The Microbiological Parameters in Establishing Stability Study … · 2010. 6. 7. · Case Study 3 other products 3 other products ––all of them topical all of them topical preparations

The Microbiological Parameters in The Microbiological Parameters in Establishing Stability Study Establishing Stability Study

ProtocolProtocolProtocolProtocol

Ngoc AnhNgoc Anh--Thu PhanThu Phan

2828thth April 2010April 2010

AMS Laboratories Pty Ltd

ContentsContents

�� IntroductionIntroduction

�� Why PE Testing?Why PE Testing?

�� Selection of Category and MethodsSelection of Category and Methods�� Selection of Category and MethodsSelection of Category and Methods

�� BP and USP comparisonBP and USP comparison

�� Environmental IsolatesEnvironmental Isolates

�� Case StudyCase Study


IntroductionIntroduction

�� Therapeutic Goods Order no 77 Therapeutic Goods Order no 77 Microbiological standards for medicines.Microbiological standards for medicines.

�� At a minimum, testing should be At a minimum, testing should be conducted at the initial and end timepoints conducted at the initial and end timepoints At a minimum, testing should be At a minimum, testing should be conducted at the initial and end timepoints conducted at the initial and end timepoints of a stability study.of a stability study.

�� Karen Longstaff Karen Longstaff --http://www.tga.gov.au/lab/presmicrobiolohttp://www.tga.gov.au/lab/presmicrobiology.pdfgy.pdf


Why PE Testing?Why PE Testing?

�� British Pharmacopoeia Appendix XVI C British Pharmacopoeia Appendix XVI C --Efficacy of Antimicrobial Preservation Efficacy of Antimicrobial Preservation

�� European Pharmacopoeia, Section 5.1.3 European Pharmacopoeia, Section 5.1.3 �� European Pharmacopoeia, Section 5.1.3 European Pharmacopoeia, Section 5.1.3 Efficacy of Antimicrobial Preservation.Efficacy of Antimicrobial Preservation.

�� United States Pharmacopoeia United States Pharmacopoeia -- chapter 51 chapter 51 Antimicrobial Effectiveness Test for Antimicrobial Effectiveness Test for Category 4 products.Category 4 products.


Why PE Testing?Why PE Testing?

�� Key Factors affecting the efficacy of the Key Factors affecting the efficacy of the antimicrobial preservative added:antimicrobial preservative added:

1.1. The active ingredient The active ingredient 2.2. The excipientsThe excipients

Storage conditions Storage conditions 3.3. Storage conditions Storage conditions 4.4. The container and its closure The container and its closure

�� The BP states for a product “The BP states for a product “it shall be it shall be demonstrated that the antimicrobial activity of the demonstrated that the antimicrobial activity of the preparation as such or if necessary, with the preparation as such or if necessary, with the addition of a suitable preservative or preservatives addition of a suitable preservative or preservatives provides adequate protection from adverse effects provides adequate protection from adverse effects that may arise from microbial contamination or that may arise from microbial contamination or proliferation during storage and use of the proliferation during storage and use of the preparationpreparation””


Selection of Category and MethodSelection of Category and Method

�� All products sent for any PE testing after All products sent for any PE testing after the prescribed stability storage conditions the prescribed stability storage conditions and times are recommended to be sent in and times are recommended to be sent in the final packaged product as distributed the final packaged product as distributed


the final packaged product as distributed the final packaged product as distributed by the sponsor.by the sponsor.

�� The BP/EP categories are forThe BP/EP categories are for�� Parenteral and ophthalmic preparationsParenteral and ophthalmic preparations

�� Topical preparationsTopical preparations

�� Oral preparationsOral preparations


�� The preparation is individually challenged with a The preparation is individually challenged with a prescribed inoculum of 10prescribed inoculum of 1055 to 10to 1066 cfu/g/ml of cfu/g/ml of preparation of bacteria and fungi and tested over preparation of bacteria and fungi and tested over 28 days.28 days.Pseudomonas aeruginosaPseudomonas aeruginosa ATCC 9027ATCC 9027�� Pseudomonas aeruginosaPseudomonas aeruginosa ATCC 9027ATCC 9027

�� Staphylococcus aureus Staphylococcus aureus ATCC 6538ATCC 6538�� Candida albicansCandida albicans ATCC 10231ATCC 10231�� Aspergillus brasiliensisAspergillus brasiliensis (formerly niger) ATCC (formerly niger) ATCC

1640416404�� Escherichia coliEscherichia coli ATCC 8739 for oral productsATCC 8739 for oral products�� Zygosacharomyces rouxiiZygosacharomyces rouxii (NCYC 381) for high (NCYC 381) for high

sugar oral productssugar oral productsAMS Laboratories Pty Ltd


�� These are tested at the initial time points These are tested at the initial time points and various time intervals depending on and various time intervals depending on the product category. the product category.

These are performed by traditional plate These are performed by traditional plate �� These are performed by traditional plate These are performed by traditional plate count methods or membrane filtration.count methods or membrane filtration.

�� The test method must be qualified for the The test method must be qualified for the product under evaluation to ensure the product under evaluation to ensure the correct diluent is used in assays for correct diluent is used in assays for surviving microorganisms.surviving microorganisms.


Selection of NeutralizerSelection of Neutralizer

Acceptance CriteriaAcceptance Criteria

�� Parenteral and ophthalmic preparations Parenteral and ophthalmic preparations 6h 24h 7day 14day 28day

Bacteria A 2 3 - - No recovery

B - 1 3 - No increase

Fungi A - - 2 - No increase

�� Topical preparationsTopical preparations



B - - - 1 No increase

2day 7day 14day 28day

Bacteria A 2 3 - No increase

B - - 3 No increase

Fungi A - - 2 No increase

B - - 1 No increase

Acceptance CriteriaAcceptance Criteria

�� Oral PreparationsOral Preparations 14day 28day

Bacteria 3 No increase

Fungi 1 No increase

�� For oral products of antacids with an For oral products of antacids with an aqueous base the above criteria must be aqueous base the above criteria must be met, however the initial inoculum must be met, however the initial inoculum must be between 1 x 10between 1 x 1033 and 1 x 10and 1 x 1044 cfu/ml of cfu/ml of product tested.product tested.


Fungi 1 No increase

BP and USP comparisonBP and USP comparison

�� Parenteral and ophthalmic preparationsParenteral and ophthalmic preparations 6h 24h 7day 14day 28day

Bacteria A 2 3 - - No recovery

B - 1 3 - No increase

USP - - 1.0 3.0 No increase

�� Topical PreparationsTopical Preparations


USP - - 1.0 3.0 No increase

from 14d


B - - - 1 No increase

USP - - NI NI NI

2day 7day 14day 28day

Bacteria A 2 3 - No increase

B - - 3 No increase

USP 2 No increase

from 14d

Fungi A - - 2 No increase

B - - 1 No increase

USP - - NI NI

BP and USP comparisonBP and USP comparison

�� Oral PreparationsOral Preparations 14day 28day

Bacteria 3 No increase

USP 1.0 No increase

from 14d

Fungi 1 No increase


Fungi 1 No increase

USP NI NI

Environmental IsolatesEnvironmental Isolates

�� Products may be more susceptible to Products may be more susceptible to environmental contamination during environmental contamination during manufacturing due to the nature of the product manufacturing due to the nature of the product and preservative system such as low pH or high and preservative system such as low pH or high and preservative system such as low pH or high and preservative system such as low pH or high water content.water content.

�� Although Although Ps. aeruginosaPs. aeruginosa is included as a test is included as a test organism, the reference culture strain may not be organism, the reference culture strain may not be as resistant to the preservative system as various as resistant to the preservative system as various strains of strains of Enterobacter, KlebsiellaEnterobacter, Klebsiella or or BurkholderiaBurkholderia..

�� Manufacturing facilities with known environmental Manufacturing facilities with known environmental contaminants are recommended to isolate and contaminants are recommended to isolate and include these strains in the PE testing. include these strains in the PE testing. AMS Laboratories Pty Ltd

Case StudyCase Study

�� The product was found to have high levels The product was found to have high levels Total Aerobic Plate count levels exceeding Total Aerobic Plate count levels exceeding the client specification. the client specification.

Identification of the isolated contaminant Identification of the isolated contaminant �� Identification of the isolated contaminant Identification of the isolated contaminant by both API and RiboPrinterby both API and RiboPrinterTMTM found it to found it to be be Enterobacter gergoviaeEnterobacter gergoviae..

�� Comparison of the strain through the Comparison of the strain through the RiboPrinterRiboPrinterTMTM database found the strain to database found the strain to be unique for that facility.be unique for that facility.







�� AMS Labs performed PE testing with the AMS Labs performed PE testing with the addition of the isolated addition of the isolated E. gergoviaeE. gergoviaestrain.strain.

Results from the PE test shows no Results from the PE test shows no �� Results from the PE test shows no Results from the PE test shows no recovery of all four test organisms, recovery of all four test organisms, however, the however, the E. gergoviaeE. gergoviae counts far counts far exceeded those of the reference cultures.exceeded those of the reference cultures.

�� This was found to be attributed to the This was found to be attributed to the lower pHof the product and the natural lower pHof the product and the natural paraben resistance paraben resistance E. gergoviaeE. gergoviae..AMS Laboratories Pty Ltd




�� 3 other products 3 other products –– all of them topical all of them topical preparations were found to show increased preparations were found to show increased growth in growth in E. gergoviae E. gergoviae over the 28 day test. over the 28 day test.

All results showed slight reductions in counts All results showed slight reductions in counts �� All results showed slight reductions in counts All results showed slight reductions in counts after 48 hours from the preservative system, after 48 hours from the preservative system, however, resistance was demonstrated by however, resistance was demonstrated by increases in counts after 14 days with only increases in counts after 14 days with only one out of the four products tested showing one out of the four products tested showing a reduction in counts at day 28 from the 14 a reduction in counts at day 28 from the 14 day count.day count.


ConclusionConclusion

�� Microbiological testing should be considered Microbiological testing should be considered throughout a stability study, as a minimum at throughout a stability study, as a minimum at the start and end of the study.the start and end of the study.

�� Parameters should be consistent with the Parameters should be consistent with the requirements of TGO 77 requirements of TGO 77 –– Microbiological Microbiological

�� Parameters should be consistent with the Parameters should be consistent with the requirements of TGO 77 requirements of TGO 77 –– Microbiological Microbiological Standards for MedicinesStandards for Medicines

�� Selection of the correct pharmacopoeia and PET Selection of the correct pharmacopoeia and PET category is critical interpretation of resultscategory is critical interpretation of results

�� Environmental isolates may be considered as an Environmental isolates may be considered as an addition to the pharmacopoeia PET methods.addition to the pharmacopoeia PET methods.

ReferenceReference

�� British Pharmacopoeia, Appendix XVI C British Pharmacopoeia, Appendix XVI C Efficacy of Antimicrobial Preservation Efficacy of Antimicrobial Preservation

�� European Pharmacopoeia, Efficacy of European Pharmacopoeia, Efficacy of Antimicrobial PreservationAntimicrobial PreservationEuropean Pharmacopoeia, Efficacy of European Pharmacopoeia, Efficacy of Antimicrobial PreservationAntimicrobial Preservation

�� United States Pharmacopoeia <51> United States Pharmacopoeia <51> Antimicrobial Effectiveness TestAntimicrobial Effectiveness Test

�� Therapeutic Goods Order no. 77 Therapeutic Goods Order no. 77 Microbiological Standards for Medicines.Microbiological Standards for Medicines.

THANK YOU!THANK YOU!

�� ANY QUESTIONS?ANY QUESTIONS?

Documents

The Microbiological Parameters in Establishing Stability Study … · 2010. 6. 7. · Case Study 3 other products 3 other products ––all of them topical all of them topical preparations