2nd Edition

Microbial Limit and Bioburden Tests Validation Approaches and Global Requirements,Second Edition

By Lucia Clontz Copyright 2009
    342 Pages 97 B/W Illustrations
    by CRC Press

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    In recent years, the field of pharmaceutical microbiology has experienced numerous technological advances, accompanied by the publication of new and harmonized compendial methods. It is therefore imperative for those who are responsible for monitoring the microbial quality of pharmaceutical/biopharmaceutical products to keep abreast of the latest changes. Microbial Limit and Bioburden Tests: Validation Approaches and Global Requirements guides readers through the various microbiological methods listed in the compendia with easy-to-follow diagrams and approaches to validations of such test methodologies.


    Includes New and Updated Material

    Now in its second edition, this work is the culmination of research and discussions with technical experts, as well as USP and FDA representatives on various topics of interest to the pharmaceutical microbiologist and those responsible for the microbial quality of products, materials, equipment, and manufacturing facilities. New in this edition is an entire chapter dedicated to the topic of biofilms and their impact on pharmaceutical and biopharmaceutical operations. The subject of rapid methods in microbiology has been expanded and includes a discussion on the validation of alternative microbiological methods and a case study on microbial identification in support of a product contamination investigation.

    Substantially updated and revised, this book assists readers in understanding the fundamental issues associated with pharmaceutical microbiology and provides them with tools to create effective microbial contamination control and microbial testing programs for the areas under their responsibility.

    I. Microbial Life and Ecology

    An Overview of Microbial Life

    Microbial Phylogeny

    Microbial Taxonomy

    Microbial Growth and Survival

    Growth Curve


    Energy Sources



    Cell Shape


    Bacterial Growth and Reproduction

    Cell Structures

    The Phyla Gram-Positive Bacteria and Proteobacteria

    Gram-Positive Bacteria


    The Gram-Staining Method

    KOH Test

    Catalase Test


    Cell Structures

    Fungal Growth and Reproduction



    Microorganisms of Interest

    Genus Staphylococcus

    Staphylococcus aureus

    Genus Pseudomonas

    Pseudomonas aeruginosa

    Genus Burkholderia

    Burkholderia cepacia

    Genus Ralstonia

    Ralstonia pickettii

    Genus Comamonas and Genus Stenotrophomonas

    Family Enterobacteriaceae

    Genus Escherichia

    Escherichia coli

    Genus Salmonella

    Genus Shigella

    Genus Serratia

    Genus Klebsiella

    Genus Bacillus

    Bacillus subtilis

    Bacillus cereus

    Genus Clostridium

    Clostridium perfringens

    Clostridium sporogenes

    Candida albicans

    Zygosaccharomyces rouxii

    Genus Aspergillus

    Aspergillus niger

    Genus Penicillium


    II. Microbial Contamination and Control

    Microbiological Contamination

    Product Recalls

    Nonsterile Products

    Microbial Limit Standards

    The Preservation of Pharmaceutical Products

    Antimicrobial Activity and Efficacy

    Types of Preservatives


    Benzalkonium Chloride

    Benzoic Acid and Salts

    Boric Acid and Salts



    Dowicil 200




    Sorbic Acid Salts

    Microbiological Control

    Risk Assessment

    Objectionable Organisms

    Sanitization and Disinfection Practices

    Definitions and Types of Chemical Products

    Factors in Choice and Use of Disinfectants

    Rotation of Disinfectants

    Qualification of Disinfectants

    In Situ Testing

    In Vitro Testing

    Expiration Date for Disinfectant Solutions

    Sanitizers Used for Equipment Cleaning

    Neutralization and Microbial Recovery Studies

    Requalification and Change Control



    III. The USP Microbial Limit Tests

    History of the Revision and Harmonization Process

    USP Chapter <61>: Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests

    Sample Preparation

    Total Aerobic Microbial Count

    Total Combined Yeasts and Molds Count

    Bioburden Tests

    Two-Media Bioburden Test

    One-Medium, Dual-Temperature Incubation Bioburden Test

    TAMC and TYMC Tests via Plate-Count Methods

    Pour-Plate Method

    Spread-Plate Method

    Incubation and Results Calculation

    Test Controls

    TAMC and TYMC Tests via Membrane Filtration Method

    Test Controls

    TAMC Test by the Multiple Tube Method


    Interpretation of the TAMC and TYMC Test Results

    USP Chapter <62>: Microbiological Examination of NonSterile Products:

    Tests for Specified Microorganisms

    Sample Preparation for Direct Inoculation Tests

    Test for Absence of Escherichia coli

    Test for Absence of Salmonella spp

    Test for Absence of Bile-Tolerant Gram-Negative Bacteria

    Test for Absence of Pseudomonas aeruginosa

    Test for Absence of Staphylococcus aureus

    Test for Absence of Candida albicans

    Test for Absence of Clostridia

    Quantitative Test for Bile-Tolerant Gram-Negative Bacteria



    IV. Pharmaceutical Waters

    Types of Water for Pharmaceutical Purposes

    Microbial Quality Attributes

    Testing of Pharmaceutical Waters

    Sampling Program

    Sample Collection and Preservation

    Bioburden Testing

    Recovery Media

    Coliform Testing

    Identification of Waterborne Microorganisms

    Establishing Alert and Action Levels

    Validation of Water Systems

    Microbial Control and Sanitization


    V. Environmental Monitoring

    Cleanroom Classification

    Occupancy State

    Routine EM Program

    Testing Frequency and Sampling Sites

    Setting Alert and Action Levels

    Test Methods and Equipment

    Surface Sampling for Viable Particles

    Active Air Sampling for Viable Particles

    Active Air Sampling for Nonviable Particles

    Microbial Identification Program

    Data Analysis

    EM During Facility Validation Activities

    Room Occupancy

    EM of Isolators

    Microbial Control in Cleanrooms


    VI. Bioburden Considerations In Equipment-Cleaning Validation

    Biocontamination Control

    Disposable and Single-Use Equipment

    Equipment-Cleaning Methods

    Validation of Cleaning Methods

    Sampling Recovery Methods

    Swabbing of Equipment

    Rinsing of Equipment

    Qualification of Sampling Methods

    Recovery Study Using the Wet Method

    Recovery Studies Using the Dry Method

    Effects of Product and/or Cleaning Agent Residue on the Recovery of Microorganisms

    Establishing Limits

    Execution of Equipment-Cleaning Validation Protocol

    Validation of Cleaned Equipment Hold Time

    Validation of Dirty Equipment Hold Time

    Ongoing Verification of Cleaning

    Validation of Holding Time/Shipping Conditions


    VII. Method Validation and Media Suitability Testing

    Suitability Test Design

    Representative Challenge Organisms

    Maintenance and Preparation of Test Organisms

    Preparation of Working Cultures

    Validation of Storage Period for Working Cultures

    Recovery of Injured Organisms

    Suitability Testing By Direct Inoculation/Plating Methods

    Validation of Screening for Specified Microorganisms

    Modifications to the Direct Inoculation Method

    Validation of the TAMC and TYMC Tests

    Approach 1

    Approach 2

    Modifications to the Plate Method

    Suitability Testing for Membrane Filtration Methods

    Validation of Screening for Specified Organisms

    Validation of TAMC and TYMC

    Modifications to the Membrane Filtration Method

    Suitability of Microbiological Media

    Growth Promotion Testing for Microbial Enumeration Media

    Growth Promotion Testing for Selective Media

    Validation of Rapid Microbiological Methods

    The Validation Package

    Validation Criteria

    Validation of Quantitative Methods




    Limit of Quantitation


    Limit of Detection




    Validation of Qualitative Methods


    Limit of Detection



    Accuracy and Precision

    Validation of Automated Microbial Identification Methods





    Final Thoughts

    Points to Consider


    VIII. Microbiological Quality of Pharmaceutical and Biopharmaceutical Products and Raw Materials

    Microbiological Testing

    Raw Materials

    Biopharmaceutical Products

    Nonsterile Finished Drug Products

    USP Chapter <1111>

    Testing Frequency

    Stability Testing

    Water Activity

    Measuring Water Activity

    The Chilled-Mirror/Dew Point Method

    Capacity Sensors

    Pharmaceutical Applications for Water Activity

    International Harmonization

    Looking Ahead


    IX. Rapid Testing and Alternative Methods in Microbiology

    Rapid Method Technology Platforms

    Impedance/Conductance Technology

    Gas Consumption or Generation

    ATP Bioluminescence

    The Celsis ATP Bioluminescence Systems

    Automated Biochemical Assays

    The VITEK® System

    The Biolog Systems

    Fatty Acid Analysis Using Gas Chromatography

    The MIDI System

    Enzyme-Linked Immunosorbent Assay (ELISA)

    The VIDAS

    Analysis of Biomolecules Using Mass Spectrometry

    Polymerase Chain Reaction (PCR)

    Detection of Microbial Contamination Using PCR Technology


    The Riboprinter®

    Fluorescent Labeling Assays

    Scan® RDI Microbial Detection


    Biosensors and Microarrays

    Laboratory-on-a-Chip Technology

    Barriers to Implementation

    Regulatory Climate

    Future Trends

    Case Study: Genotypically Similar Staphylococci

    Contaminant Isolate and Environmental Sampling

    Ribosomal Gene Sequencing

    Phenotypic Analysis

    Genetic Subtyping—PFGE

    Results and Reporting


    X. Biofilms

    Biofilm Definition

    Biofilm Structure

    The Biology of Biofilms

    Biofilm Formation

    Quorum Sensing

    Cell Adhesion

    Smooth versus Rough Surfaces

    Hydrophobic versus Hydrophilic Surfaces

    Electrostatic Charge Properties

    Low-Shear versus High-Shear Environments

    Biofilm Dispersion

    Biofilm Resistance and Phenotypes

    Pharmaceutical Production Equipment and Materials Prone to Biofilm Formation

    Water Systems

    Production Equipment

    Ultrafiltration/Diafiltration (UF/DF) Systems

    Chromatography Systems

    Miscellaneous Parts and Materials

    Biofilm Control and Prevention

    Heat Treatment

    Chemical Treatment

    Prevention of Biofilms

    Methods for Detection and Recovery of Biofilm Organisms

    Qualification of Chemical Sanitization Using Biofilm Cells

    Types of Biofilm Reactors

    Choosing a Biofilm Reactor

    Testing Sanitizers Using the CDC Biofilm Reactor

    Setting up the Biofilm Reactor

    Exposure of Biofilm to Disinfectant Solution

    Harvesting Biofilm Cells

    Sanitizer/Disinfectant Efficacy Evaluation

    Method Qualification and Test Controls

    Testing Sanitizers Using a Static Biofilm Reactor

    Industrial Significance of Biofilms

    The Future in Biofilm Research


    XI. Handling Aberrant and Out-of-Specification Microbial Data

    Historical Overview of Investigating OOS Results

    Out-of-Specification (OOS) Result

    Laboratory Investigations

    Conducting the Investigation

    Retesting and Resampling

    Testing for Outliers

    Repeat Testing

    Concluding the Investigation

    Product Lot Disposition

    OOS Investigations and FDA Citations




    Lucia Clontz