Microbiology and Sterility Assurance in Pharmaceuticals and Medical Devices

About Book

ISBN 9788190646741
Presentation Hardbound, 996 Pages
Edition 1st Edition, 2011
Weight 1.70 Kg
Author Madhu Raju Saghee,Dr. Tim Sandle,Dr. Edward C. Tidswel
Price USD 150
Availability In Stock


Injectable product manufacturing is booming because of the growth of new biopharmaceuticals and small molecule anticancer drugs. The requirements for contamination control will become even more stringent than today. Isolators or blow-fill-seal equipment have already replaced the conventional clean rooms and LAF-hoods in many production facilitities. Conventional microbiological monitoring methods, requiring 3 to 5 days of incubation will become inappropriate. Equipment is already available allowing real time, simultaneous viable and non-viable counting.

This book is a useful reference guide for the SMB (Small and Medium Business) pharmaceutical sector which does not have the resources to have access to such top-quality information in this field. This book therefore represents an unparalleled and unprecedented text in the field of pharmaceutical and medical device microbiology. Perhaps even more outstanding is the fact that this book not only covers subject matter and technical content which is established as best and expected practice, but also includes content regarded as possible, future and emerging technology or processes.

The results of 45 years of scientific and technological development are laid down in these 33 chapters. These chapters, all written by international experts, give a vivid picture of today’s pharmaceutical microbiology. The high standard of the chapters makes it an essential reference guide that should be on the shelf of everyone who is involved or interested in this field.

Experts from top pharmaceutical companies like Baxter, Johnson and Johnson, Amgen, Pfizer, Patheon, Sartorious, Gador, Catalent, British NHS, GE Healthcare and many more top experts from industry and academics have come together to create this collection of knowledge of Microbiology as related to Pharmaceuticals, Medical Devices and Biotechnology. The list of authors includes chair and members of USP expert committee on Microbiology and Sterility Assurance, president and senior experts in Parenteral Drug Association, ISPE and ASTM. Foreword by Hans van Doorne, Chair of EDQM, Committee on Microbiology.


Madhu Raju Saghee, M.Sc - Madhu Raju Saghee is working in corporate quality and sterility assurance department at Gland Pharma Limited, India.

Tim Sandle, Ph.D, CBiol, MSBiol. - Dr. Sandle is the Head of Microbiology at the UK NHS Bio Products Laboratory. His role involves overseeing a range of microbiological tests, batch review, microbiological investigation and policy development.

Edward C. Tidswell, PhD - Dr. Tidswell is the Senior Director of Sterility Assurance for Baxter Healthcare; located north of Chicago, IL (USA). Responsibilities include: leading Baxter’s research, development and implementation of new (or optimized) sterilization and aseptic technologies, qualification and validation of sterile and aseptic manufacture for parenterals and medical devices.



PART: 1     The Impact of Microorganisms in Pharmaceutical and Medical                       Device Manufacture


The Essentials of Pharmaceutical Microbiology

by Tim Sandle and Madhu Raju Saghee


  History of Microbiology

  Fundamental Characteristics of Microorganisms






    Microbial Taxonomy

    Microbial Growth

    Microbiological Culture Media

    Identification and Characterization of Microorganisms

    Types of Bacteria

Overview of Pharmaceutical Microbiology

  The Scope of Pharmaceutical Microbiology

    Product Related Testing Regimes

    Starting Materials

    In-Process Samples / Intermediate Product

    Final Product Formulations

    Finished Product

    Testing of Utilities

    Environmental Monitoring

  The Application of Pharmaceutical Microbiology



    Microorganisms Detected From Pharmaceutical Manufacturing Environments

    Contamination Control

    Other Microbiology Laboratory Tests

        Microbial Identification

        Water Activity

        Disinfectant Efficacy Testing

        Antimicrobial Susceptibility Testing

        Microbial Immersion Studies

        Cleaning Validation Studies

        Investigation of Out of Limits and Out of Trend Results

        Microorganisms Used In Pharmaceutical Microbiology Laboratories

        Other Activities







Relevance of Microorganisms in Pharmaceutical Processing


by Ossama M. El-Tayeb



Historical Review of the Involvement of Microbiology with Pharmaceutical Practice

   Implications of aseptic surgery, personal and public hygiene

   Implications of Food Microbiology

   Implications of the introduction of parenteral therapy: {earliest procedures for       

      sterilization and preservation}

   Early tests for sterility: influence of diagnostic medical microbiology

   Early pharmacopoeial guidance on sterilization and sterility testing

Historical Review of Science-Based Pharmaceutical Sterilization and Microbiological   Quality Control

   Re-definition of the pathogenicity of microorganisms

   Contributions of the study of microbial injury and death

   Contributions of the food processing technologies: the over-kill approach to                  sterilization

   Contributions of the aerospace and electronics industries.

   Hospital sterilization and Central Sterile Supply vs. on site sterilization

   Impacts of material sciences and development of cold sterilization methods

   Filtration as a breakthrough and the bio-burden approach to sterilization.

   Impact of statistical fallacies in sterility testing and population death kinetics.

The Broader Picture of Microorganisms and Pharmaceutical Manufacturing: Challenges, Solutions and Pharmacopoeial Guidance

   Microorganisms and Ophthalmic Dosage Forms

   Microorganisms and Oral Dosage Forms

   Microorganisms and Topical Dosage Forms

   Microbiological Challenges, Solutions and Pharmacopoeial Requirements

Current Pharmacopoeial Guidance

   The Fundamental Concepts

   Sampling for Microbial Quality Control and the Microbiologically Homogenous


   Quality Assurance as an Essential Element in Microbial Quality

   The Roles of the Microbial Quality Control Laboratory

   Current Pharmacopoeial Guidance {on sterilization, sterility testing and microbial

      quality of non-sterile dosage forms}

The Future

   Will all Pharmaceuticals be Aseptically Processed?

   The future of the Microbiological Quality Control Tests



Microbial Contamination and Spoilage

by David G. Allison


Sources of Microbial Contaminants

Raw Materials

Materials of Natural Origin

   Microorganisms from Plant Material

   Microorganisms from Animal Sources

   Microorganisms from Mineral-Derived Materials

Synthetic Raw Materials


   Types of Water

   Disinfection of Water

Microbial Contamination from the Manufacturing Environment

   Air supply

   Equipment and facilities


   Users / consumers

Factors affecting Microbial Spoilage of Pharmaceutical Products

   Preparation and Storage

   Nature of the Contaminant Inoculum

   Moisture Content

   Nutritional Factors

   pH and Redox

Consequences of Microbial Growth

Microbiological Control of Raw Materials




Microbiological Considerations in Medical Device Industry

by Martell Winters


The Medical Device Microbiology Life Cycle

Inherent and Accumulated Microbial Contamination

Product Design Microbiology

Raw Material and Component Microbiology

Shipping and Storage of Raw Materials and Components

Environmental Microbiology

Product Assembly Microbiology

Cleaning of Products

Packaging Process Microbiology

Sterilization Process Microbiology

Product Use Microbiology

Medical Device Microbiological Contamination

Controlling Product Bioburden

Aseptic Technique

No Magic Answers for Bioburden Control

Use of Bioburden Testing to Improve Manufacturing Microbiology

Use of Sterility Testing to Improve Manufacturing Microbiology

Microorganisms of Concern

Microbiological Challenges for Products with Tissue Components



PART: 2     Aspects of Microbiological Quality Control


Selection of Microbiological Culture Media and Testing Regimes

by Tim Sandle


Types of Culture Media

Media Manufacture

Quality Control of Culture Media


  When to Perform Quality Control?

  Establishing a Nutritive Properties Testing System

    Micro-organisms: Typed Cultures

    Micro-organisms: Environmental Isolates or ‘Wildtypes’

    Micro-organisms: The Microbial Challenge

    Micro-organisms for the Testing of Selective Media or Differential Media

    Test Method

    Test Regime

    Sterility Test of Media

    Expiry Time Assessment of Culture Media

    New Lots of Media

  Media Release and Quarantine




Microbial Identification

by Ziva Abraham

Importance of Microbial Identification

Isolation of Microorganisms

Staining Methods

  Gram Staining

  Spore Stain

  Fluorescent Stains

  India Ink (Colloidal Carbon) Stain

  Acid-Fast (Ziehl Neelsen) Stain

  Lactophenol Cotton Blue Stain


  Stereo Microscope

  Bright Field Microscopy

  Dark Field Viewing

  Phase Contrast Microscopy

  Differential Interference Contrast (DIC) Microscopy

  Florescent Microscopy

Pre-Differentiation or Confirmation

Taxonomy, Classification, Identification and Nomenclature

Phenotypic Microbial Identification Systems

  API Panels

Automation in Microbial Identification

   Vitek® Microbial Identification Systems

   BiologTM System

   MIDI Sherlock® Microbial Identification System using Fatty Acid Methyl Ester  

       (FAME) Analysis

Genotypic Systems

  Polymerase Chain Reaction (PCR)

  DiversiLab® Strain Typing

  Microarrays in Microbial Identification

Fungal Identification





Microbial Examination of Non-sterile Products

by Jaymie Tomes and Florence Wu


The Limitations and Values of Microbial Enumeration Tests

Product Sampling for Microbial Examination

    Sampling Schemes   

    Sample Sizes

    Sample Preparation

Media Growth Promotion Test

Method Suitability Test

Enumeration Methods

Media and Incubation

Colony Counting and Interpretation of the Results

Tests for Specified Microorganisms

    Staphylococcus aureus

    Pseudomonas aeruginosa

    Escherichia coli



    Candida albicans

    Bile Tolerant Gram Negative

Setting Microbial Limits for Non-Sterile Pharmaceuticals and Raw Materials




Practical Approaches to Sterility Testing

by Tim Sandle


The Sterility Test

Sterility Test Method Validation

   Validation of Culture Media

   Sterility Test Validation

     Dealing with ‘Difficult’ Products

     Membrane Filtration

         Type of Membrane Filter

         Type and Number of Rinse Solutions

    Direct Inoculation

         Type of Neutralising Agent


    Turbid Samples


    Oily Samples

    Anti-cancer Treatments


    Sterile Aerosols

    Cell Lines

    Fibrin Sealant

    Questions relating to validation that are not directly answered by the


  The Stasis Test

Training Programme

Validation Documentation

   Validation Protocol

   Validation Report




Microbial Aspects in Cleaning Validation

by Andrew Walsh


Regulatory Aspects of Cleaning

   API Manufacturing

   Finished Pharmaceuticals

   Cleaning Processes

Cleaning Agents

Cleaning Procedures

   Manual Cleaning

   Clean-Out of-Place (COP)

   Clean-in-Place (CIP)

Solid Dosage Forms

   Setting Microbial Acceptance Criteria for Solid Dosage Forms

Semi-solid Dosage Forms

   Setting Microbial Acceptance Criteria for Semi-solid Dosage Forms

Hold Time Studies

Microbial Testing in Cleaning Validation





Validation of Microbiological Methods

by Sandy Rubio



   Why is Bioburden Testing Important?

Strategy for Validation

Protocol Considerations

   Description of Product

   Manufacturing Process

   Challenge Microorganisms

   Current Validation Status

   Regulatory and Guidance Documents

   General Protocol Considerations

   Bacteriostasis/Fungistasis Evaluation

   Acceptance Criteria

   Method Development

   Sample Hold Time Determination

Maintaining Status of Validated Methods

Case Study


   Review Findings




Selection and Validation of Disinfectants

by Paul Viña, Sandy Rubio and Tim Sandle


Types of Disinfectants

   Non-Oxidizing Disinfectants




     Acid Anionics



     Quaternary Ammonium Compounds (QACs)

   Oxidizing Disinfectants


     Oxidizing Agents

Selection of Disinfectants

   Number, Type, and Location of Microorganisms


     Type of Microorganism and Disinfectant Resistance

     Location of Microorganisms



   Temperature and pH

   Interfering Substances

Validation of Disinfectants

   Test Methods

     Basic Suspension Test

     Bactericidal Suspension Test and Fungicidal Suspension Test

     Surface Test

   Test Variables

     Challenge Microorganism Selection and Inoculation of Surface Materials

     Selection of Test Surfaces

     Disinfectant Test Concentration and Preparation

     Establishing the Disinfectant Expiration Date

     Evaluating Interference from Organic Material

     Application of Disinfectant to Surface Coupons

     Contact Time

     Selection of Neutralizing Agents

Continued Evaluation of Disinfectant Effectiveness



Further Reading


Auditing a QC Microbiology Laboratory

by Andy Martin


What is an Audit and Why Should They be Performed

General Principles of the Audit Process

Auditing the QC Microbiology Laboratory, What to Look For

   Strategy for Microbiological Control

   Sample Receipt

   Product Sample Testing

   Bacterial Endotoxin Testing

   Media Control and Testing

   Incubator, Refrigerator and Freezer Control and Monitoring

   Culture Collections

   Water Testing and Water System Monitoring

   Environmental Monitoring

   Isolate Identification, Gram stain techniques and ID system validation

   Autoclave control

   Antibiotic assays and Preservative testing

   Sterilization, Cleaning and disinfection validation efficacy

   Microbiological Training of Laboratory and Production Staff

   Validation of Holding Times

   Atypical Results and Out Of Specification Procedures

Auditing Techniques to Get the Most from your Audit

Reporting the Audit

Final Thoughts



Quality Assurance in a Microbiology Laboratory

by Christophe Barcella


Quality Management, QA and QC Principles

    Quality Management

    Quality System

    Quality Assurance

    Quality Control

Laboratory Quality Manual

Laboratory Staff Qualifications and Training

        Training Policy

        Job Description

        Curriculum Vitae

        Staff Training Records

Laboratory Environment


        Air Ventilation

        Lab Sanitation and Environment Monitoring

        Environmental Hygiene and Health

        Protective Equipment

Standard Operating Procedures, Methods and Protocols

        Development, Review and Approval of SOPs

        New Method Development, Validation and Amendment

        Quality Attributes for a New Method

Laboratory Notebooks, Log Books and Records

        Minimum Acceptable Procedures for Laboratory Notebooks

        Review of Test Results

        Electronic Records

Information Technology and Computer Systems Validation

        Definition of Hardware and Software

        Computer Security and Backups

        Computer Systems Validation (CSV)

Archiving Requirements

        Archiving Physical Facilities

        Archiving Personnel

        Archiving Electronic Records

        Format of Archived Materials

        Laboratory Test Samples and Preserved Wet Materials

        Duration of the Archive

Reference Standards and Samples

        Reference Standards for Microbiological Testing

            Reference Standards Storage, Handling and Labeling

            Standard References Records

        Microbiological Reference Strains

        Management of Samples

Solutions, Reagents and Culture Media

        Reagent General Specifications

        Reagent Tracking

        Reagent Solutions

        Quality Control of Culture Media

Instruments and Equipment

        Equipment Installation and Qualification

        Equipment Operations

        Equipment Calibration and Maintenance

        Glassware and Volumetric Micropipettes

            Glassware Grades


Laboratory QA Assessments/Audits & External Quality Assurance

        QA Audit Methodology

        External Quality Assessment (EQA)

        Internal Quality Assessment (IQA)




Part: 3     Measuring and Testing for Microorganisms


Environmental Monitoring

by Tim Sandle




   Cleanroom Classification

   Physical Parameters

   Air-patterns and Air-Movement


   Air Changes

   Clean-up Times (Recovery Rate Test)

   Positive Pressure

   HEPA Filters

   Temperature, Humidity, Lighting and Room Design

Microbiological Environmental Monitoring

   Viable Monitoring Methods


     Contact Plates

     Active Air Monitoring

     Types of Active Air-Sampler

     Settle Plates

   Non-viable Monitoring Methods

Establishing an Environmental Monitoring Programme

   Sampling Plans

   Sample Frequencies

   Sample Limits


   Aseptic Technique

   Other Cleanroom Disciplines






Microbial Content Testing of Pharmaceutical and Biotechnologically Derived Products

by Dilip Ashtekar and Tim Sandle


General Methods for the Quantitative Determination of Viable Count

   Membrane Filtration

   Pour Plate

   Surface Spread plate

   Most Probable Number

Evaluation of Results by Various Enumeration Methods

   Points to Consider for Counting CFU

   Rounding and Averaging

   Significant Figures

   Countable Range

   Statistical Errors from Low Counts


Replicate Plate Counts

   Consideration of Performing of Replicate Samples in Place of Testing One Large


Selection of Method for the Enumeration of Microbial Count

Estimating Number of Micro-Organisms in Suspension for Use in Bioburden    

     Validation or Suitability Test for MLT

Bioburden Testing

   Bioburden Test Method Validation

   Validation Requirements

Microbial Limit test (MLT)

   A1: The TAMC Test

   Reporting of Enumeration Test Results


Suitability of the Enumeration Method in the Presence of Product

Test for Specified Organisms

Applied Aspects of Bioburden Testing

   Method Limitations

   Sample Expiry

     Incubation Parameters

     Influencing Factors

   Staff Proficiency Training

     Plate Counting

     Pour Plates




Bacterial Endotoxins Test

by Masakazu Tsuchiya


History of Bacterial Endotoxins Test


   Structure and Biological Activity of Endotoxin

   Stability of Endotoxin

   Standard Endotoxin

   Variability of Potency of Daily RSE Dilutions

Limulus Amebocyte Lysate



   Reactivity of LAL

   Pyrogenicity and LAL Test

Bacterial Endotoxins Test in Pharmacopoeias

Validation of Bacterial Endotoxins Test

Practical Suggestions for the Bacterial Endotoxins Test

   Uncertainty of Bacterial Endotoxins Test

   Interfering Factors


   Removal of Endotoxin/β-Glucan

   Robust LAL Methods Using an Archived Standard Curve




Appendix A (Example of Standard Operating Procedure for Gel-Clot Method)


Antimicrobial Effectiveness Testing

by Scott V. W. Sutton


The Purpose of the AET

   What the Test Means


   Pharm Eur

Development of the USP Test

   USPXVIII - The Original Test

   USP XIX - Refinements

   USP XX, XXI and XXII – Little Activity

   USP 23, 24 and 25 - Reducing Variability

   USP 23, 24 and 25 - Harmonization?

Demonstration of Method Suitability

   Importance of Preservative Neutralization

   Development of USP <1227>

   Method Suitability for Quantitative Studies

Other considerations in preservation


   In-use Testing


   Lab Investigations

   Specific Considerations for AET




Monitoring of Microbiological Quality Attributes of Water for Pharmaceutical Use

by Dilip Ashtekar


Water Types

Regulatory Requirements

Water System Description


   Activated Carbon



   Reverse Osmosis


   Ultraviolet Light


   Final Filtration


   Storage Tanks

   Distribution Systems

Water System Validation

   Installation Qualification

   System Characterization Studies and Testing

   Operational Qualification

   Performance Qualification

     Phase I PQ Testing

     Phase II PQ Testing

   Post-Validation Monitoring

Water System Monitoring

   Sampling and Frequency of Monitoring

   Sample Collection and Testing

    Alert and Action Levels

   Response to Confirmed Alert and Action Level Excursions

Water Data Trending Requirements




Investigation of Microbiological Data Deviations

by Mónica Lagomarsino


Microbiological Data Deviations

Objectives of the Investigation

Acceptance Criteria

General Process for the Investigation

Laboratory Investigation

Full-Scale Investigation

A Case Study



Recommended Bibliography


Alternative Microbiological Methods and New Pharmaceutical Microbiology Curriculum

by Claudio D. Denoya


A “Rapid” History of Microbiology

Today’s Microbiology: The Fundamentals of “Traditional” Microbiology

The New Microbiological Technology Wave for the QC Lab: Alternative and Rapid   

       Microbiological Method (ARMMs)

Metabolic or Growth Based Technologies

Viability Based Technologies

Technologies Based on Cell Component Analysis

ARMMs and the Pharmaceutical Industry

AMM, PAT, and Regulatory Status

Microbiology Curricula

The Skill Sets of a Pharmaceutical Quality Control (QC) Microbiologist

Curricula Surveys

The Disparity between the Tertiary Microbiology Curricula and the Needs of the

         Pharmaceutical Industry QC Microbiologist

An Interesting Step Forward: The United States Professional Science Master''''''''s


Conclusions and Recommendations




The Implementation of Rapid Microbiological Methods

by Michael J. Miller

Microbiology Trapped In the 19th Century

New Technologies for Pharmaceutical Manufacturing

An Introduction to Rapid Microbiological Methods

RMM Applications

Strategy for Implementation

Validating Rapid Microbiological Methods

   Initial Activities

   The Validation Strategy

     Risk Assessment

     Validation Master Plan (VMP)

     User Requirements Specifications (URS)

     Design Qualification (DQ)

     Supplier Assessment/Audit

     Functional Design Specifications (FDS)

     Requirements Traceability Matrix (RTM)

   Training and SOPs

   The Test Plan

   Installation Qualification (IQ)

   Operational Qualification (OQ)

     Validation Criteria for Quantitative Tests

     Validation Criteria for Qualitative Tests

     Validation Criteria for Microbial Identification Tests

   Performance Qualification (PQ)

   Validation Summary Report

Implementation and Secondary Site Qualification

RMMS and the Regulatory Environment

FDA Perspectives

   Pharmaceutical cGMP’s for the 21st Century: A Risk-Based Approach

   Process Analytical Technology (PAT)

   Sterile Drug Products Produced by Aseptic Processing – cGMP

   RMM’s for Sterility Testing of Cellular and Gene Therapy Products

   Encouragement from FDA’s Microbiology Review and Compliance Staff

   Strategies for Implementing RMMs with the FDA

EMA Perspectives

   EMA Revised Variations Regulation

   PDA Forum on Implementing RMMs in Europe

   Type Variations

   Scientific Advice

   RMM Validation

   The EMA PAT Team

Changing Acceptance Levels and Specifications

Regulatory Summary

Developing a Business Case for RMMS

   Where Can Savings Come From?

   Creating an Economic Analysis

     Operating Costs Associated with the Conventional Method

     Operating and Investment Costs Associated with the RMM

     Cost Savings/Cost Avoidances Associated with the RMM

   Return on Investment (ROI)

   Payback Period (PP)

   Net Present Value (NPV)

Chapter Summary




Risk Management in Pharmaceutical Microbiology

by Tim Sandle

Introduction to Risk Assessment and Risk Management Process

Regulatory Views and Guidelines on Risk Management

The Basics of Risk Assessment

Advantages and Disadvantages of Risk Assessment

Risk Assessment and Risk Management Methodologies

   HACCP: Risk Based Approach in Environmental Monitoring

     Case Study 1

     Deconstructing the Process

          Route Map

          Identification of Hazards

          Process Flow

          Environmental Monitoring

     Risk Assessment

          Perform a Simulation


   FMEA - Risk Based Approach in Sterility Testing

     Case Study 2

     Sterility Testing Isolator: The Case Study

     Description of the System


     The FMEA Study on the Sterility Testing Isolator System

     Designing the FMEA Scheme

     The FMEA Exercise

           Examination 1: The Isolator Room

           Examination 2: Potential of Sanitisation Cycle Failure

           Examination 3: Frequency of Isolator Sanitisations

           Examination 4: Compromise of Isolator Integrity

           Examination 5: Connection of Transfer Isolator to Main Isolator and

                   Transfer-in / out of Material

           Examination 6: Incomplete Transfer Isolator Sanitisation

           Examination 7: Failure of a Daily, Weekly or Six-monthly Physical

                  Parameter - HEPA filters / Pressure Leaks to Canopy

           Examination 8: Pressure Leaks to Gloves


    Other Available Tools

     Ishikawa or Fishbone Diagrams

     Risk Ranking

     Risk Filtering

     Risk Modelling

     Contradiction Tables or Matrix

Conclusion: The benefits of Risk management



Part: 4     Sterilization and Sterility Assurance




by Edward C. Tidswell


Origins of Sterilization

The Microbial Challenge

Consequences of Non-Sterility and Lack of Asepsis

What Does Sterile Mean and Can We Test It?




Process Selection for Sterile Products

by James Agalloco


Terminal Sterilization and Adjunct Processing

Aseptic Processing




Microbial Contamination Control in Pharmaceutical Manufacturing

by Matts Ramstorp

Pharmaceutical Cleanrooms and Clean Zones


    The Use of Proper Hygiene

    The Historic Development of Microbiology

    Aseptic Techniques

   Production of Medicinal Products

   Why is Pharmaceutical Production Different from Microelectronics?

   Classification of Pharmaceutical Cleanrooms

   Good Manufacturing Practice (GMP)

   Design of Pharmaceutical Cleanrooms

     How to Proceed with the Design of a Pharmaceutical Cleanroom

   Cleanroom Cleanliness

     Pharmaceutical Cleanroom Classification

   Measuring Cleanliness within Pharmaceutical Cleanrooms

     Particle Counting

     Microbial Monitoring

     Analysis of Microbiological Contaminants in the Air

   Sampling of Microorganisms

     Sampling of Microorganisms in the Air of a Cleanroom

        The Andersen Sampler


        The RCS-Sampler

         Liquid Impinger

     Surface Sampling

   Microbiological Contaminants and GMP

   Pharmaceutical Cleanroom Ventilations Systems

     Conventional Ventilated Cleanrooms

     Unidirectional Flow Cleanrooms

   Ventilation Filter for Pharmaceutical Cleanrooms

   Cleanrooms and Clean Zones

     Working in Clean Zones

   Pharmaceutical Cleanroom Production Systems

     Production using Terminal Sterilization

     Aseptic Production

Cleaning and Disinfection of a Pharmaceutical Cleanroom


   What is the Purpose of Cleaning?

   What is Cleaning?

         How to Work with Preventive Cleaning

         Active Cleaning

   Classification of Surfaces in a Cleanroom

   Cleaning Program for Cleanrooms

         Example of a Cleaning Program

   How to Analyse and Control Cleaning

   Cleaning Techniques

         Cleaning Methods

            Dry Cleaning Methods

            Wet Cleaning Methods

   Cleaning Solution

   The Zinner Circle


   Potential Risk Factors Associated with Cleanroom Decontamination

   Standards and Practices

Cleanroom Garments


   What is the Purpose of the Cleanroom Garment?

   Garment Systems

   Single-use versus Washable Garments

   Choice of Material


   Construction of Garment Systems

   The EU GMP and Cleanroom Garment Systems

   Using Cleanroom Garments

   Clothing, Personal Items and Underwear

   Processing of Clothing and Change Frequency

   Risk Factors Associated With Cleanroom Clothing


Personal Hygiene and Personal Responsibility


   Humans as Particle Generators

         Particles and Fibres

         The Normal Flora of Man

   Good Manufacturing Practice in Relation to Personnel

   Personal Hygiene

   The Interconnection between Personnel and the Process

   Contamination Hazards Connected to Personnel

   General Rules when Working in Cleanrooms and other Controlled Environments




Aseptic Process Simulations/Media Fills

by Marco Budini and Francesco Boschi



Media Selection

Frequency and Number of Runs

Size and Duration of Runs

Line Speed and Container Size

Closure Type

Process/Line Configuration and Set-up

Fill Volume

Number of Persons and Activities


Line Clearance

Environmental and Personnel Monitoring

Other Worst Case Scenarios

Cleaning after Process Simulation

Incubation and Inspection of Filled Units

Growth Promotion Test

Interpretation of Results – Acceptance Criteria

Invalidation and Abortion of Process Simulation Runs

Investigation of Process Simulation Contaminations/Failures




Biological Indicators for Sterilization

by Russ Nyberg


Performance Qualification of Biological Indicators

BI Use in Parenteral Product Loads

Use of a Process Challenge Device (PCD)

Selection of BIs

Contracting for 3rd Party D-Value Testing


   Test Method Used

   Recovery Media

   Technique and Lab Utensils/Personnel

Rapid Read-out Biological Indicators




Moist Heat Sterilization

by Michael Sadowski


Sterile Products

History of Sterilization with Heat

Mechanisms of Spore Heat Resistance And Inactivation

Inactivation of Spores Using Moist Heat Sterilization Processes

   Use of Semilogarithmic Survivor Curve Model for Characterizing and Predicting Microbiological Inactivation

Pharmaceutical Products

Selection of a Moist Heat Sterilization Process Type

   Saturated Steam Sterilization Processes

   Gravity Displacement Processes

    Pre-vacuum Processes

   Air Overpressure Processes

   Steam/Air Mixture Processes (SAM)

   Superheated Water Air Overpressure Processes

Product or Item Loading Patterns

 Biological Indicators for the Development and Qualification of Moist Heat Sterilization Processes

Determination of The Hardest to Sterilize Solution Formulation or Item/Component

The Liquid Products Master Solution Approach

The Process Challenge Device (PCD) Approach for Porous/Hard Goods

Determination of Hardest To Sterilize Locations or Cold Spots Used For Penetration Probes

Use of Chemical Indicators

Development Moist Heat Sterilization Processes

   Development of the Sterilization Process Heat Up/Conditioning Phase

   Development of the Sterilization Process Cooling/Drying Phase

   Development of the Exposure Phase

   Determination of the Minimum Physical Lethality Value

   Minimum Physical Lethality Values Prescribed in Regulatory Standards

   Minimum Physical Lethality Values Required for the Overkill Cycle Design Approach

    Determination of Minimum Physical Lethality Values for the Product Specific Approach

    Determination of Exposure Time to Meet Minimum Physical Lethality Requirements

   Use of Biological Lethality in the Determination of Exposure Time

   Use of the Fractional Exposure Approach in the Determination of Exposure Time

Validation of The Moist Heat Sterilization Process

   Installation Qualification

   Operational Qualification

     Pre-vacuum Sterilizer Vacuum Leak Rate Test

     Steam Penetration or Bowie Dick Type Test

     Steam Quality Tests

   Performance Qualification

    Use of Physical Lethality in the Qualification of Moist Heat Sterilization Processes

    Use of Biological Lethality in the Qualification of Moist Heat Sterilization Processes

Routine Monitoring and Control of the Moist Heat Sterilization Process

   Preventive and Unplanned Maintenance

    Calibration Program

   Ongoing Sterilizer and Utility System Functionality Tests

Product and Process Change Control

Sterile Product Release Process

   Bioburden Testing

   Sterilizer Functionality

   Sterilization Cycle Parameters

   Chemical Indicator and Biological Indicator Results

   The Sterility Test

   Parametric Release





Sterilization and Depyrogenation by Dry Heat

by Madhu Raju Saghee and Gary R. Mitchel


Functions of Dry Heat

Thermodynamical Aspects of Heat Transfer in Dry Heat Processes

Destruction of Microorganisms and Endotoxins by Dry Heat

   Spectrum of Activity on Microbial Populations

      Effect of Microbial Water Content in Dry Heat Sterilization Processes

      Effect of Temperature and Time

   Destruction of Endotoxins

Types of Dry Heat Sterilization/Depyrogenation Processes

   Batch Process

   Continuous Process

Validation and Ongoing  Control

   Temperature Measurement Errors

   Validation Approach

   Installation Qualification

   Operational Qualification

   Performance Qualification

   Routine Monitoring and Control




Appendix A (Example of an outline for an Installation Qualification protocol)

Appendix B (Example of an outline for Operational and Performance Qualification protocols)


Radiation Sterilization

by Mark A. Seybold and John A. Williams


History of Radiation Sterilization

Types of Radiation Sterilization

   Gamma Sterilization

   Electron Beam Sterilization

   X-Ray Sterilization

   Radiation Sterilization Applications

Microbial Inactivation

   Mode of Action

   Microbial Resistance to Ionizing Radiation

   Radiation Resistance Factors

      Environmental Factors

      Organism Characteristics

   Inactivation Kinetics


   Installation Qualification

   Operational Qualification

    Performance Qualification

   Microbiological Validation of Radiation Sterilization Processes

     Dose Setting

     Method 1

     Method 2

     Method VDmax

   Materials Qualification

   Process Validation

Routine Monitoring And Control

Maintaining Process Effectiveness

   Biorburden Monitoring Program

   Dose Audits

   Radiation Equipment




Sterilization by Filtration

by Maik W. Jornitz and Theodore H. Meltzer

Introduction to Sterilizing Filtration

Filtration Parameters

    Contamination Removal

    Rate of Flow

    Total Throughput

    Unspecific Adsorption

Filter Types

Filter Materials

Filter Construction

Filter Validation

Filter Integrity Testing

    Bubble Point Test

    Diffusion Test

    Pressure Hold Test

    Water Intrusion Test

    Product Wet Integrity Testing

Filtration Applications


         Solvent (API) Filtration

         Ophthalmics Filtration

         Cell Culture Media

         Buffer Filtration


         Fermentor Inlet Air

         Fermentor Off-Gas

         Vent Filters on Tanks

         Autoclave and Lyophilizer Vent Filter

         Filtration of Service Gases



Sterilization by Ethylene Oxide

by Gerry A. O’Dell


Factors Affecting the Lethality of EO Sterilization

   Gas Concentration



   Inherent resistance of microorganisms

   Conferred resistance

    Combination of factors

The EO Sterilization Process


   Sterilization Cycle


Product and Process Definition for EO Sterilization

   Product definition

   Process definition

EO Sterilization Validation

   Installation Qualification

   Operational Qualification

   Performance Qualification

       Microbiological PQ

       Physical PQ   

Routine monitoring of the EO sterilization process

   Conventional Release

   Parametric Release

   Use of tests for sterility

Demonstrating the Ongoing Effectiveness of the EO Sterilization Process

    Addressing changes (change control)

    Process equivalence

    Product adoption

    Periodic requalification




Maintaining Sterility

by Michelle A. Luebke & Bonnie J. Heredia

Introduction to Maintaining Sterility

Medical Product Packaging System Development

   Packaging Development Phase

   Routine Manufacturing/In-Process Control Phase

   Stability/Product Testing Phase

Microbiological Versus Physical Integrity Test Methods

Test Method Correlation

   Direct Approach

   Indirect Approach

Test Method Sensitivity

Test Method Validation

   Method and Vendor Requirements

   Validation Plan

   Installation Qualification

   Operational Qualification

   Performance Qualification

Sterile Medical Product Classification

   Medical Devices

     Medical Devices - Sterile Designation

     Medical Devices - Sterile Fluid Path Device Designation

   Needleless Medical Devices

   Drugs and Biologics

Sterile Barrier System Classification



Sterile Barrier Integrity Test Methods

   Physical Methods

     Visual Inspection

     Bubble Test

     Pressure/Vacuum Decay

     Dye Test

     Tracer Gas Leak Detection

   Microbiological Methods

     Microbial Challenge - Liquid Immersion

     Microbial Challenge - Aerosolization

     Static Aerosol Challenge

     Dynamic Aerosol Challenge

     Simulated Clinical Use Test Method

   Miscellaneous Methods




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