NPCB
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OUTLINE
Introduction
Certificate of Analysis
Media Validation
Tests Methods
Method Validation
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Sterility Test -Introduction
Definition : The sterility of a product is defined by the
absence of viable and actively multiplying
microorganisms when tested in specified culture
media.
The test is applied to substance, preparations or
articles which, according to the Pharmacopoeia, are
required to be sterile.
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Sterility Test -Introduction
Turbidity in the broth media uSually indicates
contamination.
DTest is performed on the end -product and is one of the
quality control tests specified for release of a batch of
sterile product.
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Certificate of Analysis
Specification and Result
DAs per British Pharmacopoeia or USP
DBP - Appendix XVIA. Sterility
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Sterility lest - Media Validation
Media types
aFluid Thioglycollate medium (FTM)
D Soybean Casein Digest Medium (SCD TSB)
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Sterility Test -Media Validatíon
Prior to test, make sure that:
DMedia is sterile
D
Media supports growth of microorganisms
2 components in Media validation :
DMedia sterility Test
DGrowth Promotion Test
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Sterility Test -Media Validation
Media sterility
DNegative Control - may be used to identify a "false
positive" test result
D Incubate for 14days prior to use, may be conducted
concurrently with test
30- 35°C for Fluid Thioglycollate medium (FTM)
20 - 25°C for Soybean Casein Digest Medium (SCD/TSB)
Acceptance criteria:
DShould be sterile, no growth observed
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Sterility Test -Media Validation
Growth Promotion Test
DTo test the ability of media to support the growth of micro
organisms
DThe media should be inoculated with <100 cfu of challenge
organisms. The challenge inoculum should be verified by
concurrent viable plate counts
DGrowth promotion challenge organisms should show clearly visible
growth in the test media within 3 days for bacteria and 5 days
for fungi.
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Sterility Test - Media Validation
Table 2.6.1-1- Strains of the test micro-organisms suitable for use in the Grouth Promotion Test and the Validation Test
Aerobic bacteria
ATCC 6538, CIP 4.83, NCTC 10788, NCIMB 9518
Staphylococus aureus
Bacillus subtilis ATCC 6633, CIP 52.62, NCIMB 8054
ATCC 9027. NCIMB 8626, CIP 82.118
Pseudomonas aeruginosa
Anaerobic bacterium
ATCC19404, CIP 79.3. NCTC 532 or ATCC I437
Clostridium sporogenes
Fungi
ATCC 10231, IP 48.72, NCPF 3179
Candida albicans
ATCC 16404, IP 143183, IMI 149007
Aspergillus niger
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Sterility Test -Test Methods
Methods are defined in Pharmacopoeia:
DMembrane Filtration Method
(open or a closed system)
DDirect Inoculation Method
*When the preparation to be tested has an antimicrobial
effects, these effects must be reduced or neutralised by
adding an appropriate substance to the specified test
media, to diluents or solvents, or to the preparation prior
to testing.
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Sterility Test -est Methods t (cont)
" Membrane Filtration Method (open Funnel Method)
Sample been Membrane
Membrane
filtered and filter is cut Incubate
rinsed into medium
into half
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Sterility Test - Test Methods (cont.)
Membrane Filtration Method (closed System Method)
Sample been Adding medium
Filtered and (FTM/SCD) into Incubate
rinsed apparatus
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Sterility Test - Test Methods (cont)
Direct lnoculation of the culture medium
a Transfer the preparation directly into the culture medium
a Volume of the product is not more than 10% of the volume
of the medium.
Table 2.6.1.-2 Minimum quantity to be used for each mediumn
Quantity per container Minimum quantity to be used for each medium unless
otherwise justified and authorised
Liquids
less than l ml The whole contents of each container
140 ml Half the contents of each container but not less than l ml
- greater than 40 ml and not greater than 100 ml 20 ml
- greater than 100 ml 10 per cent of the contents of the container but not less than 20 nl
Antibiotic liquids 1ml
Other preparations soluble in water or in isopropyl nyristate The whole contents of each container to provide not less than 200 mg
Insoluble preparations, creams and ointments to be suspended or
emulsified
The whole contents of each container to provide not less than 200 mg
Solids
less than 50 mg The whole contents of each container
50mg or more but less than 300 mg
Half the contents of each container but not less than 50 mg
300 mg to 5 g 150 mg
- greater than 5 g 500 mg
Catgut and other surgical sutures for veterinary use 3 sections of a strand (each 30 cm long)
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Sterility Test -Test Methods (cont)
Incubation
G Period : At least 14 days incubation
DTemperature : 30-35Cfor FTM
20-25C for SCD/TSB
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Sterility Test - Test Methods (cont)
Incubation and Examination
DAlltest & sterility control containers - incubated for
at least 14days (unless microbial contamination
detected earlier)
D
Examine for evidence growth
aPreparation not readily seen (turbid/cloudy due to
its nature) - after 14 days of incubation transfer
asuitable portion (2-5% of contents) to fresh, same
medium incubate for 7days
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Sterility Test - Interpretation of results
No evidence of microbial growth is found.
If turbidity or other evidence of growth is seen:
Streak on solid media
DExamine the suspected growth microscopically by
Gram stain
Dldentifythe isolates, as far as the genus and
preferably species level
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Sterility Test - Method Validation
Validation (bacteriostasis & fungistasis) Test
DThe test should be validated by inoculation with <100
cfu of challenge organism strains to the media/product
container at the beginning of the test incubation period.
DThe challenge inoculum should be verified by concurrent
viable plate counts.
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Sterility lest - Method Validation emns
Validation (bacteriostasis & fungistasis)Test
aThe challenge organisms, preferably, should be added
directly to the product prior to membrane filtration or direct
inoculation. If this is not practicable, the challenge organisms
should be added to the last rinse solution (membrane filtration)
or directly to media containing the product (direct inoculation).
Validation done should mimic the test proper in every detail.
aPerform a growth promotion test as a positive control. Incubate
all the containers containing medium for not more than 5 days.
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Sterility Test - Method Validation
Interpretation of results
Challenge organisms should clearly show visible growth
of bacteria within 3 days, and fungi within 5 days in
the test media containing product.
DVisually comparable to that in the control vessel without
product.
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Sterility Test - Method Validation (eont)
Validation (bacteriostasis &fungistasis)Test
alIf performed concurrently with ST should
confirmed validation
tests as sUCcessful before the results of the ST
are interpreted
Validation to be performed on all new product and repeated
whenever there is a change in the experimental conditions.
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Checklist
Resulis
Test Document Required Method (Raw
data)
CoA 1. Specification and Results
|1. Sterility Test (Bacteriostasis and
Validation Test
Fungistasis Test)
1. Sterility Test
Routine Test 2. Growth Promotion Test
3. Test for Media Sterility
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Comments for ST:
Ujian Steriliti(ST):
1.Sila kemukakan tatacara pengujian(SOP) untuk yang berikut, berserta keputusan ujian (raw data) bagisatu
kelompok keluaran siap:
Growth Promotion Test dan Media Sterility Test untuk semua media yang digunakan.
- Ujian steriliti.
- Validasi untuk ujian steriliti (Bacteriostasis & Fungistasis Test)
Tatacara hendaklah spesifik kepada produk. Salinan terus dari farmakopoeia tidak diterima.
2. Tatacara ujiansteriliti dan Validasi ujian steriliti perlu lengkapkan dengan butiran seperti di bawah bagi:
i) Bilangan sample/ volum produk yang diguna untuk vjian.
i) Tatacara yang diguna (Membrane Filtration/ Direct Inoculation)
ii) Composition rinsing buffer.
iv) Volum rinsing buffer yang diguna untuk setiap membrane.
v) Cara penyediaan sampel
3. Kesemua raw data yang dikemukakan perlu mengandungi nama dan nombor kelompok bagi Finished
Product, tarikh mula dan selesai pengujian, keputusan pemerhatian setiap hari (contoh: pemerhatian selama
14 hari bagi Media Sterility Test dan ujian steriliti, berserta pemerhatian selama 3-5 hari bagi Growth
Promotion Test dan Validasi ujian steriliti) & tandatangan/ nama penganalisis.
4. Sila kemukakan terjemahan bahasa Inggeris sekiranya data adalah dalam bahasa negara asing. 23
