Microbiology for Oral and Topical Products - The Basics

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Microbiology for Oral and Topical Products - The basicsScott ColbourneBusiness Manager NSW – ALS Food & Pharmaceutical

Microbiology for Oral and Topical Products - The Basics

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Contents

• TGO 77 - Introduction• Tests Performed

– Common Tests– Preservative Efficacy– Endotoxin– Bioburden

• Method Turn Around Times (TAT)• The Confirmation Process• Method Validation• Causes of counts being high initially, then low when retested• Topical Products


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TGO 77 - General

• Therapeutic Goods Order No. 77 – Microbiological Standards for Medicines (TGO 77)

• This Order specifies the minimum microbiological requirements with which a medicine must comply throughout its shelf life in Australia.

• On and from 1 January 2010, each medicine to which this Order applies must comply with this Order.


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TGO 77 - General

• Acceptance criteria is the same as used in the BP, Ph.Eur. and the USP:

• A medicine is considered to be non-compliant with a microbial count acceptance criterion if the CFU (colony forming unit) count exceeds two (2) times the stated limit.

• When the Order establishes a limit of 102 CFU, the maximum acceptable count is 200 (which allows for microbial testing variability)


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TGO 77

• Section 7: Sterility and Bacterial Endotoxin testing

– A medicine must comply when these tests are required by an individual or general monograph

– Where a sponsor labels or packages a medicine in a way that states or implies that the medicine is sterile


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TGO 77

• Section 8: Efficacy of antimicrobial preservation of multidose medicines

– Applies to multidose sterile medicines (e.g. eye drops)

– Applies to multidose non-sterile medicines (e.g. oral liquids)


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TGO 77

• Subsection 9(1): – Applies to prescription, OTC and many complementary

medicines– Acceptance Criteria

• BP Appendix XVI (Ph. Eur. 5.1.4): Limits defined by route of administration

• USP chapter <1111>: Almost identical to the BP criteria– Testing consists of some/all of the following:

Total Aerobic Microbial Count

Total CombinedYeasts/MouldsCount

Escherichia coli Staphylococcus aureus

Pseudomonas aeruginosa

Candida albicans

Bile-tolerant Gram-negative bacteria


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TGO 77

• Subsection 9(2): – Applies to a complementary medicine oral dosage form containing raw

material of natural origin

* A herbal medicinal product consisting solely of one or more herbal substances (whole, reduced or powdered) to which boiling water is added before use

Microbiological Quality Units Schedule 1:Containing

Schedule 2:Solely herbal substances *

Total aerobic microbial count CFU per g or mL ≤ 104 ≤ 107

Total yeast and mould count CFU per g or mL ≤ 102 ≤ 105

Bile-tolerant Gram negative bacteria

CFU per g or mL ≤ 102 ≤ 102

Salmonella in 10g or 10mL Absent Absent

Escherichia coli in 1g or 1mL Absent Absent

Staphylococcus aureus in 1g or 1mL Absent Not applicable


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Common Tests – what and why?

• Total Aerobic Microbial Count (TAMC)What?A generic count of micro-organisms present. The results are non-specific Why?Gives a general indication of the level of micro-organisms present in the sample testedCan be referred to as…Total Microbial Count (TMC), Standard Plate Count (SPC), TVC, TVAC


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• Yeast and MouldWhat?Generic test for the levels of these present. Non-specific.Why?• They cause biodegradation of natural materials• Some yeasts are useful in fermentation (e.g. Bread and

Beer) • However some (e.g. Candida albicans) are opportunistic

pathogens and can cause infections


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• Bile Tolerant gram negative bacteriaWhat?• The name comes from their gram staining method of bacterial

differentiation. Basically they do not retain crystal violet.• Large family of bacteria including Enterobacteriaceae, Salmonella and E.Coli

Why?• In humans, disease is produced by both invasive action and production of

toxin. • Species not normally associated with disease are often opportunistic

pathogens. • Enterobacteriaceae have been responsible for up to half of hospital

infections annually in the United States


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• SalmonellaWhat?Pathogen found in cold/warm blooded animals and the environment

Why?• It causes food poisoning, such as gastrointestinal issues• To ensure there is no presence of pathogens


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• E. coliWhat?A coliform almost exclusively of faecal origin

Why?• E. coli presence is an effective confirmation of faecal

contamination. • Most strains of E. coli are harmless, but some can cause

serious illness in humans.


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• Staphylococcus AureusWhat?There are over 30 Staphylococcus types of bacteria, but Staphylococcus aureus causes most staph infections

Why?• Can cause skin infections (most common), Pneumonia, food

poisoning, toxic shock syndrome and blood poisoning (bacteremia)

• A staph skin infection is more likely if you have a cut or scratch


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• Pseudomonas aeruginosaWhat?It is found in soil, water and most man-made environments

Why?• An opportunistic human pathogen of

immunocompromised individuals• Because it thrives on moist surfaces, this bacterium is also

found on and in medical equipment, including catheters, causing cross-infections in hospitals and clinics


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• Candida AlbicansWhat?• A fungus that is a constituent of the normal gut flora that live in the

human mouth and gastrointestinal tract. • Present in 80% of the human population without causing harmful effects

Why?• Causes opportunistic oral and genital infections in humans• Has emerged as an important cause of morbidity and mortality in

immunocompromised patients (e.g., AIDS, and cancer chemotherapy)• Routinely required for Vaginal use medicines


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Preservative Efficacy Testing (PET)

• What is preservative efficacy?• The test consists of challenging the preparation by inoculating with suitable

micro-organisms, at a prescribed temperature, withdrawing samples at specified intervals and counting the organisms present.

• The preservative properties of the preparation are adequate if there is a significant fall (or no increase)

• Acceptance criteria vary for different types of preparations


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• Acceptance criteria vary for different types of preparations • What samples are tested ?

• Samples with preservative systems e.g. liquid products, creams and lotions


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• Samples with preservative systems e.g. liquid products, creams and lotions• How long does it take?

• Samples are tested at intervals such as 0, 1, 2, 7, 14 and 28 days.• Therefore the completed full PET will be 5-7 days after the final time point.


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• Samples with preservative systems e.g. liquid products, creams and lotions• How long does it take?

• Samples are tested at intervals such as 0, 1, 2, 7, 14 and 28 days.• Therefore the completed full PET will be 5-7 days after the final time point.

• How often is it performed?• Generally not required to release batches of product.• When finalising the formulation and to check the preservative system.


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Endotoxin (LAL)

• What is endotoxin testing?• The outer membrane of a Gram-negative bacterium is composed of

lipopolysaccharide (an endotoxin).• The qualitative determination of bacterial endotoxin concentration is

performed by the Limulus Amebocyte Lysate (LAL) gel clot test


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Endotoxin (LAL)



performed by the Limulus Amebocyte Lysate (LAL) gel clot test• What samples are tested ?

• The primary application for LAL is the testing of parenteral pharmaceuticals and medical devices that contact blood or cerebrospinal fluid


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Endotoxin (LAL)



performed by the Limulus Amebocyte Lysate (LAL) gel clot test• What samples are tested ?

• The primary application for LAL is the testing of parenteral pharmaceuticals and medical devices that contact blood or cerebrospinal fluid

• Why is it performed?• When the endotoxin is at a significant level it produces a pyrogenic effect• A pyrogen is an agent which elevates the body temperature of an animal

or human


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Bioburden

• What is Bioburden testing?• This testing does not constitute sterility testing, but is a method

used to determine the microbial bioburden of the sample.• Bioburden is normally defined as the number of bacteria living on a

surface that has not been sterilised


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Bioburden



surface that has not been sterilised• What samples are tested?

• Medical Devices


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Bioburden




• Medical Devices• How long does it take?

• The test requires 5 days of incubation


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Bioburden




• Medical Devices• How long does it take?

• The test requires 5 days of incubation• Why is it performed?

• The aim of bioburden testing is to measure the total number of viable micro-organisms on a medical device prior to its final sterilisation before implantation or use.


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Method Turn Around Times

MethodTurnaround Time (Days)

If Clean If Confirmation is Required

Total aerobic microbial count 5 n/a

Yeast and Mould 5-7 n/a

Bile tolerant Gram negative bacteria 2-3 n/a

Salmonella 3-4 5-6

E. coli 3-6 4-8

Staphylococcus Aureus 2-4 4-6

Extras not listed in TGO 77 section 9(2)

Pseudomonas aeruginosa 2-4 4-6

Candida albicans 6-7 10+


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Confirmation Process

Hi John,Please note that the below sample is suspect for E.Coli:

Lot: 12345 sample A, DOM 15/05/2015

The sample is under confirmation and the final result will be updated once complete

Thank you and best regards

Jenny BloggsMicrobiologist

• Have you ever received an email like this?


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Lot: 12345 sample A, DOM 15/05/2015





For a number of microbiological tests the initial test is generic and any positive or SUSPECT result requires more testing. A suspect may be:• The micro-organism being analysed• Other micro-organisms present that interfere• Matrix interference

Synonyms: Presumptive


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Lot: 12345 sample A, DOM 15/05/2015






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Lot: 12345 sample A, DOM 15/05/2015





What is confirmation?• Carrying out further steps of a test for identifying a particular type of bacteria.• Therefore CONFIRMING its presence.• When the steps have been completed we say that the test has been “Confirmed”.• After confirmation, the result will be finalised.

Synonyms: Follow up


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• What tests can require confirmation?




Yeast and Mould 5 n/a

Bile tolerant Gram negative bacteria 3 n/a

Salmonella 4 2-3

E. coli 5 7

Staphylococcus Aureus 3 6


Pseudomonas aeruginosa 3 5



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• What tests can require confirmation?




Yeast and Mould 5 n/a

Bile tolerant Gram negative bacteria 3 n/a

Salmonella 4 2-3

E. coli 5 7

Staphylococcus Aureus 3 6


Pseudomonas aeruginosa 3 5



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Method Validation

• What is microbiological method validation?• Verifying that the method used including media and sample

dilution are suitable to recover the micro organism


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Method Validation


dilution are suitable to recover the micro organism• How is it done?

• By inoculating the sample with the bacteria, incubating and recovering >70% of the micro organism


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Method Validation




• How long does it take?• The validation is the same duration as the test


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Method Validation




• How long does it take?• The validation is the same duration as the test

• How often is it performed?• Once per product every 5 years


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Method Validation

• Why do they fail?• Due to antimicrobial efficacy of the product• Presence of preservatives• <70% recovery of the validation test


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Method Validation

• Why do they fail?• Due to antimicrobial efficacy of the product• Presence of preservatives• <70% recovery of the validation test

• How can I avoid validation delays on new products?• By sending in samples for validation in advance, e.g.

process validation or pilot scale batches


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High to Low Counts

• How can a result be high in one test and then low when retested?

• This can be caused by a number of factors– The sample may not be completely homogenous– Lab error– The sample has anti-bacterial properties– Preservative taking effect


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High to Low Counts: Sample Not Homogeneous

• The laboratory generally uses 10g of the sample in the required media, e.g. 90 mL


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• Of that 90 mL, 0.1ml to 1ml is removed for each test


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• A reported result of 500 cfu/mL, may only be 5 colonies on an agar plate. This is a low number and with repeat testing small variations will affect the result by 100’s of cfu/mL


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• A reported result of 500 cfu/mL, may only be 5 colonies on an agar plate. This is a low number and with repeat testing small variations will affect the result by 100’s of cfu/mL

• In general, acceptable variation would be within half a log, e.g. Result Possible Variation

5,000 (or 5 x 103) ± 500

3,000,000 (or 3 x 106) ± 500,000


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High to Low Counts: Lab Error

• This is always a possibility and our quality procedures and controls minimise this risk. However it can never be 100% removed.


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• If we believe lab error is a possibility we will try to look for a pattern, e.g.• Do all the similar samples have a high count?• Does the sample have a history of high counts?• Do unrelated samples tested concurrently have high counts?


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• If we believe lab error is a possibility we will try to look for a pattern, e.g.• Do all the similar samples have a high count?• Does the sample have a history of high counts?• Do unrelated samples tested concurrently have high counts?

• We also look at other factors, e.g.• Experience of the technician• Environmental monitoring results• Media testing


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High to Low Counts: Anti-Bacterial Properties

• The sample may contain substances that are anti-bacterial (e.g. preservative systems)

• Samples are stored for up to 5 days before a retest can occur and substances in the sample itself may lower the bacterial count

• Therefore possibly high levels upon initial testing have been killed off or had their numbers lowered before the retest.


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Topical Products

• What should I test?


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Topical Products

• What should I test?• Based on Cutaneous acceptance criteria in the Pharmacopoeias

(BP, USP, Ph. Eur)Microbiological Quality Units Pass Criteria

Total aerobic microbial count CFU per g or mL ≤ 102

Total yeast and mould count CFU per g or mL ≤ 101

Staphylococcus aureus in 1g or 1mL Absent

Pseudomonas aeruginosa in 10g or 10mL Absent


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Topical Products







• Preservative Efficacy Testing (PET) – for liquid, multi-use products, using Pharmacopoeial methodologies


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Topical Products







• Preservative Efficacy Testing (PET) – for liquid, multi-use products, using Pharmacopoeial methodologies

• Method Validation?• This is critical to ensure the method used is suitable to recover

the micro organisms. Per product (formulation) every 5 years


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Further Reading

British Pharmacopoeia (2015): Appendix XVI D. Microbiological Quality of Non-sterile Pharmaceutical Preparations and Substances for Pharmaceutical Use

United States Pharmacopoeia – National Formulary (38): <1111> MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS: ACCEPTANCE CRITERIA FOR PHARMACEUTICAL PREPARATIONS AND SUBSTANCES FOR PHARMACEUTICAL USE

Therapeutic Goods Order No. 77 - Microbiological Standards for Medicines (22/09/2008)

http://www.comlaw.gov.au/Details/F2008L03574

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Microbiology for Oral and Topical Products - The Basics

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