Microbiology for Oral and Topical Products - The basics Scott Colbourne Business Manager NSW ALS Food & Pharmaceutical

Microbiology for Oral and Topical Products - The basics Scott Colbourne Business Manager NSW ALS Food & Pharmaceutical RIGHT S O L U T I O N S RIGHT PARTNER

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 2

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. 3

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 10 2 CFU, the maximum acceptable count is 200 (which allows for microbial testing variability) 4

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 5

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) 6

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 Combined Yeasts/Moulds Count Escherichia coli Staphylococcus aureus Pseudomonas aeruginosa Candida albicans Bile-tolerant Gram-negative bacteria 7

TGO 77 Subsection 9(2): Applies to a complementary medicine oral dosage form containing raw material of natural origin Microbiological Quality Units Schedule 1: Containing Total aerobic microbial count CFU per g or ml 10 4 10 7 Total yeast and mould count CFU per g or ml 10 2 10 5 Bile-tolerant Gram negative bacteria CFU per g or ml 10 2 10 2 Salmonella in 10g or 10mL Absent Absent Escherichia coli in 1g or 1mL Absent Absent * A herbal medicinal product consisting solely of one or more herbal substances (whole, reduced or powdered) to which boiling water is added before use Schedule 2: Solely herbal substances * Staphylococcus aureus in 1g or 1mL Absent Not applicable 8

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 tested Can be referred to as Total Microbial Count (TMC), Standard Plate Count (SPC), TVC, TVAC 9

Common Tests what and why? 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 10

Common Tests what and why? 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 11

Common Tests what and why? 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 12

Common Tests what and why? 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. 13

Common Tests what and why? 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 14

Common Tests what and why? 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 15

Common Tests what and why? 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 16

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. 19

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 What samples are tested? The primary application for LAL is the testing of parenteral pharmaceuticals and medical devices that contact blood or cerebrospinal fluid 22

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 What samples are tested? 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. 27

Method Turn Around Times Method If Clean Turnaround Time (Days) 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+ 28

Confirmation Process Have you ever received an email like this? 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 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 30

Confirmation Process Have you ever received an email like this? 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 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 32

Confirmation Process What tests can require confirmation? Method If Clean Turnaround Time (Days) If Confirmation is Required Total aerobic microbial count 5 n/a 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 Extras not listed in TGO 77 section 9(2) Pseudomonas aeruginosa 3 5 Candida albicans 6-7 10+ 33

Method Validation What is microbiological method validation? Verifying that the method used including media and sample dilution are suitable to recover the micro organism 35

Method Validation What is microbiological method validation? Verifying that the method used including media and sample 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 How long does it take? The validation is the same duration as the test 37

Method Validation Why do they fail? Due to antimicrobial efficacy of the product Presence of preservatives <70% recovery of the validation test 39

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 40

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 41

High to Low Counts: Sample Not Homogeneous The laboratory generally uses 10g of the sample in the required media, e.g. 90 ml 42

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 43

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 In general, acceptable variation would be within half a log, e.g. Result Possible Variation 5,000 (or 5 x 10 3 ) ± 500 3,000,000 (or 3 x 10 6 ) ± 500,000 45

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. 46

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? 47

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. 49

Topical Products What should I test? 50

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 10 2 Total yeast and mould count CFU per g or ml 10 1 Staphylococcus aureus in 1g or 1mL Absent Pseudomonas aeruginosa in 10g or 10mL Absent Preservative Efficacy Testing (PET) for liquid, multi-use products, using Pharmacopoeial methodologies 52

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) 54