Pharmaceutical Microbiology

By
Gemmechu H (MSc, Assistant Professor)
 Course Contents
• Introduction to pharmaceutical microbiology
• Microbiological testing methods
• Antimicrobial susceptibility testing;
• Sterility test;
• Bacterial endotoxin test;
• Microbial enumeration methods, such as the microbial limits test;
• Tests for specified microorganisms;
• Methods and limits for testing pharmaceutical grade water;
• Disinfectant efficacy;
• Pyrogen and abnormal toxicity tests;
• Environmental monitoring;
• Biological indicators

• Current and Future Challenges in Pharmaceutical Microbiology

Introduction to Pharmaceutical
Microbiology
 Introduction

• Microbiology?
 Cont’d….

• Pharmaceutical microbiology?
• The basics?
• Microorganisms and microbial by-products.
• Other Aspects
• Research and development of anti-infective agents,
• Mutagenic and carcinogenic activity
• Insulin and human growth hormone.
 Cont’d….

• Risk assessment….centers
• Likelihood of Product Contamination
• Severity of Contamination
• Minimizing Contamination
• Detection Methods
 Cont’d…
• Understanding the type of product, its intended use, and the nature and
numbers of contaminants are crucial.
• Sterile injectable products Vs non-sterile products!!!
• Microorganisms of concern depend upon the type of product and route of
administration.
 Cont’d…
• Use of culture media.

• Foundation and overriding technique

• Aseptic technique.
 Scope of Pharmaceutical Microbiology

• The manufacture of pharmaceutical and healthcare preparations and

medical devices.
• risk assessment (both proactive and reactive),
• testing materials and monitoring environments and utilities.
The essential tests are described in the United States, European, and
Japanese.

• Antimicrobial susceptibility testing;

• Sterility test;
• Bacterial endotoxin test;
• Microbial enumeration methods, such as the microbial limits test;
• Tests for specified microorganisms;
• Methods and limits for testing pharmaceutical grade water;
• Disinfectant efficacy;
• Pyrogen and abnormal toxicity tests;
• Environmental monitoring;
• Biological indicators.
 Cont’d….
• Contemporary developments!
• Confined to a range of laboratory tests.
• The concept of “testing to compliance” is outdated.
• Involves testing and the assessment of data.
• Risk assessment…design system
• Contamination control strategy!
• Twins areas should be considered.
 Microbiological Test Methods
• Qualitative methods—“Is there something there?”

• Quantitative methods— “How many are there?,”

• Identification methods—“What are they?”

• Starting materials
• In-process samples/intermediate product
• Final product formulations
The key test areas to assess
• Finished product
• Testing of utilities
• Environmental monitoring
• Other tests
 Starting materials

• Pharmacopeial monographs.
• Decision of appropriate testing regime.
• Microbial limits tests
• Microbial enumeration.
• Specified Microorganisms.
USP <61>/Ph. Eur. And USP <62>/Ph. Eur.
 Cont’d…
• bile-tolerant Gram-negative bacteria;
• Salmonella;
• Escherichia coli;
• Pseudomonas aeruginosa;
• Staphylococcus aureus;
• Candida albicans; Clostridia
 Cont’d…
• Not every test organism is applicable to each material.
• Some materials require examination of endotoxin
• Bacterial endotoxin testing.

USP <85>/Ph. Eur. 2.6.14

In-process Samples/Intermediate Product
• Microbiology laboratory for bioburden testing
• The number of bacteria living on a surface or in a material.
• The total viable count (TVC) method.
• a quantitative idea about microorganisms in or on a sample.

• The result obtained represents the number of colony-forming units (CFUs) per gram (or
per milliliter)
 Cont’d…
• Granulation solutions, coating solutions, suspensions for spray drying,
buffers, water rinses, and so on.
• Endotoxin testing should also be considered.

• A risk assessment should be conducted for all samples submitted.

• A further consideration is the validation of sample hold times.
• Typically maximum hold times are 24h,
• put in place to avoid over-growth of microorganisms.
 Final Product Formulations

• For aseptic manufacturing the sterile bulk is tested.

• The extent of the testing will varies.

• As a minimum, a sample should be taken for bioburden testing.

 Finished Products

• For sterile products.

• Sterility test.
• Endotoxin or pyrogen test.
• Abnormal toxicity test, using an animal model (some organization).

• For nonsterile products

• Test for specified microorganisms (maximum permitted microbial count).
 Testing of Utilities
• Utilities that support production processes.
• Compressed gases (microbial count and particulates),
• Steam (bacterial endotoxin), and
• Pharmaceutical grade water.
• water-for-injection, purified water and feed water.
• total aerobic microbial count;
• bacterial endotoxins;
• specific microorganisms.
 Environmental Monitoring
• A key part of the assessment of pharmaceutical manufacturing facilities.
• Environmental monitoring data indicates
• if cleanrooms are operating correctly.

• An assessment of the heating, ventilation, and air conditioning (HVAC)

parameters,
• which contribute toward making the cleanroom “clean.”

• Temperature, humidity, pressure, room air changes, and air-flow patterns.

 Others
• Cleaning validation studies;
• Maintenance of microbiological cultures;
• Advising on process and equipment design;
• Conducting risk assessments;
• Investigation of out of limits results;
• Maintenance of laboratory equipment;
• Laboratory training
 The application or activities of Pharmaceutical Microbiology

• Counting
• Sampling
• Microorganisms detected from pharmaceutical manufacturing
environments
• Contamination control strategy
 Counting
• The ability to enumerate microorganisms.
• The microbial quality of water, in-process bioburden samples, of
raw materials
• Total cell count and the viable count
• Microscopic examination,
• Turbidity of a suspension (using a nephelometer or
spectrophotometer).
• spread plate, pour plate, spiral plating, and membrane filtration.
 Sampling
• An aseptic manner, and the containers and storage conditions of the
sample.
• The hygiene of the sample is maintained (aseptic technique).
• In-process and water samples are normally required to be placed at 2–8°C
within 1 or 2 h of sampling.
• The containers selected must normally be sterile
• should not leech out any chemicals that might have antimicrobial properties.
 Microorganisms detected from Pharmaceutical
Manufacturing Environments
• Two aspects of screening microbiota are to note possible resistant strains and
objectionable microorganisms.
• Gram-positive sporing rods and certain Gram-negative rods.
• Microorganisms pose a contamination risk to pharmaceutical processing.
• Adequate cleaning and disinfection to reduce the risk of microorganisms proliferating.
• Disinfection agents may be used depending on the types of microorganisms.
 Cont’d…
• When objectionable microorganisms occur the risk assessment should be conducted.
• Risk assessment criteria may include;
• Absolute numbers of organisms seen
• The type of microorganisms and the characteristics of the microorganism
• Considering if the microorganism can survive
• pH, salt concentrations; sugar concentrations; available water; temperature.
• Product characteristics
• Potential impact on patients.
 Contamination Control Strategy

• A Path for Quality & Safety

 Conclusion
• The more detailed aspects of pharmaceutical micro microbiology.
• An overview of some of the specifics of pharmaceutical microbiology.
• The more common and essential elements.
 Microbiology Laboratory Techniques
• Quality assurance represents a major consideration.
• Microbiological contamination
 Cont’d…

• Contamination risks remain an ever-present threat.

• The microbiological contamination control of pharmaceuticals
should be evaluated.
• This requires a range of microbiological tests to be conducted.
• Such tests focus on the number and type of microorganisms.
• National and international pharmacopeia.
• BP, Ph. Eur, USP, JP, WHO
• The tests described constitute the set of basic microbiological laboratory
techniques
• in relation to pharmaceuticals and healthcare.
• Alternative tests to the pharmacopeia described methods can be validated
and employed
• but the pharmacopoeial method remains the referee test.

• Good laboratory practice!

Microbiology test methods described in Pharmacopeia

• Antimicrobial susceptibility testing;

• Sterility test;
• Bacterial endotoxin test;
• Microbial enumeration methods, such as the microbial limits test;
• Tests for specified microorganisms;
• Methods and limits for testing pharmaceutical grade water;
• Disinfectant efficacy;
• Pyrogen and abnormal toxicity tests;
• Environmental monitoring; Biological indicators.
 Antimicrobial Effectiveness Testing
• What is an antimicrobial or preservative?
• Microbial contamination.
• Multi-doses formulas of drug products.
• Preservative Effectiveness Testing (PET).
• Microbial challenge.

USP <51> Antimicrobial Effectiveness Testing,

ISO 11930 – Evaluation of the Antimicrobial Protection of a Cosmetic Product, and
Personal Care Products Council Methods M-3 to M-7.
 Cont’d…
• An Invaluable Product Development Tool.
• Sterile drugs,
• Multi-use drug products
• Personal care and cosmetic products.

• Microbiological risk assessment.

• Product pH, water activity or nature of the product’s raw materials.
• An environment conducive to microbial growth.

• AETs are often included in stability study protocols.

Cont’d….
• Challenging the product with a panel of
microorganisms.
• Staphylococcus aureus,
• Escherichia coli,
• Pseudomonas aeruginosa,
• Candida albicans, and
• Aspergillus brasiliensis.

USP <51> Antimicrobial Effectiveness Testing

 Cont’d…
Media
• For the cultivation of the test organisms, select agar medium that is favorable to the
rigorous growth of the respective stock culture.
• The recommended media are Soybean Casein Digest Agar/Broth and Sabouraud’s
Dextrose Agar/Broth.
• Media should be test for growth promotion.
 Cont’d…
• Test Organisms
• All cultures must 5 passages removed from the original stock culture.
• Candida albicans (ATCC No. 10231)
• Aspergillus brasiliensis (ATCC No. 16404)
• Escherichia coli (ATCC No. 8739)
• Pseudomonas aeruginosa (ATCC No. 9027)
• Staphylococcus aureus (ATCC No. 6538)
 Preparation of Inoculum
• A panel of five challenge organisms are used in USP <51>, including
• Candida albicans (yeast),
• Aspergillus niger (mold),
• Escherichia coil (Gram-negative enterobacillus),
• Pseudomonas aeruginosa (Gram negative bacillus),
• Staphylococcus aureus (Gram-posit ive coccus).

• Fresh cultures of each organism are harvested in sterile saline and standardized to about 10 8 cfu/mL.
• Extensive propagating of microbial cells is discouraged because it could lead to changes in
phenotypic expression and antimicrobial susceptibility.
 Cont’d…
• Therefore, seed-stock techniques are recommended for long-term storage, and stock
cultures of each organism are limited to no more than five passages removed from the
original seed stock.
• The microbial enumeration test is performed to determine the number of viable cells in
each cell suspension.
• Bacteria are grown at 30°C to 35°C on Soybean-Casein Digest Agar, while yeast and
mold are grown at 20°C to 25°C on Sabouraud Dextrose Agar.
 Procedure/Challenge test
• The standardized cell suspensions are added to the test product in five separate containers, one
container for each challenge organism.
• The concentration of challenge organisms in product Categories 1 through 3 is between 105 and 106
cfu/mL.
• The products in Category 4 (antacids) contain between 103 to 104 cfu/mL of each challenge
organism.
• The inoculum volume should not exceed 1% of the total volume of the product to be tested
• Inoculated samples are incubated at 20°C to 25°C for 28 days.
• The microbial enumeration test is performed at days 7, 14, and 28 by the validated method.
• The acceptance criteria are specified for each drug product categories.

• In general, 1 to 3 log reduction in bacteria from the initial level should occur
in one to two weeks, with no further increase in bacteria thereafter at 28
days.

• For yeast and mold, no increase from the initial inoculums level is permitted
at all sampling intervals.
• Pharmaceutical products are divided into four categories based on product risk, shorter sampling
intervals during a 28-day period, and more stringent criteria are associated with
• Category 1 products, which includes sterile parenteral in aqueous base or emulsions (e.g.. injections,
optic products, ophthalmic products, nasal products). Adequate preservation is indicated by not less
than 1 and 3 log reduction in bacterial count from the initial value at day 7 and day 14. respectively.
Subsequently, bacterial counts at day 28 should not increase from counts at day 14.
• Less stringent criteria are applied to topical and oral products in categories 2 and 3. For oral and
topical products, at least 1 log (oral products) and 2 log (topical products) reduction from initial
bacterial count at day 14 should be observed, and no increase relative to day-14 counts at day-28
testing.
• Antacid products are qualified by separate criteria in Category 4 due to the
inherent issues with this product (e.g. high pH. interactions of
preservatives with formulation ingredients).
• Effective antimicrobial activities in antacid products are indicated by no
increase in bacterial, yeast, and mold from initial counts when tested at
day 14 and day 28.
 Assignment

S/N Title Stu Name

1 Bacterial endotoxin test; Alemu Bedada

2 Microbial enumeration methods Addisu Afrassa

3 Methods and limits for testing pharmaceutical grade water Duresa Dedefo

4 Environmental monitoring Gebremariam Genet

5 Sterility test Mastewal Abebe

6 Particulate Matter Wude Deressa