GPT and AET – Methods and Best Practices

Objectives

Review the regulations supporting quality control in

Pharmaceutical laboratories

– Growth Promotion Testing (GPT)

– Antimicrobial Effectiveness Testing (AET)

Best practices

Where are the Standards for Pharmaceutical Quality Control Found?

The USP, JP, and Ph. Eur. have

harmonized for Growth

Promotion Testing

The USP, ISO and others offer

guidelines for AET/PEC

The FDA, USP, and others have

guidelines for Environmental

Monitoring

Pharmaceutical Manufacturing

Non-Sterile

Multi-use

Solutions, suspensions, creams, capsules, tablets

, ,

Sterile

Injections & Infusions

Single Use

Non-Sterile Products: Microbial Enumeration Tests USP

Purpose – to count the number of aerobic

microorganisms present in a product sample

Monographs put a limit on the number of

microorganisms

The product is inoculated onto Tryptic Soy

Agar/Broth or Sabouraud Dextrose Agar

The spread plate, pour plate or membrane

filtration method is used

Products for GPT in Microbial Enumeration Tests: USP

Staphylococcus aureus ATCC® 6538™*

Pseudomonas aeruginosa ATCC® 9027™*

Bacillus subtilis ATCC® 6633™*

Candida albicans ATCC® 10231™*

Aspergillus brasiliensis ATCC® 16404™*

*The ATCC Licensed Derivative Emblem, the ATCC Licensed Derivative word mark and the ATCC catalog marks are trademarks of ATCC. Microbiologics, Inc. is licensed to use these trademarks and to sell products derived from ATCC® cultures.

Non-Sterile Products: Tests for Specified Microorganisms USP

Purpose – to detect pathogenic organisms like

Salmonella or indicator organisms like E. coli

Monographs put limit on types of allowable

organisms – for example, Clostridium in purified

honey in products for children

Products for GPT in Tests for Specified Microorganisms USP

Staphylococcus aureus ATCC® 6538™*

Pseudomonas aeruginosa ATCC® 9027™*

Escherichia coli ATCC® 8739™*

Salmonella Typhimurium ATCC® 14028™*

Candida albicans ATCC® 10231™*

Clostridium sporogenes ATCC® 19404™*

or ATCC® 11437™*

*The ATCC Licensed Derivative Emblem, the ATCC Licensed Derivative word mark and the ATCC catalog marks are trademarks of ATCC. Microbiologics, Inc. is licensed to use these trademarks and to sell products derived from ATCC® cultures.

Sterile Products: Sterility Tests USP

Purpose – to detect the presence or absence

of contamination in a sample of a product

Sterile product is inoculated into Tryptic Soy

Broth and/or Thioglycollate Broth

Incubated and checked for growth

Alternative methods can be used if

they are validated

Staphylococcus aureus ATCC® 6538™*

Pseudomonas aeruginosa ATCC® 9027™*

Bacillus subtilis ATCC® 6633™*

Candida albicans ATCC® 10231™*

Clostridium sporogenes ATCC® 19404™* or ATCC® 11437™*

Aspergillus brasiliensis ATCC® 16404™*

*The ATCC Licensed Derivative Emblem, the ATCC Licensed Derivative word mark and the ATCC catalog marks are trademarks of ATCC. Microbiologics, Inc. is licensed to use these trademarks and to sell products derived from ATCC® cultures.

Products for GPT in Sterility Tests:

USP

Environmental Testing

Multiple industries, including pharma and

cosmetics

Cleanroom surfaces and air are monitored

Surfaces are checked using contact plates

Air is checked with air monitors or by exposing

open agar plates to the air in the room

Test Methods

Growth Promotion Testing of Media

Performed on media used in:

– Microbial Enumeration Tests

– Tests for Specified Microorganisms

– Sterility Tests

– Antimicrobial Effectiveness Test

– Environmental Monitoring

Four Rules to GPT Testing

1.Test each new batch of media

2. Inoculate media with small number of organisms

3. Organisms should be 5 passages or less from Reference Culture

4. Use organisms listed in USP

Microbial Enumeration Test: Agar

1. Prepare inoculum (≤100 CFU)

2. Inoculate previously approved

medium and new medium with

same suspension

3. Distribute suspension

4. Incubate

Previously

approved

medium

New

medium

Acceptance Criteria for Agar

Compare number of colonies on

previously approved medium to number

on new medium

The number must be within a factor of 2

Example:

• previously approved medium = 40

• new batch of medium must = 20 to 80

Previously

approved

medium

New

medium

Microbial Enumeration Test and Sterility Test: Broth

1. Prepare inoculum (≤100 CFU)

2. Inoculate new and previously approved broth

3. Inoculate non-selective agar as a control

4. Incubate

Soybean-Casein

Digest Broth

Soybean-Casein Digest

Agar

Acceptance Criteria for Broth

Compare turbidity in new medium to

turbidity in the previously approved

medium

Turbidity should be “comparable”

Count colonies on non-selective

agar: there should be ≤ 100

Previously

approved

broth

New

broth

Control Plates Soybean-Casein Digest Agar

Tests for Specified Microorganisms: Selective Medium

Tested for one or all of the

following criteria:

– Growth promoting

properties

– Indicative properties

– Inhibitory properties

Property Microorganism Inoculum

Growth-promoting Staphylococcus

aureus

- good growth

≤ 100 CFU

Indicative Staphylococcus will

have yellow or white

colonies with yellow

zone

≤ 100 CFU

Inhibitory E. coli will not grow ≥ 100 CFU

Example: Mannitol Salt Agar

Tests for Specified Microorganisms: Growth Promotion, Indicative

and Inhibitory Properties

1. Growth Promoting and Indicative Properties:

– Prepare inoculum of S. aureus (≤100 CFU)

2. Inhibitory Properties:

– Prepare inoculum of E.coli (≥100 CFU)

3. Inoculate Mannitol Salt Agar and TSA

4. Incubate

Acceptance Criteria: Mannitol Salt Agar

Growth of S. aureus on the new medium should be

comparable to growth on the previously approved

medium

E. coli should not grow on the Mannitol Salt Agar

Soybean-Casein Digest Agar

Mannitol Salt Agar

Soybean-Casein Digest Agar

Mannitol Salt Agar

S.aureus E.coli

Biggest Challenges

Factor of 2

“…growth obtained must not differ by a factor

greater than 2 from the calculated value for a

standardized inoculum.”

Growth on Selective Media and Acceptance criteria

“… Growth of the microorganism comparable to

that previously obtained with a previously tested and

approved batch of medium occurs.”

Solution – Factor of 2

The value of the standardized inoculum should be obtained by

counting the number of colonies on the previously approved media

Perform side-by-side testing for direct comparison of new and

previously approved media

Only one variable: the medium

Other variables eliminated,

including:

• Temperature

• Atmosphere

• Technique

• Method

• Incubation time

Why Side-by-Side Testing?

What If There Is No Previously Approved Media?

Use historical data

– it’s best to use data from

the same microorganism

lot

Challenge: Selective Medium

Problem: Recovery on selective media can be low

Pictures below show results when same microorganism suspension plated to TSA and MacConkey

0.1 ml on TSA

25 CFU

0.1 ml on MacConkey

6 CFU

Solutions for Selective Agar

Test non-selective agar such as TSA in parallel with the

selective agar

The number of colonies on the non-selective agar is the true

value of the inoculum

Other factors…

Incubation Temperature

Medium

Technician Technique

Incubation times

Variable - Temperature

Candida albicans after 48 hours incubation

25°C 20°C

Variable-Medium

Variances in Media Manufacturer– GPT was performed on various brands of Soybean-Casein Digest Agar using the same

suspension of EZ-Accu Shot™, Pseudomonas aeruginosa, 0484A. The test was performed in duplicate.

Conclusions• CFU results can vary between media manufacturers• GPT test is important to determine if medium is suitable

36

45

57

38

21

10

Brand A Brand B Brand C Brand D Brand E Brand F

1. Test in parallel

2. Double the inoculum for selective medium when necessary

3. Use non-selective agar as a control when testing liquid media

4. Routinely calibrate pipettes

5. Bring pellets to room temperature before opening pellet packaging.

6. Use correct vial of hydrating fluid when reconstituting pellets

7. Mix, mix, mix

8. Use environmental conditions required by species

9. Keep organisms warm, but not TOO warm

10. Make certain you can find all objectionable microorganisms in your

product

Top 10 Tips for Growth Promotion Testing

Antimicrobial Effectiveness Testing (AET)

Purpose of the Antimicrobial Effectiveness Test (AET/PET/PEC)

To find the lowest concentration of antimicrobial or

preservative which is effective in controlling microbial

growth in products

There are Two Goals:

The first goal is efficacy. The preservative must prevent the

growth of microorganisms.

The second goal is safety. The preservative must be used

at a concentration below a level toxic to humans.

What is an Antimicrobial?

Antimicrobials and preservatives are agents that kill or inhibit

microbial growth

Common preservatives for pharmaceutical products

– Thimerosal

– Benzoic acid

– EDTA

– Benzalkonium chloride

Test Method Overview

Product CategoryPreservative

Testing?Comments

Single-use pharmaceuticals No Aseptic manufacturing

Multi-dose pharmaceuticalsYes; USP ,

EP 5.1.3

Compendial strains; environmental

isolates

Cosmetics

Yes; USP ,

AOAC, PCPC, EP

5.1.3

Compendial strains; environmental

isolates

Household products GMP Strains vary

Industries That Use the AET

Pharmaceutical

Personal Care

Products/Cosmetics

Biotechnology

Household Products

Food

Nutraceutical

Textiles

Methods and Guidelines

USP

EP 5.1.3

Japanese Pharmacopeia

ISO 11930

CTFA – guidelines written by Personal Care Products Council

BAM – Bacteriological Analytical Manual

SCCS – Notes of Guidance for the Testing of Cosmetic Ingredients and their Safety Evaluation

ASEAN – Association of South East Asian Nations Cosmetics Association

AOAC – American Society of Analytical Chemists

The Methods Differ Slightly In:

Product sample volume

Size of inoculum per ml of product

CFU concentration of inoculum

CFU concentration in product

Strains required

The Basic USP Test

1. Add microorganism to

product.

-The concentration in

product should be 105 to

106 CFU/ml

3. Sample product at

intervals to see how many

organisms remain over a

period of time:

for example; 7, 14, 28

days.

2. Incubate at

20°C - 25°C

ProductProduct

When is the AET Performed?

During development of a new

product

When the formulation of a

product has changed

When product ingredients

have changed

Best Practices

1. Qualify media used in test

2. In addition to compendial strains, also test environmental isolates,

objectionable organisms

3. Determine the initial CFU concentration in product

4. Validate that you can recover microorganisms from the product

(also known as suitability testing)

1. Qualify Media Used in Test

The media used to enumerate the CFU in the product should be pre-

qualified using the growth promotion test

2. Test With Environmental Isolates and Objectionable Organisms

Consider testing other reference

cultures as well as environmental

isolates

B. cepacia in alcohol – free

mouthwash sickened 12 ICU

patients at a German hospital

Mold was found on Acetaminophen

tablets and Folic Acid Liquid

Pseudomonas putida was found in

mud mask

3. Determine the Initial CFU Concentration in Product

Why is this important? Because the initial count

must be compared to counts after 7, 14, and 28

days

But, the initial count in the product may be too

low because the preservative may kill many

microorganisms immediately

4. Validate Microorganisms Can Be Recovered

Inoculated product needs to be tested at

specified intervals but the preservative or

antimicrobial in the product may prevent the

organisms from growing

Lab must validate it can neutralize the

preservative (Suitability Test)

EZ-PEC™

Made Specifically for the

Antimicrobial Effectiveness

Test

20 lyophilized pellets of a

single enumerated strain

10 vials of hydrating fluid –

2 ml each

Online Certificate of

Analysis

USP Strains

The ATCC Licensed Derivative Emblem, the ATCC Licensed Derivative word mark and the ATCC catalog marks are trademarks of ATCC.

Microbiologics, Inc. is licensed to use these trademarks and to sell products derived from ATCC® cultures.

Microorganism Reference # Microbiologics #

Bacillus subtilis subsp. spizizenii ATCC® 6633™* 0486-PEC

Candida albicans ATCC® 10231™* 0443-PEC

Escherichia coli ATCC® 8739™* 0483-PEC

Escherichia coli NCIMB 8745 0581-PEC

Pseudomonas aeruginosa ATCC® 9027™* 0484-PEC

Staphylococcus aureus subsp. aureus ATCC® 6538™* 0485-PEC

Aspergillus brasiliensis ATCC® 16404™* 0392-PEC

Additional Add-Ons

GPT Products for Media Testing

GPT Products and Epower for Suitability Testing

Custom Solutions for AET, Media Testing and Suitability Testing

Take-Aways

AET is performed on non-sterile products and multi-dose sterile products during product development, ingredient changes, manufacturing changes

AET can be a difficult test; EZ-PEC makes it faster and easier!

Custom Solutions should always be discussed as an option!