automate Counting System(Micro-Measurements, Saffron Walden,Essex, United Kingdom)

Scan RDI (Chemunex, MonmouthJunction, NJ)

DCount (Chemunex, Monmouth Junc-tion, NJ)


Water monitoring,Bioburden monitoring,Environmental monitoring(E)Sterility testing (P)


Direct EpifluorescentFilter Microscopy

Membrane LaserScanning FluorescenceCytometry

Fluorescence FlowCytometry


2.5 Artifact-based Technologies

2.5.1 Fatty Acid Profiles

Fatty acid analysis by high resolution gas chro-matographs determines the fatty acid compositionof unknown isolates. Databases are then searchedfor a match with known isolates. Cellular fattyacid compositions are stable and conserved. Thistechnology is able to be utilized because of thelarge number of fatty acids which occur in bacte-ria and the reproducibility of fatty acid composi-tion within a taxonomic group. Samples are sa-ponified, methylated, and extracted manually be-fore being placed onto the gas chromatogram andthe fatty acid methyl esters (FAMEs).

2.5.2 Mass Spectrometry

Matrix-assisted laser desorption ionization – timeof flight (MALDI-TOF) mass spectrometry maybe used to provide rapid identification and char-acterization of intact microbial cells. Different mi-croorganisms generate different charged molecu-lar weight patterns or spectra that can be used toidentify organisms to genus, species, and in somecases strains.

2.5.3 ELISA

Enzyme linked immunosorbent assay (ELISA) isa labeling technique for demonstrating the presence,or absence, of an antibody or antigen. The methoddepends on the use of immunoreagents coupled toan enzyme. A separation step of free and boundconjugate is then necessary to obtain a result. Thereare several components involved to carry out anELISA: solid phase, which is necessary to allowfor separation of the bound and free conjugate; en-zyme, which is used in the conjugate where it islabeled to an antigen or antibody; and substrate,used to detect end product.

2.5.4 Fluorescent Probe Detection

Fluorescent probes are designed to bind to specifictarget sites on or in cells, e.g., antibodies or nucleicacid and contain a molecule capable of fluorescingwhen stimulated by an energy source such as a co-herent light (laser).

2.5.5 Bacterial Endotoxin-Limulus Amebocyte Lysate Test

The lysate of the blood cells of the American horse-shoe crab, Limulis polyphemus has a very sensitiveclotting system triggered by the lipopolysaccharide ofthe cell well of gram-negative bacteria. This test re-placed the rabbit pyrogen test and is an example ofwhere an alternate test became the compendial test.


Sherlock Microbial IdentificationSystem (MIDI, Newark, DE)

Kratos Analytical Systems(Manchester, UK)Perseptive Biosystems Voyager –DE (Framingham, MA)

RBD-2000 (Advanced AnalyticalTechnologies, Ames, IA)

VIDAS & Mini-VIDAS ELISA Systems(bioMerieux, Hazelwood, MO), Tecra -Salmonella ELISA (InternationalBioProducts, Redmond, WA), Salmo-nella-Tek ELISA (Organon-Teknika,Durham, NC)

Bactigen Salmonella-Shigella Test(Wampole Laboratories, Cranbury, NJ)

Pyrogent Gel Clot (BioWhittaker,Walkerville, MN) & Pyrotell (Associ-ates of Cape Cod, Falmouth, MA)

Conventional

Table 3: Artifact-based Microbiological Methods


Microbialidentification (I)


Pathogen monitoring(P, E & I)

Pathogen monitoring (I)

Pathogen monitoring(P & E)

Pyrogen monitoring(P & E)

Differentiates bacteriaby Gram reaction (I)

Fatty Acid Profiles

MALDI-TOF MassSpectrometry

Fluorescence AntibodyTechniques

Enzyme-linkedImmunosorbent Assay

Latex Agglutination

Limulus AmebocyteLysate-EndotoxinAssay

Gram’s Stain

2.6 Nucleic Acid-based Technologies

2.6.1 DNA Probe

DNA hybridization assays specifically designed forthe detection of target organisms utilize solid sup-port devices for hybridization chemistry and colo-rimetric detection via an enzyme. Steps involvedare sample lysis, hybridization, hybrid capture, en-zyme label, color development, and detection.

2.6.2 Ribotyping/Molecular Typing

This technique characterizes and definitively iden-tifies organisms using restriction fragmentsofnucleic acids from bacterial genomes. The size-separated DNA restriction fragments are hybrid-ized to a ribosomal RNA probe. A chemilumines-cent substrate is applied. A camera converts theluminescing DNA fragments to digital informa-tion. This digital information is captured and data


Table 4: Nucleic Acid-based Microbiological Methods


are extracted. A pattern is then generated. Thispattern is compared to a database of patterns fromknown bacterial isolates. The ribotype pattern isa stable epidemiological marker that provides de-finitive taxonomic information as well as speciesdiscrimination.

2.6.3 Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) targets a spe-cific fragment of DNA which is highly conservedand therefore stable. This provides a highly reli-able indicator of the organism’s presence. PCRquickly (2-3 hours) provides millions of copiesof this specific fragment by combining sampleDNA with polymerase, nucleotides, and primersthat are specific for a given nucleotide sequence.

The mixture is placed into a thermocycler and isheated and cooled through many cycles. Heatingdenatures the DNA into single strands. As coolingtakes place, the primers anneal to the targetDNAsequence. The polymerase enzyme then usesthe nucleotides to extend the primers. These stepscreate two copies of the target fragment during onecycle. Repeated cycles of denaturing, annealing, andextending allow for exponential production of tar-get DNA fragments. If the target sequence is notpresent in the sample, detectable amplification doesnot occur. Electrophoresis of the PCR product isusually used to detect for the presence of the spe-cific fragment. PCR technology allows for far morerapid, sensitive, and accurate detection than con-ventional methods.

Gene-Trak System (Gene-Trak Systems,Hopkinton, MA) & Gene-Probe System,(Gene-Probe, San Diego, CA)

BAX ® Microbial Identification System(Qualicon, Wilmington, DE)Probelia System (BioControl Systems,Bellevue, WA)

MicroSeq. 16S rDNA Bacterial Identifi-cation System (PE Applied Biosystems,Foster City, CA)

RiboPrinter ® System (Qualicon,Wilmington, DE)

Pathogen monitoring (P)

Pathogen monitoring (P)


DNA Fingerprinting formicrobial characterization& identification (I)

Nucleic Acid Probe

Polymerase ChainReaction - DNAAmplification

16S rRNASequencingTechniques

AutomatedRiboprinting


3.0 REGULATORY REVIEW

3.1 General Classification of Microbiological Testing

There is a range of microbial tests conducted in themicrobiological testing laboratory, and their regu-latory requirements differ, based on the criticalityof the test to the final pharmaceutical drug productand the intended route of administration of the prod-uct. The range of tests can be classified as follows:

3.1.1 In-process Tests

Most in-process testing is to assure the adequacyof components, environments, equipment and pro-cessing steps to control microbial contamination.These tests are often required to demonstrate con-formance to current Good Manufacturing Practices(cGMPs). Changes to microbiological tests basedon new technology will be evaluated scientifically.However, this evaluation is less likely to be doneunless the change is filed in the product applica-tion. Many microbiological test methods used inin-process monitoring are not included in the origi-nal product application files, and changes in thesemethods would not be filed when the new methodsare introduced. It is recommended that changes tonew microbiology technologies be filed as changesin the product application. It should be noted thatmost in-process microbiological tests are quantita-tive.

When evaluating test data, comparisons are validonly when the methods remain consistent. Thisshould not, however, prevent new technology frombeing considered as a replacement for an oldermethod. During test method validation, a periodof comparison may permit the transition from oldto new microbiological methods. New acceptancecriteria may need to be established, e.g., a viabil-ity-based enumeration may give higher recoveriesthan a membrane filtration method.

3.1.2 Product Tests

Microbiological tests are conducted for productrelease and also as part of the stability protocol.

These tests may provide qualitative or quantita-tive results that may be used for different pur-poses, e.g., to demonstrate that the product theabsence of an objectionable microorganism ormeets a Microbial Limit.

3.1.3 Qualitative Tests

Product release microbiology tests are oftenqualitative and assess a limit for conformance toestablished acceptance criteria. Comparisons ofnew and old technology for qualitative tests arebased on the ability of the test to detect the quali-tative attribute. For example, sterility tests evalu-ate a qualitative attribute and may sacrifice quan-titative results in order to enhance sensitivity ofthe test.

3.2 Compendial Microbiological Test Method References

3.2.1 Water for Pharmaceutical Purposes

Types of water, methods and specifications fortesting them are listed in U.S. Pharmacopeial In-formational Chapter <1231> Water for Pharma-ceutical Purposes (5). The United States Phar-macopeia (USP) references Standard Methods forthe Examination of Water and Waste Water,(APHA) 20th edition, 1998 (6) for informationon specific test methods. In supplement 2000 ofthe Pharmacopeia Europa (Ph. Eur.) there will betwo monographs on water. One is water for in-jections, subdivided into sections on water forinjection in bulk and a section for sterilized wa-ter for injections. The second monograph is onpurified water. This monograph is also dividedinto two sections: purified water in bulk and pu-rified water in containers. For the microbiologi-cal examination (if relevant) soybean-casein di-gest agar and not R2A or Plate Count agar is pre-scribed.

3.2.2 Antimicrobial Effectiveness Testing

Antimicrobial preservatives are substances addedto multi-use products to protect them from mi-


crobial contamination that may be introduced in-advertently during use of the product (post manu-facturing). The test for Antimicrobial Effective-ness is used to demonstrate the effectiveness ofany added antimicrobial preservative(s).Compendial References include: USP 24 Chap-ter <51>, Antimicrobial Effectiveness Test (7),Japanese Pharmacopeia (JP) XIII., General Infor-mation 3. Preservatives-Effectiveness Tests, andthe Ph. Eur. 3rd Edition., Biological tests, 5.1.3.Efficacy of Antimicrobial Preservation.

3.2.3 Microbial Limit Testing

The tests for microbial limits and recommenda-tions for microbial quality criteria of raw materi-als, excipients (inactive pharmaceutical ingredi-ents) , drug substances (active pharmaceutical in-gredients) and pharmaceutical products have beenestablished in compendia for over 30 years. Thesetests are listed in the USP 24 Chapter <61> Mi-crobial Limits Tests (8), the Ph. Eur. 3rd Edition.,Biological tests 2.6.12 and 2.6.13.Microbial Con-tamination of Products Not Required to Complywith the Test for Sterility (Total Viable Count2.6.12, Test for specified Micro-Organisms2.6.13) , and the JP XIII 30. Microbial Limit Test.

3.2.4 Sterility Testing

The Sterility Test is applicable for determiningwhether drug substances, preparations or otherPharmacopeial articles are sterile as defined bythe Compendial method. A satisfactory result onlyindicates that no contaminating microorganismshave been found in the sample examined in theconditions of the test. Therefore, the result is afunction of the efficiency of the adopted samplingplan. Compendial references to sterility testinginclude: USP 24 Chapter <71> Sterility Tests (9),the Ph. Eur. 3rd Edition. Biological Tests 2.6.1.Sterility, and JP XIII 45. Sterility Test.

3.2.5 Environmental Monitoring

The microbiological monitoring of air, surfaces andpersonnel in facilities used for sterile pharmaceuti-

cal manufacturing is discussed in the USP 24 Infor-mational Chapter <1116> Microbiological Evaluationof Clean Rooms and Other Controlled Environments(10) and the PDA Technical Report No. 13 Funda-mentals of a Microbiological Environmental Monitor-ing Program (11). The documents also covers thedesign and implementation of a microbiological moni-toring program, suggests monitoring frequencies andmicrobiological acceptance criteria.

3.2.6 Microbial Identification

The compendial identification methods for microor-ganisms are described in the Microbial Limit test chap-ters (see Microbial Limit Testing references).

3.3 Changing a Microbiological Test Method

3.3.1 The Regulatory Perspective on the Introduction of New Microbiological Methods

The FDA Center for Biologics Evaluation and Re-search (CBER), Center for Drug Evaluation and Re-search (CDER) and Center for Devices and Radio-logical Health (CDRH) do not approve test meth-ods, but do approve applications for new productsor supplements to the filings for existing products.An important part of these applications includes es-tablishment of analytical procedures and acceptancelimits for products, components and processes. Ina New Drug Application (NDA) or AbbreviatedNew Drug Application (ANDA) the compendial testmethods for microbiological testing are typicallylisted as found in Pharmacopeial Testing Chapters,as referenced in Section 3.2 of this guidance.

A regulatory analytical procedure is the test methodproposed by the applicant and accepted by FDA forevaluation of a defined characteristic of the drugsubstance or drug product. The analytical proce-dures in the U.S. Pharmacopeia/National Formu-lary (USP/NF) are those legally recognized underSection 501(b) of the United States Federal Food,Drug, and Cosmetic Act, as the regulatory analyti-cal procedures for monograph items. A drug appli-cation may include an alternative procedure to theapproved regulatory procedure for testing the drug


substance and drug product. However, for purposes of determining compliance with the Act, the regu-latory analytical procedure is used. A test methodthat is accepted for a product application may ormay not be acceptable for tests of another product.

A specification is the quality standard (i.e., tests,analytical procedures, and acceptance criteria) pro-vided in an approved application to confirm thequality of drug substances, drug products, interme-diates, raw materials, reagents, and other compo-nents including container and closure systems, andin-process materials.

Acceptance criteria are numerical limits, ranges, orother criteria for the tests described. Unless other-wise exempted by regulation or guidance, allchanges in specifications from those in the approvedapplication must be submitted in a prior approvalsupplement. Unless otherwise exempted by regu-lation or guidance, these recommendations alsoapply to specifications associated with monitoringof the production environment (e.g., environmen-tal monitoring for particulates and/or microorgan-isms) that are included in NDA and ANDA submis-sions.

To change a regulatory microbiology test for a prod-uct or component, it is necessary to submit to thefile a description of the test and acceptance limitfor the drug product, as well as information dem-onstrating, the appropriateness of the test. Theo-retical and practical demonstrations of the tests ap-propriateness, are part of the validation of the newmethod. Validation criteria are contained in USPInformational Chapter <1225> Validation ofCompendial Methods. The Informational Chapterincludes sections detailing the validation param-eters: Accuracy, Precision, Specificity, Limit ofDetection, Limit of Quantitation, Linearity andRange. For a more detailed discussion on the ap-plication of parameters for new microbiological test-ing methods, refer to Part 2 of this document. Alsohelpful in the area of statistics is the American So-ciety for Testing and Materials (ASTM) StandardPractice for Comparing Test Methods D 4855 (12)and draft USP General Informational Chapter

<1010> Analytical Data - Interpretation andTreatment (13).

To establish a new regulatory method in an ap-proved drug application, one should file a supple-ment for approval prior to using the method. Touse an alternate analytical method to thecompendial method, prior FDA approval is notnecessary, and documentation to an application’sfile may be made in an annual report (21 CFR314.70.d). However, a validation report is neces-sary to document that the alternate method isequivalent to the regulatory or compendial testmethod. This validation report should be avail-able at the manufacturing site for examination bya regulatory investigator. Although an alternateanalytical method may be used, the officially rec-ognized analytical test method (referee method)for that drug product remains the regulatorymethod. Whenever results are disputed, the regu-latory method is conclusive.

When introducing novel microbiological testingmethods it is advisable for the instrument manu-facturer to file a Drug Master File (Type 5) andfor the pharmaceutical manufacturer to submit thevalidation of the method in a supplement to thedrug application for a selected product. This willinitiate a formal review of the method by the FDA.After approval of the supplemental NDA, a com-pany may discuss with the FDA the options forsubmitting similar changes in supplements or an-nual reports for other NDAs.

3.3.2 The Compendial Perspective

The USP position can be summarized as follows:The USP 24 General Notices states that alterna-tive methods may be used to determine that prod-ucts comply with the Pharmacopeial standards forthe advantages in accuracy, sensitivity, precision,selectivity, adaptability to automation or comput-erized data reduction, or any other special circum-stances. Such alternative or automated methodsshall be validated; however, when disputed, thecompendial method is conclusive as it is the offi-cial or referee test. In addition, USP Chapter <61>


Microbial Limit Tests states that automated meth-ods may be substituted provided they are validatedand give equivalent or better results, while USPChapter <71> Sterility Tests states alternative pro-cedures may be employed to demonstrate an ar-ticle is sterile, provided the results obtained areat least of equivalent reliability.

Although the Code of Federal Regulations (CFR)does provide detailed methods for performing spe-cific microbiological tests, the CFR also recognizesthat equivalent methods are acceptable (see 21 CFR610.9). Similarly, various guidance documents pro-vide details concerning procedures for various tests,but these are not intended to address all test condi-tions and are not to be considered as binding. Also,the International Committee on HarmonizationGuideline, Q6A, provides the following definition:“alternative procedures are those which may be usedto measure an attribute when such procedures con-trol the quality of the drug substance or drug prod-uct to an extent which is comparable or superior tothe official procedure.” Regulators will evaluate testmethods based on the product being tested and theappropriateness of the methods to assess a specificattribute.

What are the other compendial requirements? TheEur. Ph. General Notices Assays and Tests states,“The assays and tests described are the official meth-ods upon which the standards of the Pharmacopeiadepend. The analyst is not precluded from employ-ing alternative methods, in any assay or test if it isknown that the method used will give a result ofequivalent accuracy. In the event of doubt or dis-pute the methods of analysis of the Pharmacopoeiaare alone authoritative.” Similarly, the JP GeneralNotices state, “The test methods of the JapanesePharmacopoeia can be replaced by alternative meth-ods which give better accuracy and precision. How-ever, where a difference is suspected, only the re-sults obtained by the procedure given in this Phar-macopoeia is effective for the final judgment.”

The JP General Informational Chapter Validation ofAnalytical Procedures emphasizes that a limit test,such as the presence or absence of microorganisms

in the product is determined, only require the evalua-tion of specificity and detection limit of the test.

Other useful documents include the ICH Guideline forthe Validation of Analytical Methods and the ISOGuideline for the Determination of the Precision ofTest Methods.

3.4 Obtaining Compendial Change

The U.S. Pharmacopoeia (USP) is recognized asan official compendium by the Food, Drug andCosmetic (FDC) Act. The USP standards are usedto determine the identity, strength, quality and pu-rity of pharmaceutical articles. The “General Chap-ters” section of the USP includes requirements fortests and assays is numbered from <1> to <999>.Any proposed change in an existing test or a newtest proposal must f irst be published as aPharmacopeial Preview for comment inPharmacopeial Forum (PF), the USP Journal ofStandards Development and Revision. The article,including all supporting data, is reviewed by theMicrobiology Subcommittee of the USP Commit-tee of Revision prior to publication in PF. Subse-quently, all comments on the article are sent to thatSubcommittee and shared with the authors. A re-vised article is then published as an In-Process Re-vision for further comment, as needed, prior toadoption by USP as a Supplement to the currentUSP. In addition to publication in PF, any inter-ested party can request that the USP hold an OpenConference to discuss new microbiological meth-ods.

In Europe proposals for revisions are prepared byExpert Group 1. The drafts are published inPharmeuropa and a process of revisions starts fromthis point. The document is accepted by the expertgroup, and, after editing, send the PharmacopoeiaCommission which officially adopts the new mono-graph. Proposals for something that is on theagenda of international harmonization must be pub-l ished simultaneously in PF, JP Forum andPharmeuropa.


3.5 Regulators Assessment of New Microbiological Methods

Specific tests are provided in the product applica-tion file, along with methods and acceptance crite-ria. Methods using a new technology may be sub-mitted as changes to the application, and these willbe evaluated scientifically in relation to the prod-uct. New methods which have been described inscientific journals may be supported in the applica-tion by references to those publications. Techniqueswhich have not been described in peer reviewedjournals, may require more detailed discussion anddata submitted to the application file.

Submissions providing for new test methods shoulddescribe the attributes to be tested, and should com-pare the old and new methods for the test. Themethod should be validated by the applicant to dem-onstrate that it is equivalent or better than themethod used in approved application. Ultimately,the new microbiological method must be suitable

for assessing the specific product attribute, e.g.,sterility, microbial limit or absence of objection-able microorganisms.

Criteria for determining whether a new microbio-logical test method is equivalent or better thanthose methods currently used, should be estab-lished and tested in the conventional style of ascientific study. These studies should comparethe old and new methods, and include definedacceptance criteria (testing the hypothesis) for theexperiment. Experimental controls should dem-onstrate the accuracy and precision of the test.Data should be presented and discussed showingthe appropriateness of the method for tests of theproduct. Findings should be presented in a for-mat similar to a scientific journal article or note.Improved precision or accuracy may be adequatejustification for making a change in methods. Areduction in false negative results is particularlydesirable to regulatory scientists.


PART TWO:How to Validate and Implement a NewMicrobiological Test Method

4.0 THE VALIDATION PROCESS

Validation is defined in the FDA Guideline on Gen-eral Principles of Process Validation, May 1987 (14)as: Establishing documented evidence which pro-vides a high degree of assurance that a specific pro-cess will consistently produce a product meeting itspredetermined specifications and quality attributes.

The two critical components of any definition ofvalidation are appropriateness of a specific productor process (it does what it purports to do) and repro-ducibility (it continues to perform).

Therefore, for a new test method, it is important tobe able to demonstrate the appropriateness of themethod for the intended analytical application andthat there are procedures in place to show it contin-ues to perform to the same standard of quality overtime.

Validation should be more than a study conductedon a new method or product. Instead, it should en-compass the entire process that commences with thedecision to change some aspect of the microbiologi-cal testing program and continues through ongoingroutine use. It follows, therefore, that validationstarts from the outset, and the validation plan is de-signed to include each stage of the process that isrequired to implement a new test method. Adoptionof this approach is intended to streamline and expe-dite the introduction of the new method by ensuringthat each step in the process is considered in depthand documented before moving onto a subsequentstage.

A useful tool that can be applied to this process isthe Equipment Qualification Model.

4.1 The Equipment Qualification Model

The Equipment Qualification Model is a well-estab-lished and documented method for validation. The

model was used initially in computerized process con-trol and later adapted for analytical instrumentation.It is a useful framework that can be applied to the vali-dation of a complete system, i.e., all the componentsof the new test method including any instrumentation,software, firmware, and chemical reagents. It guidesthe assessor through the process steps involved in thedecision making and practical work required whenimplementing a new test method.

Analytical methods validation comprises the threesections of the equipment qualification model, i.e.,Installation, Operational and Performance Qualifi-cation. Two other sections, specification and de-sign qualification, may be used, but they are nor-mally reserved for the employment of large-scaleprocess equipment used in pharmaceutical manu-facturing and therefore are not considered here.However, there is considerable merit in ensuringthat before validation commences, adequate testmethod specifications have been provided for andan appropriate validation plan designed.

Breaking down the validation process into the fivesections, as described below, enables the processto become more manageable, since specific activi-ties are assigned to each section. The activities ineach section need not be carried out in serial; par-allel path activities can occur. For example, dataderived from one activity can be applied to the plan-ning and documentation of another. It is importantto note that the individual sections have been de-fined to apply to the validation of a new microbio-logical test system based on a consensus of the TaskForce members and may differ from the definitionsthat you may have encountered previously. Note:Individual companies may choose to define the vali-dation process differently in accordance with theirinternal policies and procedures and still achievethe same outcome in terms of meeting the overallvalidation requirements.

Method/Vendor RequirementsDefine vendor and test method specifications as partof the pre-validation activities.


Validation Plan DesignDesign and document plan objectives, methods andacceptance criteria.Note: This may be used as an executive summarywithin the validation protocol to aid auditing of thedocument.

Installation Qualification (IQ)Verify and document that the system was suppliedand installed as specified in an appropriate labora-tory environment. Note: IQ is instrument specificand portions may need to be repeated if the equip-ment is moved within the laboratory or to anothersite.

Operational Qualification (OQ)Verify and document that the system, i.e., methodand instrumentation, works for selected compendialorganisms in the selected environment.

Performance Qualification (PQ)Verify and document that the system performs asspecified using selected compendial organisms andenvironmental isolates for routine testing of batchesof product.

4.1.1 Vendor/Specification Requirements

Method/vendor selection commences when the de-cision is made to improve some aspect of the mi-crobiological testing program. The objective is toconsider all the pertinent information and the im-plications of change to the test method. From thisreview, arrive at a comprehensive specification ofwhat the new method should be capable of achiev-ing and what attributes are required of a vendor thatsupplies the new test system.

Consider economic and regulatory aspects before afinal decision is made to purchase the instrumenta-tion.

Economic considerations might include:

Is the unit cost of a test more or less with the newmethod than the current method? Note: Rememberto include all the variables when making this deter-mination, e.g., the new test method may cut downthe amount of testing required, reduce the sample/

media preparation requirements, reduce growth-promotion testing of microbiological media, pro-vide labor efficiencies in the reading, recordingand analyzing of data, etc.

Can the organization take advantage of the re-duced testing cycle time to reduce the productrelease cycle time? Is the microbiological test-ing the rate-limiting step?

What is the return on investment in equipment,method development and validation for the newmicrobiological testing method based on testingcosts, reduced product failure, product releasecycle time and reduced inventory holding costs?

Will the testing instrumentation PC or LIMS in-terface reduce the product release cycle time,improve data management and test result trend-ing?

Regulatory considerations might include:

What are the filing requirements for the new testmethod? For example, is the testing method de-tailed in a regulatory filing or named genericallyso that an alternate method may be substitutedfor the compendial test in the Annual Report?

Is the new test method required to demonstratecomparable test data to a compendial method asper the current stipulations of the Compendia?

Is the microbiological testing method so novel thatit would need to be validated as a new methodand not an alternate method?

Does the new method demonstrate improved pre-cision, accuracy and selectivity? If so, the regu-lators may view the new test method more favor-ably.

Is the microbiological testing method so novel thatit is prudent to file NDA supplements for the newtesting method although it may be filed as an al-ternate test in an Annual Report?


Is the technology widely enough understood sothat regulatory investigators will accept it as anew microbial testing method?

4.1.1.1 Test Method Selection

At this stage, candidate test methods are selectedbased on factors that might include the follow-ing: (Note: These factors may not apply to cer-tain methods, e.g., microbial characterization andidentification.)

Number and type of samplesWhat type of samples are to be tested and howmany per work shift? Can the new test method,once implemented, be extended to test other typesof samples in the process once implemented?Samples may be chosen based on their value orvolume, i.e., the implementation of a new testmethod may be justified solely based on the fasterrelease of a particular product or group of prod-ucts alone.

Sensitivity (limit of detection)What is the required level of sensitivity? Thiswill depend on the current specification for thetest method you are trying to replace, e.g., thedetection limit for a plate count method may be <10 cfu per mL. Will the new method have a lowerdetection limit? Is this a good or bad attribute?

Specificity (organism detection)What organisms does the new test method needto detect or identify? This should be based on thehistorical data generated from the current testmethod and complemented by information sup-plied by the vendor of the new method.

Comparable test dataHow important is it to be able to demonstrate com-parable test data? If the new test method is basedon novel, more sensitive technology, then the vali-dation studies and acceptance criteria will needto be designed to reflect this. If, on the other hand,the new method is based on technology that canreadily demonstrate comparable data, then the

validation studies and acceptance criteria detailedin Section 4.2.3 of this report can be used.

Degree of operator qualificationHow complicated is the new test method to per-form? What skill level is required to run an assayand interpret results? Do the laboratory personnelhave the right educational background? Do theynow need a background in analytical chemistry,nucleic acid biochemistry and computer skills?

Data management capabilitiesDoes the new instrumentation need to have LIMSinterface capability? What data management toolsare desirable or required? Evidence of softwarevalidation and functional testing reports will be re-quired to support each part of the software and firm-ware functions.

4.1.1.2 Vendor Selection

Once candidate test methods have been identified,a canvassing of potential vendors can be made toensure they are suitable long-term suppliers of in-strumentation and test reagents to your company.Considerations include:

Quality Assurance Procedures and StandardsDoes the potential vendor have the required qual-ity assurance certifications required to do businesswith your organization? What quality assurancestandards, e.g., ISO 9000, are employed by the ven-dor to manage their products, processes and proce-dures, e.g., change control. Has the vendor beenaudited by the regulators or by pharmaceuticalcompanies using the new test method?

Economic ViabilityIs the potential vendor economically viable and inbusiness for the long haul?

ReferencesWhat references exist that lend credibility to thenew test method? For example, an extensive cur-rent user list, refereed scientific publications, orthird party accreditation, such as AOAC Interna-tional.


Support ServicesWhat technical support services is the vendor ableto offer? Do these cover the appropriate geographi-cal locations? How many hours of the day are cov-ered? What and how much training is offered?What service and maintenance programs are pro-vided and at what cost?

DocumentationDoes the vendor supply evaluation and validationdocumentation?

4.1.2 Validation Plan Design

This is the planning part of the process, where theprocedures and protocols for evaluation of the newtest method are formulated and documented in thevalidation plan. It is the responsibility of the userto ensure that the Validation Plan is appropriate andcorrectly documented, although it is likely to beprepared jointly by the vendor and the user.

A candidate test method(s) and vendor are selectedbased on the requirements defined within theMethod/Vendor Selection. It is likely that a com-promise between the ideal requirements of the userand practical deliverables of a commercial testmethod will have to be reached. This should alsobe documented.

Prior to formal validation, a brief evaluation orproof-of-concept phase should be considered. Theinstrumentation might be loaned or rented from thevendor for interim period in order to minimize riskassociated with a large capital expenditure on a tech-nology that does not work out.

The products for evaluation are selected in consul-tation with your management and in considerationof the number and types of organisms chosen againstwhich to challenge the new test method. Valida-tion protocols should be written complying withindustry practice, defining the requirements fordemonstrating successful completion of installation,operation and performance qualification. Accep-tance criteria should be established and a contin-gency plan agreed upon to allow for discrepanciesto be documented.

To be able to demonstrate successful completionof Operational Qualification, validation criteria(see Section 4.2.3) are required. It is the respon-sibility of the new test method vendor to supplydata against these validation criteria that demon-strate the new test method is appropriate for theintended analytical application. After a thoroughreview of the data provided, a decision can bereached as to which of the experimental studiesneed to be repeated to a particular degree andwhich can simply be referenced in the user’s vali-dation documentation.

Note: A key objective throughout should be tokeep the amount of practical work to a minimum.This should be borne in mind when assessing ven-dor data. When a large volume of supporting datais provided, some confidence should be placed inthe information and only some critical elementsrepeated on site. For example, data indicating thata wide range of microorganisms have been de-tected in a large number of products may trans-late to a user validation protocol involving veri-fication that the test method works for a few criti-cal microorganisms that may be objectionable inthe your products.

4.1.3 Installation Qualification

Installation Qualification (IQ) studies should es-tablish that the equipment is properly and safelyinstalled with the right utilities in an appropriatelaboratory environment. A significant part of in-stallation qualification is a verification that incom-ing new equipment meets the specifications forthe equipment ordered. Any exceptions to theoriginal specifications should be documented,showing the corrected specification, and ap-proved.

Installation qualification studies should be per-formed in accordance with the approved proto-col. The key types of information to be includedin an IQ document are identification information,utility requirements, operating environmental con-ditions, safety features, and supporting documen-tation (e.g., technical manuals, blueprints, draw-


ings, etc.). It is impossible to verify every criticalfeature associated with a piece of equipment.Therefore, a judgment needs to be made regard-ing the relevance of testing those features of thesystem which will not be used during the routineuse of the new test method. One way to addressthis type of concern is to obtain a certificate ofconformance for those particular features withinthe instrument received from the manufacturer ofthe system.

Typical information to be included in the in-stallation qualification document include:

Purpose:

Verification that all key aspects of the installa-tion adhere to the appropriate installation codesand approved design specifications.

Scope:

Identification of the equipment to be qualified.For some applications, more than one piece ofequipment or system may be included in a singleprotocol.

Responsibilities:

Identification of the person/departments respon-sible for performing, reviewing, and approvingthe work. CGMPs require that the Quality Unitapprove all validation protocols.

References:

Identification of the pertinent procedures, poli-cies, and methods used in the qualification proto-col, inspection, and testing of the equipment.

Procedure:

Description of how the installation qualificationis to be performed, methods to perform the veri-fications, and how results should be documented.

For example the procedural steps may include:

Unpack the equipment, confirm there was no dam-

age in transit and check all parts against the pur-chase order.

Record serial numbers.

Ensure that adequate safety precautions are in place.

Site the equipment correctly in an appropriate labo-ratory environment and label as unvalidated equip-ment not for routine testing.

Connect to the specified utilities and power-up theequipment.

Perform key test to verify the performance of theequipment supplied.

Recalibrate the equipment, if necessary.

Acceptance Criteria:Definition of the requirements to be satisfied in or-der to deem the piece of equipment qualified.

Installation Qualification Report:Summary of the work performed, results of the in-spections and results obtained.

Approvals:Documentation that all appropriate departments in-cluding the Quality Unit have approved the quali-fication.

Computerized or microprocessor controlled systemsshould also document important features such as,dip switch settings, cabling connections, micropro-cessor chips utilized, the computer configuration,any special features of the equipment required,printer connections, buffers, files, and memory re-quirements. It is also important to document soft-ware required and appropriate version numbers.This includes any operating systems used by thecomputer.

Installation will often be carried out by the sup-plier and witnessed throughout by the user.


4.1.4 Operation Qualification

Operation Qualification forms the main practicalpart of validation, verifying that the new test methodperforms in the laboratory of the user with theirproducts within defined limits and tolerances. Op-eration qualification should be tackled in manage-able sections, perhaps in a phased approach wheregroups of up to five products head towards releaseby the new test method.

The first phase may be viewed as a confirmation ofproof of concept or principle, should one be re-quired. Testing of up to five products to prove thatthey are compatible with the technology and per-haps demonstrating the feasibility of the system withlow counts of a single microorganism may be indi-cated. This proof of principle phase is most appro-priate if the vendor has no supporting data on simi-lar products and may be conducted prior to finalpurchase of the equipment. This phase may be per-formed prior to large amounts of the documenta-tion being put in place, hence preventing possiblewasted effort.

Once a decision is made to commit to the valida-tion of the new test method, the data generated arestill valid if performed on the user site with a sys-tem conforming to the installation qualification partof the protocol.

The second phase can be termed verification, wherecritical parts of the supplier’s supporting data arerepeated to show that the test method is appropri-ate for its intended application. Base the workaround the validation criteria but include referenceto as much of the supporting data as possible. Forexample: for enumeration methods, choose a lim-ited panel of microorganisms including compendialmicroorganisms and a couple of isolates from yourpharmaceutical manufacturing plant. Demonstrateaccuracy, precision, linearity, limits of detection,and quantitation using these organisms in a simplediluent. Whenever possible, compare your resultswith those published by the supplier in his manualor published in the technical literature. The greaterthe availabil i ty of supporting data, the less

microorganisms can be used to demonstrate com-parable results.

The user must exercise judgement in determininghow much data need to be generated, but give athought to the perils of overvalidation. A balanceshould be struck between getting the new methodinto routine use and demonstrating that it is fitfor all purposes.

During the OQ, the computer system is validated.It would be demonstrated that the menu com-mands operate the equipment in a predictablefashion and this is documented in any instrumentinput.

4.1.5 Performance Qualification

Performance Qualification provides confirmationthat the system will perform to the specified stan-dard using your products and will include workto meet the compendial requirements, e.g., USPPreparatory Testing for Microbial Limits and Bac-teriostasis and Fungistasis testing for SterilityTesting.

It prescribes and documents the schedule of con-trols, maintenance, and calibration procedures tobe performed on an ongoing basis.

In general, the manufacturer’s recommended test-ing should be performed. If it is not performed, atechnical justification for not performing the test-ing should be provided.

Performance Qualification considerations also in-clude:

Change Control procedures should be designedand implemented. This should be part of a de-fined and controlled process. All changes madeshould address the impact of the change upon thevalidation status of the piece of equipment.

Standard Operating Procedures should be writ-ten covering each aspect of the operation and


maintenance of the new test method. The need foreffective instructions is of utmost importance in or-der that personnel can understand exactly what todo. SOPs should be appropriate, clear and accu-rate, and approved by appropriate individuals.

Often the supplier will provide the instructionsfor the system electronically so they can be in-corporated into your internal SOPs with minimumeffort.

A special area of concern is the typical on-goingpreventive maintenance program, frequentlyhandled by the equipment manufacturer. Manyprograms include updating system software withthe current software versions and periodic cali-bration checks. It is important to ensure that ap-propriate validation testing is performed beforeplacing the unit back into use and that the techni-cian performing the maintenance accurately docu-ments the maintenance performed.

A training log should be maintained specifyingand documenting the training requirements. Suc-cessful completion of training by all personnel re-sponsible for operating the new test system shouldbe recorded.

The PQ is, in fact, no more that a protocol withrecord forms, which will be carried throughoutthe lifetime of the test method. It demonstratesthe uniformity, consistency, and reliability of testresults over time.

Implementation of the new test method by run-ning it in parallel with the current method is thefinal phase. At first, release product followingthe results of the existing method exclusively,while comparing the results to those generated bythe new method. As confidence in the new methodincreases, perhaps after a pre-specified period oftime or number of production batches showingequivalence between the two methods, switch toreleasing product from the results of the newmethod. Initially, the old method may continueto be run in parallel as an ‘insurance’ but, after

a pre-specified period of equivalence the old methodshould be dropped. The judgement is required fordetermining the number of batches required for par-allel testing. Regulators expect to see data from atleast three batches, and the user has to decidewhether this number is sufficient.

Prior to the switch to the new method, a brief re-view of the final phase of validation should be per-formed and the change authorized.

Some products may require, in order to release fromthe results of the new method, a product licenseamendment (PLA) or New Drug Application (NDA)Supplement be sought. In other instances, no priorapproval is required, however, the new method willhave to be justified to a regulatory inspector. If inany doubt, contact your district office for more in-formation.

Periodic ReviewOnce the alternate test method is in routine use, aformal mechanism should be put in place to peri-odically review its performance. This can be assimple as running the older compendial method pe-riodically (for example, once every six months) inorder to verify that the new method is still perform-ing as expected.

Validation SummaryAs described at the beginning of this document, eachphase of validation should be reviewed and ap-proved before moving on to the next. Thepenultimate stage is the preparation of the Valida-tion Summary, which provides a synopsis of thevalidation objectives and data, showing the accep-tance criteria have been met and making the rec-ommendation that the test method should be imple-mented.

4.2 Validation Criteria

Broadly speaking, the validation criteria which needto be satisfied for microbiological testing methodscan be divided, with the exception of microbial iden-


tification, into two categories: quantitative andqualitative. USP Chapter <1225> Validation ofCompendial Methods, indicates which criteriashould be satisfied for a qualitative or limit test (atest with two outcomes either positive or negative)and which criteria should be satisfied for a quanti-tative test.

It is recommended to use the criteria detailed in theUSP <1225> with the following refinements. First,that the criteria are redefined in microbiologicalterms so as to have greater relevance to our appli-cations. Second, we have added an additional

Table 5: Microbiological Method Validation Criteria

Validation Parameter

Accuracy

Precision

Specificity

Limit of Quantification/Limit of Detection

Linearity

Range

Ruggedness/Repeatability

Equivalence

Quantitative Test

Yes

Yes

Yes

Yes/Yes

Yes

Yes

Yes/Yes

Yes

Qualitative (Limit) Test

No

No

Yes

No/Yes

No

No

Yes/Yes

Yes

All of the parameters listed, with the exception ofLimit of Quantification, need to be proven with themicrobiological equivalent to a standard chemicalsolution, which is a suspension of a laboratory cul-ture. Equivalence of the new test method with thecurrent recommended method should be demon-

strated for each parameter using the same testsample. Equivalence may also be demonstratedby the use of “real” samples with “cross-over”studies in the routine microbial testing laboratory.However, this approach may not be appropriateto microbial counts when the typical result is zero,

category – equivalence as well as acceptance cri-teria – for each. Third, we recommend acknowl-edgment that there needs to be a division of re-sponsibility for demonstrating attainment of thesecriteria between the vendor and the potential userof the new test system. For example, robustnesscan be much more extensively tested in the labo-ratories of the vendor than by each potential newuser.

The criteria that should be considered during thevalidation of microbiological methods are foundin Table 5.


e.g., Water for Injection (WFI) monitoring, airmonitoring in class 100 areas, or presence/absencetests when the typical result in Sterility Testingand Absence of Objectionable MicroorganismTesting is negative.

4.2.1 Preparation of Test Samples

Unlike chemical analytes where we can accuratelyweigh out a quantity of a chemical of known pu-rity and dissolve it in a solvent such as water toobtain a standard solution, it is more difficult toconsistently prepare a bacterial inoculum with auniform cell count per unit volume of water. Toprepare a one molar chemical solution, dissolve1 mole, i.e., the molecular weight in gram, in 1liter of water. This solution contains oneAvogadro’s number, i.e., 6.023 x 1023 moleculesper liter.

In contrast to chemical analytes, to prepare aninoculum of a bacterial suspension, grow up a pureculture of the organism for 18 to 24 hours at 35°C in soybean-casein digest broth, adjust the tur-bidity of the culture, serially dilute the culture toobtain the desired cell count, and verify the countby plating or direct cell counting using a hemocy-tometer or another type of counting chamber.Other variations on the bacterial inocula prepara-tion include adjusting the turbidity of the over-night culture using a McFarland Standard or spec-trophotometric readings at, for example, a wave-length of 550 nm prior to dilution into the desiredinocula range.

The recovery of the test organism will depend onthe specific organism, inoculum state, culturemedia, and incubation conditions. For example,the microorganisms recommended by the differ-ent compendia for qualifying testing methods maynot be appropriate for the media used for watertesting. However, for the water microflora, it iswell known that lower nutrient levels, lower in-cubation temperatures and extended incubationtimes result in higher counts.

Errors are associated with sampling, dilution, plat-ing, incubation, counting, and calculation. The lit-erature suggests that the 95% confidence limitsabout the mean for plate counts is of the order of ±0.5 log for fungi and ± 0.3 log for bacteria.

If you have an ideally mixed suspension of a pureculture, then the counts in aliquots taken from thatsuspension will follow a Poisson distribution. Thestandard deviation of a Poisson distribution is thesquare root of the average. For counts ranging from36 to 289, the standard deviation will be 6 to 17;the relative standard deviation will be 16% to 6%respectively. The 95% confidence limits for suchexperiments (taking twice the standard deviation)will be 32% and 12%. The major source of varia-tion between counts from a product may result fromthe non-uniform distribution of the microorganismsin the product. Other errors are associated withsampling (weighing, etc.), dilution, plating, incu-bation, counting and calculation with the last twobeing essentially be negligible.

It should be noted that the precision increases asnumber of colonies counted per plate increases, e.g.,confidence limits are 4 to 16, 80 to 120 and 455 to545 for 10, 100 and 500 colonies respectively. Inthe colony range of 30 to 300 per plate, 60 and 88%of six technicians counts are within 5 and 10% of astandardized photo-count method, with a tendencyto undercount as the number increases.

An important issue is the determination of the num-ber of replicates that are used in a validation proto-col. The number of replicates required to declare astatistically significant difference between two mi-crobial counting methods differs with a stated levelof confidence. The number of replicates dependson the true percent difference (10, 20, 25, 50, and100%) that you want to detect, the probability (50,70, 90%) of being able to detect the difference, andthe target concentration (1, 10, 50 and 100 cfu) ofthe sample (see Table 6). The results assume a 5%risk of declaring a difference between the methods,when in fact they are equivalent. (After ChristophMaier, 1999, personal communication.)


Table 6: Number of replicates needed to make a statistical claim of a probability of 50 to 90% todetect difference of 10, 20, 25, 50 and 100% between two microbial enumeration methods in thecounting range of 1 to 100 cfu.

50% Probability (Power 0.5)

% Difference 1 cfu 10 cfu 50 cfu 100 cfu

10 596 60 12 6

20 163 16 3 2

25 109 11 2 1

50 33 3 1 1

100 12 1 1 1



10 1037 104 21 10

20 284 28 6 3

25 190 19 4 2

50 58 6 1 1

100 20 2 1 1



10 1887 189 38 19

20 517 52 10 5

25 345 35 7 3

50 106 11 2 1

100 37 4 1 1


4.2.2 Variability of Microbiological Methods: Special Note

An important point to consider during the valida-tion of a new microbiological test method is theinherent variability in microbiology. There arethree sources of variation: sample distributionerror, cell morphology, and metabolic activity.

For any given test procedure, the relative impor-tance and contribution from these sources willdepend on the principle of the test method andmust be carefully considered.

4.2.2.1 Sample Distribution Error

Distribution error is the biggest source of errorcontributing to the variation in microbiologicaltest results. The natural distribution of microor-ganisms is heterogeneous and rarely follows nor-mal Gaussian distribution even after log transfor-mation. The distribution tends towards a nega-tive binomial or Poisson distribution and is ex-tremely difficult to assess and predict, particularlyat the low contamination levels experienced inclean room environmental samples and purifiedwater systems. These facts are frequently over-looked in our assumptions and interpretation ofthe results from microbiological tests.

The level of assurance provided by results from amicrobiological test is questionable and a func-tion of both the efficiency of the adopted sam-pling plan and the test method. Extrapolation ofthe results to the whole batch of a product requiresassurance that the product was produced underhomogeneous conditions. Care should be exer-cised in the design of validation experiments toaccount for such distribution error.

4.2.2.2 Cell Morphology

Traditional culture methods detect and /or enu-merate microorganisms by monitoring changes inturbidity or by counting colonies visible to thenaked eye. For enumeration, the assumption isthat one colony forming unit is derived from a

single organism that was uniformly distributedwithin the test aliquot. Yet microorganisms have avariety of morphologies and can occur singly andin pairs, tetrads, or irregular clusters. Microorgan-isms also have a tendency to colonize surfaces andform biofilms, which may also affect results. Thenumber of colony forming units (cell density) in aplated sample directly affects the colonial morphol-ogy and the accuracy of the viable count estimate.

Consequently, the coefficient of variance for mi-crobiological methods is large. To validate any al-ternative method, the variability must be determinedand compared with that of an equivalent traditionalmethod or test procedures recommended by thePharmacopoeiae.

4.2.2.3 Metabolic Activity

Successful detection and/or enumeration of micro-organisms is influenced by their metabolic activ-ity, genotype, and readiness for growth. In anymicrobiological test method, the presence of inter-fering factors must also be considered. Microor-ganisms may be stressed due to exposure to pro-cessing, environmental and experimental condi-tions, or inhibitory components in the product it-self. Stressed cells may require a period of resus-citation and repair before they can be detected bycultural methods. Inactivating agents added to themedia can neutralize some products which stresscells and inhibit their growth. Conversely, someproducts may contain nutrients sufficient to sup-port microbial survival or even growth.

The validation of alternative methods should in-clude, therefore, an evaluation of real samples takenfrom a variety of environmental and process andproduct types relevant to the test application. Theresults must be compared to the recommendedmethod. For non-cultural methods where the de-tection principle is based on metabolic activity, theeffect of these stresses on the detection principleand test results should be evaluated in terms of theperformance characteristics and assessed against therecommended method.


4.2.3 Protocol Design Using Recommended Validation CriteriaNote: the strict definitions of the USP validationcriteria are found in the Appendix. Details of thestandard statistical methods may be found in USPGeneral Informational chapter <1010> AnalyticalData – Interpretation and Treatment as well as stan-dard texts on statistical analyses.

[i] Accuracy

Definition - The accuracy of a microbiologicalmethod is the closeness of the actual test resultsobtained by the test method to the results predictedfrom the dilution of the microbial suspension or theresult obtained by the current compendial method.Accuracy should be demonstrated across the prac-tical range of the test. With a Plate Count this willbe limited by the countable numbers of colonies ona plate, i.e., 30 to 300 colonies for a particular dilu-tion. Accuracy is usually expressed as the percent-age recovery of microorganisms by the assaymethod. Accuracy is a measure of the exactness ofthe microbiological method that is true for all prac-tical purposes.

Determination - Prepare a suspension of microor-ganisms at the upper end of the range of the testand serially dilute down to the lower end of therange of the test. At least five suspensions acrossthe range of the test should be analyzed. Calculateeach of the suspensions as a percentage dilution ofthe original. The result obtained by the sample atthe upper range of the test should be referred to as100%. Compare the result, i.e., actual versus ex-pected, obtained by each of the other suspensions,i.e., 100%, 75%, 50%, 25% and 10% of the origi-nal culture, against the result expected from the di-lution; present these as percentage recoveries.

Acceptance criteria - The new method should giveequivalent or better results than the current method.Percentage recoveries of the order of ± 30% can beexpected for microbiological methods. The accep-tance criterion is at least 70% recovery.

The comparison of the accuracy of the compendialand alternate method establishes whether the al-

ternate method is more or less accurate than thecurrent method. If the methods generate data thatare normally distributed and have equal variances,then the simplest approach is to apply a Studentt-test. Analysis of variance (ANOVA) can be usedfor more complex models. If the confidence in-terval for the differences between the true meansof the compendial and alternate microbial count-ing methods contains zero, i.e., the upper limit isa positive number and the lower limit is a nega-tive number, then there is no statistically signifi-cant difference between the two methods. How-ever, it should be noted that the two methods maybe statistically significantly different, but this dif-ference is of no practical consequence. In otherwords, the results should be reviewed against therequirements of the test. Since microbiologicalcounts tend to follow a Poisson distribution, theratio of the means may be used with the confi-dence limit containing one and not zero.

If the alternate method gives a more accurate, i.e.,higher recovery across the range of the microbialcount, then the use of the alternate method is more“conservative” than the compendial method.

[ii] Precision

Definition - The precision of a microbiologicalmethod is the degree of agreement among indi-vidual test results when the procedure is appliedrepeatedly to multiple samplings of suspensionsof laboratory microorganisms across the range ofthe test. The precision of a microbiologicalmethod is usually expressed as the standard de-viation or relative standard deviation (coefficientof variation). Precision may be a measure of ei-ther the degree of reproducibility or repeatabilityof the microbiological method under normal op-erating conditions.

Repeatability refers to the use of the microbio-logical method within the same laboratory over ashort period of time using the same analyst withthe same equipment. Reproducibility refers to theuse of the microbiological method within


Acceptance criteria - All microorganisms selectedas representative are successfully isolated and enu-merated from the sample matrices.

[iv] Limit of Detection

Definition - The limit of detection is a parameterof a limit test. It is the lowest number of microor-ganisms in a sample that can be detected, but notnecessarily quantified, under the stated experimen-tal conditions. A microbiological limit test deter-mines the presence or absence of microorganisms.Due to the nature of microbiology, the limit of de-tection refers to the number of organisms presentin the original sample, before any incubation step,not the number of organisms present at the point ofassay. Also, the amount of sample tested and thedilution of that sample may determine the Limit ofDetection. For example, when 10 grams of testmaterial is diluted in 90 mL of diluent and 1 mL isplated, the absence of colonies on the plate wouldbe reported as <10 cfu per g.

Determination - As it is not possible to consistentlyobtain a reliable sample containing a single micro-organism, it is essential that the limit of detectionof an assay is determined from a number of repli-cates (n ≥ 5) for the standard compendial microor-ganisms.

Acceptance criteria - The best statement that canbe made is that if a single organism is present inthe sample, it will be detected during the time frameof the assay. The ability of the two methods to de-tect the presence of single organisms can be dem-onstrated using the chi square test.

[v] Limit of Quantification

Definition - Limit of quantification is a parameterof quantitative assays for low levels of microorgan-isms in sample matrices. It is the lowest number ofmicroorganisms which can be determined with ac-ceptable precision and accuracy under the statedexperimental conditions.

the same laboratory over a short period of timeusing different analysts with the same equipment.

Determination - Prepare a suspension of micro-organisms at the upper end of the range of thetest and serially dilute down to the lower end ofthe range of the test. At least 2 suspensions acrossthe range of the test should be analyzed. For eachsuspension at least 10 replicates should be assayedin order to calculate statistically significant esti-mates of the standard deviation or relative stan-dard deviation (coefficient of variation).

Acceptance criteria - Generally, a coefficient ofvariation (relative standard deviation) in the 15to 30% range is acceptable for microbial counts.

For an alternate method to be equivalent to thecompendial method, e.g., the plate count method,the method precision should not be significantlylower. The recommended statistical method ofcomparing the precision of the two methods is theapplication of the F-test. In this test, the varianceof each method is estimated, and the ratio of thelargest to the smallest variance is calculated andcompared to the tabulated values for an F distri-bution. The critical value from the table dependson the degrees of freedom and the desired confi-dence level. If the calculated ratio for that de-gree of freedom exceeds the value in the F distri-bution table, a significant difference exists be-tween the precision of the two methods.

[iii] Specificity

Definition - The specificity of a microbiologicalmethod is its ability to detect a range of microor-ganisms which demonstrates that the method isfit for purpose. Method compatibility with thetypes of sample matrices with which the methodwill be used should also be proven.

Determination - Screen the method against a rep-resentative range of microorganisms appropriateto the method. Screen the method against a rep-resentative range of sample types.


Determination - As it is not possible to obtain areliable sample containing a known number of mi-croorganisms, it is essential that the limit of quan-tification of an assay is determined from a numberof replicates (n ≥ 5) at each of at least five differ-ent points across the range of the assay.

Acceptance criteria - The best statement that canbe made is that if a single organism is present in thesample, it will be quantified during the time frameof the assay.Note: Limit of Qualification of the new methodshould be equivalent or better than the existingmethod.

[vi] Linearity

Definition - The linearity of a microbiological testmethod is its ability to elicit results which are pro-portional to the concentration of microorganismspresent in the sample within a given range.

Determination - As it is not possible to obtain areliable sample containing a known number of mi-croorganisms, it is essential that the linearity of anassay is determined from at least duplicates at eachof at least five different points across the range ofthe assay.

Acceptance criteria - Correlation coefficient r2 =0.9 or better with the slope not diverging more than20% from 1.0, i.e., r2 = 0.8 to 1.2. Another statisti-cal tool that could be used is the test for goodnessof fit.

[vii] Range

Definition - The range is the interval between theupper and lower levels of microorganisms that havebeen demonstrated to be determined with precision,accuracy, and linearity using the method as writ-ten.

Determination - The range of the method is vali-dated by verifying that the analytical method pro-vides acceptable precision, accuracy and linearitywhen applied to samples containing analyte at theextremes of the range as well as within the range.

Acceptance criteria - This will depend on the per-formance characteristics of the method.

[viii] Ruggedness

Definition - The ruggedness is the degree of re-producibility of test results obtained by analysisof the same samples under a variety of normaltest conditions, such as different analysts, differ-ent instruments, different lots of reagents, etc.Ruggedness is normally expressed as the lack ofinfluence on test results of operational and envi-ronmental variables of the microbiologicalmethod. Ruggedness is a validation parameterbest suited to determination by the supplier of thetest method with easy access to multiple instru-ments and batches of components. Data suppliedby the test method manufacturer are entirely ad-missible to prove validation of ruggedness.

Determination - Prepare a suspension of micro-organisms and test at least 5 replicates againsteach assay variable in order to be able to calcu-late statistically significant estimates of the stan-dard deviation or relative standard deviation (co-efficient of variation). For a quantitative method,the range of the test method should also be cov-ered. Attention should be paid to the inherentinstability of microbiological suspensions andexperimental protocols randomized to eliminatebias.

Acceptance criteria - Generally a coefficient ofvariation in the 10 to 15% range is acceptable.

[ix] Robustness

Definition - The robustness of a microbiologicalmethod is a measure of its capacity to remain un-affected by small but deliberate variations inmethod parameters. It provides an indication ofits reliability during normal usage. Robustnessis a validation parameter best determined by thesupplier of the test method. Data supplied by thetest method manufacturer are entirely admissibleto prove validation of ruggedness.


Determination - The manufacturer investigateschanges up to 20% on the critical reagent con-centrations, instrument operation parameters andincubation temperatures.

Acceptance criteria - The results need to be re-viewed against the manufacturer’s quality assur-ance requirements and the instruction for use.

[x] Equivalence

Definition - The equivalence is a measure of howsimilar the test results are to those of the methodit is intended to replace.

Determination - Equivalence should first bedemonstrated in pure culture work for each of therelevant validation criteria. The variability ofmicrobiological samples is the limiting factor, andhence validation in microbiology requires that thetwo methods are run in parallel to prove each ofthe validation criteria discussed above. It is es-sential that experimental designs are randomizedbecause of the inherently unstable nature of mi-crobiological samples. A number of replicatesshould also be tested to obtain some assessmentof sample variability.

In addition, ‘real’ samples need to be tested byboth methods to show that the results are equiva-lent. In order to demonstrate the equivalence with‘real’ samples, samples need to be identified, someof which are expected to contain microorganismsand some which are not. Parallel testing shouldbe carried out for at least three batches.

The critical element in the validation of alternatemicrobial testing methods is demonstrating thatthe method is equivalent to the compendialmethod. How can equivalence be demonstrated?

The ASTM has a good statistical approach to thisissue in their Statistical Handbook (D4855).There are a number of considerations when com-paring two test methods. You need to decidewhether the precision, sensitivity, accuracy and/or bias of the two methods is to be compared. Then

specify the values of the probability of the type Iand II errors, e.g., alpha = 0.05 and beta = 0.10 isusually used. Then determine the least differenceof practical importance. This is the most criticalstep that calls for a judgment and will strongly in-fluence the number of replicates. Choose the ap-propriate statistical test. Collect and analyze data,calculate the test statistic, compare the result to thatin the F-value or t-value table and decide whetherthe methods are significantly different.

Two recent stimuli articles in the PharmacopeialForum by major suppliers of new microbiologicalenumeration equipment and a future publication co-authored by the Chair of this Task Force are citedas useful examples of microbial method validation(15,16, and 17).

4.3 Special Considerations for the Validation of Microbiological Methods

4.3.1 Use of Multiple Pieces of Same Equipment within the Laboratory and Company

When more than one instrument is purchased for alaboratory, and the equipment is exactly the sameas the unit validated, i.e., the exact same compo-nents with the exact same version numbers for allsoftware, microprocessors, computers, etc., manycompanies perform reduced testing on subsequentunits. The testing required includes completion ofthe installation qualification, which verifies that theequipment is equivalent to the validated piece ofequipment. Some companies also choose to repeata reduced operating qualification study, e.g., usinga reference solution or standard.

When the equipment is identical but used in mul-tiple production facilities, i.e., not at the same geo-graphical location, many companies will share somequalification data, but other parts of the qualifica-tion must be repeated. The installation qualifica-tion is always repeated. Any testing of the envi-ronmental conditions, personnel, methods and utili-ties must be performed for the new site.


4.3.2 Unique Testing Requirements for Microbiological Equipment

Although the USP specifies parameters for consid-eration in validation of chemical testing equipment,many of the definitions do not really address theconcerns of microbiological equipment.

Validation testing requirements are influenced bysome factors not always considered. If your com-pany is the first company to use the piece of equip-ment, more testing will probably be required thanfor the two hundredth company validating the pieceof equipment. Additional testing is also requiredwhen the type of system used for the testing is anew technology.

Several pieces of laboratory equipment have vali-dation requirements specified by FDA and othergovernmental agencies. The following list is notmeant to be all-inclusive, but is provided as an ex-ample of equipment requirements.

Automated Endotoxin Detection/Assay Equipment

This equipment must meet the FDA Guideline forLimulus Amoebocyte Lysate-Endotoxin testingqualification as specified in the document. Thisincludes validation of the laboratory and the ana-lyst. Validation of reader systems usually involvesverification that standard solutions yield the speci-fied standard curves.

If the system also includes a database function andcustomized reports, it is important to verify thatthese functions operate and perform as expected.For example, if a summary report is requested, areall of the data present and correct? What factorscan damage the accuracy of the results, e.g., howmuch data are lost in the event of a power loss?Are there typical occurrences which can corrupt thefile of data? For spectrophotometric and light sys-tems, it is important to assess the filters transmis-sion wavelengths and calibration of the system.

Automated Microbial Identification Systems

Evaluation should include assessment in a mannerand proportion that reflect actual microbial test-

ing conditions at the site. This would includerepresentative flora from the manufacturing en-vironment, pharmaceutical ingredients, water forpharmaceutical purposes, product, personnel, etc.Additionally, if product must be free of objection-able microorganisms, the characterization andidentification system should be challenged withthese organisms as well. Typical microflorashould be represented in the validation. If 80% ofthe isolates are non-fermenting, gram-negativebacteria, then they should be well represented inthe validation. These typical isolates should pro-vide reproducible results with a high degree ofprobability when repeatedly tested to character-ize and identify the microorganisms.

Validation must also include an assessment of themanual procedures, e.g., isolation of pure cultures,inoculum preparation, Gram’s staining, oxidasetesting, etc., required to support the accurate char-acterization and identification of test organisms.

Sterility Testing Equipment

A special area of concern in this type of equip-ment includes the ability of the equipment to as-sess the sterility of a product. Critical elementsinclude the ability to manipulate the test materi-als aseptically, detect a full range of microorgan-isms, i.e., bacterial cells, bacterial spores, aerobesand anaerobes, fungi and yeast that have beenexposed to the stress of contact with products thathave inherent antimicrobial activity and containpreservative systems, and obtain equivalent re-sults to the USP Sterility Test. The false positiverate should be not more than with the compendialsterility test, i.e., less than 0.1% when conduct-ing manipulation controls.

The equivalency of the current and proposedmethods should be demonstrated using 1) inoculaof the range of USP Bacteriostasis and Fungistasistest microorganisms with counts below 100 cfufor enumeration, and 2) inocula of the same mi-croorganisms diluted to extinction where 20 to80% of the series of inocula will contain at lessone microbial cell for presence/absence testing.


The equivalency of the enumeration using the pro-posed method and standard counting method, i.e.,plate count or membrane filtration, can be dem-onstrated for accuracy and precision while rela-tive proportions of positive sterility test resultsfor the current and proposed sterility test meth-ods can be tested for a statistically significant dif-ference using a Fisher’s exact test or the continu-ity-corrected chi square test.

The use of parallel or split sample testing with ac-tual product to show the equivalency of two steril-ity test methods is not generally recommended. Tohave a 95% probability of detecting a difference intwo sterility test methods when the product batcheshave a 0.1% microbial contamination rate with a95% assurance, it is necessary to run parallel testson 10,600 batches. This means that the use of paral-lel testing to demonstrate equivalency is not a prac-tical option. The following is an example of howequivalency can be demonstrated.

To demonstrate that two sterility testing methodsgive equivalent results, prepare a dilution of anovernight culture of the growth-promotion micro-organisms, so that a 1 mL inoculum of the USPmicroorganisms will give a positive sterility testusing either of the methods at a set proportion (P)of the time, i.e., P = 0.8. If you conduct a series ofsterility tests by taking 1 mL aliquots from the di-lution, i.e., 100 tests, using both sterility test meth-ods; they are equivalent when the proportion ofpositive and negative test results using each methodare not statistically different using a chi square orWilicoxen paired data test (18).

For example, prepare 10-5 and 10-6 dilutions from a

microbial cell suspension of 2 x 105 organisms permL. Aliquots from these dilutions should containcells 87% and 18% of the time (see Table 7).

Table 7: Dilution, inoculum count, expected proportion of inocula showing no microbial growth(negative) or microbial growth (positive), and the expected number of positive tests when N =100tests are conducted from 10-4, 10-5, 10-6 , and 10-7 dilutions of a microbial cell suspension of 2 x 105 cfuper mL.

DilutionTested

10-4

10-5

10-6

10-7

Meaninoculum level

per test

20

2

0.2

0.02

Expectedproportionnegative

<0.001

0.135

0.819

0.980

Expectedproportion

positive

>0.999

0.865

0.181

0.020

Number oftests positive

for N=100

100

87

18

2


5.0 Glossary of Terms

Compendial TestsCompendial or official tests as defined by the UnitedStates, Japanese, or European Pharmacopeia for de-termining compliance with the pharmacopeial stan-dards for identity, strength, quality and purity. Ev-ery compendial article in commerce, i.e., marketedproducts labeled USP, when examined in accordancewith the official assays and test procedures, shallmeet all of the requirements of the monograph de-fining the product; hence, the tests are also termedreferee tests. Microbiological tests included in theUSP General Tests and Assays chapters are <51>Antimicrobial Effectiveness Test, <61> MicrobialLimit Tests and <71> Sterility Tests. Methods forwater monitoring and environmental monitoring ofclean rooms are found in USP General InformationalChapters <1231> Water for Pharmaceutical Pur-poses and <1116> Microbiological Evaluation ofClean Rooms and other Controlled Environments.

Other Standard MethodsUSP General Informational chapter <1231> Waterfor Pharmaceutical Purposes cites the AmericanPublic Health Association publication as a sourceof suitable standard methods.

AOAC International publishes official methods forthe chemical and microbiological analyses of com-modities related to food, agriculture, public healthand safety, and the environment. These methodsare validated, subject to collaborative studies andscientific review of the performance results (19).AOAC International has published methods for themicrobial assay of vitamins, isolation and identifi-cation of human pathogens, sterility testing of lowacid canned food, etc.

Validation of Compendial MethodsAs stated in the USP General Informational Chap-ter <1225>, the analytical parameters used in assayvalidation are accuracy, precision, specificity, lim-its of detection and quantification, linearity andrange.

Validation of a microbiological method is the pro-cess by which it is established, by laboratory stud-

ies, that the performance characteristics of themethod meet the requirements for the intendedmicrobiological applications. The parameters thatshould be considered during the validation of mi-crobiological methods are listed below.

Accuracy:The agreement between the official or recom-mended method of analysis and the alternatemethod that would be stated in terms of statisti-cal significance. With a chemical assay, a stan-dard solution may be accurately prepared and as-signed a potency of 100% and the recovery deter-mined at 100, 75, 50, 25 and 10%. In contrast, amicrobial suspension with an appropriate countin cfu per mL or g is assigned a value of 100%and would be diluted to five levels, i.e., 100, 75,50, 25 & 10%, to determine the microbial countrecovery.

Linearity:The linearity of a microbiological test method isits ability to elicit results which are proportionalto the concentration of microorganisms presentin the sample within a given range. As it is notpossible to obtain a reliable sample containing aknown number of microorganisms, it is essentialthat the linearity of an assay is determined from anumber of replicates (n ≥ 5 at each of at leastfive different points across the range of the as-say). The measure of the linearity is the correla-tion coefficient, r. For microbiological methods,r should be 0.9 or better.

Precision:The degree of agreement between repeated mea-surements of the same sample. The precision ofa microbiological method is the degree of agree-ment among individual test results when the pro-cedure is applied repeatedly to multiple samplingsof suspensions of laboratory microorganismsacross the range of the test. The usual index ofprecision is the standard deviation or relative stan-dard deviation of the log (10) cfu for methods ofmicrobiological enumeration or the standard de-viation for normally distributed measurement such


as a chemical assay. For example, the HPLC sys-tem suitability requirements for six repeat injec-tions of a standard solution is a RSD of NMT2.0%. With a microbiological method an RSD ofthe order of 15 to 30% is acceptable.

Specificity:The ability of the method to distinguish a testmicroorganism from the sample background orfrom other organisms in the sample. Method com-patibility with the types of sample matrices themethod will be used for testing should also beproven.

Limits of detection and quantification:The ability of the method to provide accurate re-sults under the conditions of low sample or ex-cess sample. For plate counts, the limit of detec-tion is 1 cfu per mL, while the greatest degree ofaccuracy is found counting between 30 and 300colonies per plate. With membrane filtration, thelimit of detection is 1 cfu in the volume filtered,e.g., 1 cfu per 1000 mL.

The limit of detection is a parameter of a limittest. It is the lowest number of microorganismsin a sample that can be detected, but not neces-sarily quantified, under the stated experimentalconditions. A microbial limit test determines thepresence or absence of microorganisms, e.g., ab-sence of indicator organisms in 10 g of materialor coliform count per g of material. Due to thenature of microbiology, the limit of detection re-fers to the number of organisms present in theoriginal sample, before any incubation step, notthe number of organisms present at the point ofassay. For example, the test material may be di-luted and an aliquot taken from that dilution thatwill limit the sensitivity or limit of detection ofthe microbiological method.

Repeatability and Reproducibility:Repeatability refers to the use of the microbio-logical method within the same laboratory over ashort period of time using the same analyst withthe same equipment. Reproducibility refers to theuse of the microbiological methods within the

same laboratory over a short period of time usingdifferent analysts with the same equipment.

Ruggedness:The ruggedness of a microbiological method is thedegree of precision of test results obtained by analy-sis of the same samples under a variety of normaltest conditions, such as different analysts, differentinstruments, different lots of reagents, etc. Rug-gedness is normally expressed as the lack of influ-ence on test results of operational and environmen-tal variables of the microbiological method. Rug-gedness is a validation parameter best suited to de-termination by the supplier of the test method witheasy access to multiple instruments and batches ofcomponents. Data supplied by the test methodmanufacturer are entirely admissible to prove vali-dation of ruggedness.

Robustness:The robustness of a microbiological method is ameasure of its capacity to remain unaffected bysmall but deliberate variations, e.g., variationswithin the order of ±10%, in method parametersand provides an indication of its reliability duringnormal usage. Robustness is a validation param-eter best suited to determination by the supplier ofthe test method. Data supplied by the test methodmanufacturer are entirely admissible to prove vali-dation of ruggedness.

Equivalence:The equivalence of a microbiological method is ameasure of how similar the test results are to thecompendial method that the alternate method is in-tended to replace. The variability of microbiologi-cal samples may be the limiting factor and hencevalidation in microbiology requires that the twomethods are run in parallel with pure cultures ofrepresentative microorganisms to prove each of thevalidation criteria discussed above. In addition,‘real’ samples need to be tested by both methods toshow that the results are equivalent.

When comparing two methods for accuracy, preci-sion and /or sensitivity on the basis of data collected,statistical tests of significance should be used. For


example, when comparing the precision of the twotest methods, calculate the average and the standarddeviation for each of the five levels and eachmethod. The F-statistic as the ratio of the two vari-ances for each level for each test. Determine thesignificance of each of the calculated F-values bycomparing them to a critical value selected from atable of the F-distribution for the 1 or 5% level andthe appropriate degrees of freedom to determine ifthe variance of compendial test is different from thealternate method. If the F-ratio for the precision isgreater than a critical value, then conclude that thetwo methods have significantly different precision.Details can be obtained in ASTM D 4855 StandardPractice for Comparing Test Methods.

Alternate TestsThe USP 23 General Notices states that alternativemethods may be used to determine that productscomply with the pharmacopeial standards of iden-tity, strength, quality, and purity, for the advantagesin accuracy, sensitivity, precision, selectively, adapt-ability to automation or computerized data reduc-tion or any other special circumstances. Such alter-native or automated methods shall be validated.However, when disputed the compendial method isconclusive. Furthermore, USP chapter <61> Mi-crobial Limit Tests states that automated methodsmay be substituted provided they are validated andgive equivalent or better results, while USP <71>Sterility Tests states alternative procedures may beemployed to demonstrate an article is sterile, pro-vided the results obtained are at least of equivalentreliability.

Regulatory Approval for the Use of AlternateMethodsAccording to 21 CFR 314.70 “Supplements andother changes to an approved application,” the ad-dition or deletion of an alternate analytical methoddoes not require prior approval and may be filed inthe Annual Product Report.

Rapid Microbial Test MethodsNew microbiological testing methods that achievethe same results as the classical tests within a shortertime period, i.e., less than 7 days for sterility test

ing and 24 hours or less for microbial counts andmicrobial identification. The ultimate rapid testsare completed in real time, i.e., 1 to 3 hours.

ATP Bioluminescence:The estimation of the microbial population by themeasurement of light in a luminometer producedby the reaction of the American firefly enzymeLuciferase and substrate Luciferin and the energycontained in the energy-rich phosphate bond ofadenosine triphosphate (ATP) found in all livingorganisms. A single molecule of ATP produces aphoton of green light so that the signal is propor-tional to the amount of ATP in the sample. Thethreshold for detection is 103 bacteria and 102

yeast. A lower number of microorganisms maybe detected by allowing them to grow into theseranges.

Immunological Detection Methods:The presence of an antibody or antigen in a sampleassociated with a specific microorganism may bequantitatively or qualitatively determined by aspecific antibody-antigen reaction which is de-tected by agglutination, color, or fluorescence de-velopment from a linked enzyme reaction. Withthe advent of monoclonal antibodies to specificantigens associated with targeted microorganisms,immunological detection methods to specificpathogens have been developed. Examples fromthe food industry are the detection of Salmonella,Listeria and E. coli O157 by enzyme-linkedimmunosorbent assay (ELISA).

Nucleic Acid Probes:Nucleic acid probes consist of a short segment ofDNA or ribosomal RNA complimentary to thetarget sequence. A portion of an enrichment cul-ture is treated to liberate the nucleic acid that ishybridized with the enzyme-labeled probe and isthen detected colorimetrically.

Polymerase Chain Reaction (PCR) Methods:From an enrichment culture, specific rDNA seg-ments from lyzed bacterial cells are amplified toa sufficient quantity to be detected. Detection isbased on viewing a target-specific band using gel


electrophoresis. PCR allows for highly sensitive,accurate and rapid detection of target organisms.

Impedance and Conductance Methods:Changes in the electric impedance or conductiv-ity of microbiological media as growing micro-organisms break down large, relatively unchargedmolecules (such as proteins) to smaller, moreabundant, highly charged molecules (such asamino acids) are used to detect microorganisms.Larger inoculum levels give rise to shorter detec-tion times, and specific pathogens may be detectedby the use of selective media.

Good Manufacturing Practices

The US Food and Drug Administration publishedregulations describing the minimum current goodmanufacturing practices for the preparation ofdrug products.

21 CFR Part 211.113 Control of microbiologicalcontamination states a) Appropriate written pro-cedures, designed to prevent objectionable micro-organisms in drug products not required to be ster-ile, shall be established and followed, and b) Ap-propriate written procedures, designed to preventmicrobiological contamination of drug productspurporting to be sterile, shall include validationof any sterilization process.

21 CFR Part 211.165 Testing and release for dis-tribution states b) There shall be appropriate labo-ratory testing, as necessary, of each batch of drugproduct required to be free of objectionable or-ganisms.

21 CFR Part 211.167 Special testing requirementsstates a) For each batch of drug product purport-ing to be sterile and/or pyrogen-free, there shallbe appropriate laboratory testing to determineconformance to such requirements.

21 CFR Part 211.194 Laboratory records states(2) A statement of each method used in the test-ing of the sample. The statement shall indicatethe location of data that establish that the meth-

ods used in the testing of the sample meet properstandards of accuracy and reliability as applied tothe product tested. (Note: USP, NF and AOACmethods need not be validated, but the suitabilityof test method verified under actual use conditions).

FDA Guidelines

FDA guidelines are published under the proceduralprovisions of 21 CFR Part 10.90 to permit com-ments from interested individuals and companies.This regulation provides for the establishment ofguidelines that are of general applicability toCGMPs but are not legal requirements. If a com-pany follows the guidelines to achieve CGMP com-pliance then this will generally be considered ac-ceptable to the FDA. Firms are not obligated tofollow the guidelines, but alternative approaches tothose listed in the guideline will require FDA ap-proval. For example, the 1987 FDA Guideline onSterile Drug Products Produced by Aseptic Process-ing (20) that discusses clean room air quality mi-crobial levels and the conditions under which ste-rility tests may be repeated.

FDA Inspectional GuidesGuides issued by the Division of Field Investi-gations, Office of Regulatory Affairs, FDA forthe FDA field investigators. For example, Guideto Inspections of Microbiological Pharmaceu-tical Quality Control Laboratories, July, 1993which addresses facilities, equipment, media,microbiological testing of non-sterile products,sterility testing, methodology and validation oftest procedures, data storage, management re-view and contract testing laboratories.


Is currentcompendial method

acceptable?

Determine requirementsof the test, i.e., cost,

cycle time, specification, etc.

Does thecandidate alternate

method meetrequirements?

Perform proof of concept

Does method work?

Write validation protocol

Wasvalidation protocol

fulfilled?

Implement test

IMPLEMENTATION OF ALTERNATE MICROBIAL TEST METHODS

Yes

Yes

Yes

No

STOP

Reject

candidate andselect another

No

No

No

Yes


Is new methodvalidated as an

alternative method?

Is currentmethod describedas compendial?

File as alternatemethod in annual report

Qualify lab and beginusing test

REGULATORY APPROVAL FOR ALTERNATE MICROBIAL TEST METHOD

Yes

Yes

STOP

File as asupplement

No

No


This page intentionally left blank.

6.0 REFERENCES

General Information Chapter <1225> Valida-tion of Compendial Methods, USP 24, USPC,Inc., Rockville, MD, 2000, p. 2149.

International Conference on Harmonization(ICH) Validation of Analytical Methods.

ASM, Cumitech, No. 31, Verification and Vali-dation of Procedures in the Clinical Microbi-ology Laboratory, February 1997.

General Information Chapter <1227> Valida-tion of Microbial Recovery fromPharmacopeial Articles, USP 24, USPC, Inc.,Rockville, MD, 2000, p. 2152.

General Informational Chapter <1231> Waterfor Pharmaceutical Purposes, USP 24, USPC,Inc., Rockville, MD, 2000, p. 2154.

Standard Methods for the Examination of Wa-ter and Wastewater, American Public HealthAssociation, R. T. Marshall (editor), Washing-ton D. C., USA 20th Edition, 1998.

Chapter <51>, Antimicrobial EffectivenessTesting, USP 24, USPC, Inc., Rockville, MD,2000, p. 1809.

Chapter <61> Microbial Limit Tests, USP 24,USPC, Inc., Rockville, MD, 2000, p. 1814.

Chapter <71> Sterility Tests, USP 24, USPC,Inc., Rockville, MD, 2000, p. 1818.

General Information Chapter <1116> Micro-biological Evaluation of Clean Rooms andOther Controlled Environments, USP 24,USPC, Inc., Rockville, MD, 2000, p. 2099.

PDA Technical Report No. 13, Fundamentalsof a Microbiological Environmental Monitor-ing Program, J. Parent. Sci. Tech., 44, No. S1(1990).

American Society for Testing and Materials(ASTM) Standard Practice for Comparing TestMethods D, pp. 225-232, ASTM, Philadelphia,PA, 1991.

In-process Revision, General InformationChapter <1010> Analytical Data - Interpre-tation and Treatment, Pharmacopeial Forum,Vol. 25, No. 5, pp. 8900-8909, 1999.

“Guideline on General Principles of ProcessValidation,” FDA, May 1987.

K. Willis, H. Woods, L. Gerdes, A. Hearn,N. Kyle, P. Meighan, N. Foote, K. Layte, andM. Easter, “Satisfying Microbiological Con-cerns for Pharmaceutical Purified WatersUsing a Validated Rapid Test Method,”Pharmacopeial Forum, Vol. 24, No. 1, pp.5645-5664, 1998.

D. L. Jones, M. A. Brailsford, and J-LDrocount, “Solid Phase Cytometry: A NovelTwo-Hour Method for the Rapid MicrobialAnalysis of Pharmaceutical Water,”Pharmacopeial Forum, Vol. 25, No. 1, pp.7626-7645, 1999.

Marino, G.C. Maier, and A.M. Cundell, “AComparison of the MicroCount Digital Sys-tem to Plate Count and Membrane FiltrationMethods for the Enumeration of Microorgan-isms in Water for Pharmaceutical Purposes,”PDA Journal of Pharmaceutical Sciencesand Technology (In Press).

B. Javis, Statistical Aspects of the Microbio-logical Analysis of Food In Progress in In-dustrial Microbiology, Volume 12, pp.117-121, Elsevier Science Publishers B.V.,Amsterdam, The Netherlands, 1989.

Cunniff, P. (Editor), Official Methods ofAnalysis of the AOAC International, AOACInternational, Gaithersburg, MD, 16th Edi-tion, 5th Revision, 1999.

“Guideline on Sterile Drug Products Pro-duced by Aseptic Processing,” FDA, June1987.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.


NOTES

PDA Journal ofPharmaceutical Science and Technology

Supplement TR33 Volume 54

May/June 2000 EDITOR: Lee Kirsch No. 3

c/o The University of IowaPharmacy Building, S223Iowa City, IA 52242, USA(319) [email protected] Assistant: Anjali Joshi

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ADVISORY BOARDMichael AkersFrederick J. CarletonPatrick DeLuca, University of KentuckyBarry Garfinkle, Merck Sharp & DohrneMichael Groves, University of IllinoisJoseph Robinson, University of WisconsinTheodore Roseman, Baxter Healthcare

2000 OFFICERS AND DIRECTORSChair: Robert B. MyersChair-Elect: Floyd BenjaminSecretary: Jennie AllewellTreasurer: Nikki V. MehringerImmediate Past Chair: Joyce H. Aydlett

Vince R. AnicettiStephanie R. GrayHenry K. Kwan, Ph.D.Suzanne LevesqueRichard V. Levy, Ph.DP. Michael Masterson, P.E.Robert J. Mello, Ph.D.Taiichi Mizuta, Ph.DRobert F. Morrissey, Ph.DGeorg Roessling, Ph.DKenneth B. Seamon, Ph.DGlenn E. Wright

President: Edmund M. Fry

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