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Transcript of GPT and AET Methods and Best Practices - bdcint.com.do › wp-content › uploads › 2018 › 10...
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GPT and AET – Methods and Best Practices
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Objectives
Review the regulations supporting quality control in
Pharmaceutical laboratories
– Growth Promotion Testing (GPT)
– Antimicrobial Effectiveness Testing (AET)
Best practices
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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
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Pharmaceutical Manufacturing
Non-Sterile
Multi-use
Solutions, suspensions, creams, capsules, tablets
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Sterile
Injections & Infusions
Single Use
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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
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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.
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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
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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.
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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
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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
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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
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Test Methods
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Growth Promotion Testing of Media
Performed on media used in:
– Microbial Enumeration Tests
– Tests for Specified Microorganisms
– Sterility Tests
– Antimicrobial Effectiveness Test
– Environmental Monitoring
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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
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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
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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
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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
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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
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Tests for Specified Microorganisms: Selective Medium
Tested for one or all of the
following criteria:
– Growth promoting
properties
– Indicative properties
– Inhibitory properties
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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
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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
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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
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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.”
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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
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Only one variable: the medium
Other variables eliminated,
including:
• Temperature
• Atmosphere
• Technique
• Method
• Incubation time
Why Side-by-Side Testing?
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What If There Is No Previously Approved Media?
Use historical data
– it’s best to use data from
the same microorganism
lot
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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
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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
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Other factors…
Incubation Temperature
Medium
Technician Technique
Incubation times
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Variable - Temperature
Candida albicans after 48 hours incubation
25°C 20°C
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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
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21
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Brand A Brand B Brand C Brand D Brand E Brand F
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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
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Antimicrobial Effectiveness Testing (AET)
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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
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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.
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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
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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
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Industries That Use the AET
Pharmaceutical
Personal Care
Products/Cosmetics
Biotechnology
Household Products
Food
Nutraceutical
Textiles
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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
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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
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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
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When is the AET Performed?
During development of a new
product
When the formulation of a
product has changed
When product ingredients
have changed
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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)
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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
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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
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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
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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)
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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
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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
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Additional Add-Ons
GPT Products for Media Testing
GPT Products and Epower for Suitability Testing
Custom Solutions for AET, Media Testing and Suitability Testing
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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!