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Microbiology for Oral and Topical Products - The basics

Scott Colbourne

Business Manager NSW – ALS Food & Pharmaceutical

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

2Microbiology for Oral and Topical

Products - The Basics

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.



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)



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



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)



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

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

Common Tests – what and why?

Microbiology for Oral and Topical

Products - The Basics9

• 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 tested

Can be referred to as…

Total Microbial Count (TMC), Standard Plate Count (SPC), TVC,

TVAC


• Yeast and Mould

What?

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




• Bile Tolerant gram negative bacteria

What?

• 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




• Salmonella

What?

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




• E. coli

What?

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.




• Staphylococcus Aureus

What?

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




• Pseudomonas aeruginosa

What?

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




• Candida Albicans

What?

• 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



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











• What samples are tested ?

• 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.














• 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.

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

Endotoxin (LAL)









• The primary application for LAL is the testing of parenteral pharmaceuticals

and medical devices that contact blood or cerebrospinal fluid

Endotoxin (LAL)









• 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

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

Bioburden








• What samples are tested?

• Medical Devices

Bioburden









• Medical Devices


• The test requires 5 days of incubation

Bioburden









• Medical Devices


• 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.

Method Turn Around Times



Method

Turnaround Time (Days)

If CleanIf 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+

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 Bloggs

Microbiologist

• Have you ever received an email like this?




Hi John,


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

The sample is under confirmation and the final result will

be updated once complete


Jenny Bloggs

Microbiologist


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

Synonyms: Presumptive




Hi John,


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

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


Jenny Bloggs

Microbiologist





Hi John,


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

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


Jenny Bloggs

Microbiologist


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


• What tests can require confirmation?



Method



Required


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



• What tests can require confirmation?



Method



Required


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


Method Validation

• What is microbiological method validation?

• Verifying that the method used including media and sample

dilution are suitable to recover the micro organism



Method Validation




• How is it done?

• By inoculating the sample with the bacteria, incubating and

recovering >70% of the micro organism



Method Validation




• How is it done?




• The validation is the same duration as the test



Method Validation




• How is it done?




• The validation is the same duration as the test

• How often is it performed?

• Once per product every 5 years



Method Validation

• Why do they fail?

• Due to antimicrobial efficacy of the product

• Presence of preservatives

• <70% recovery of the validation test



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



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



High to Low Counts: Sample Not Homogeneous

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





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






• 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






• 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

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.






• 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?






• 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



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.



Topical Products

• What should I test?



Topical Products


• 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

Topical Products



(BP, USP, Ph. Eur)








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

using Pharmacopoeial methodologies

Topical Products



(BP, USP, Ph. Eur)








• 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

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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