Sterility Testing

Question 1

Sterility Testing

Answer 1

Quality control test to ensure sterility of end product
End product is labelled sterile
1. Sampling from batch
2. Select culture media (test culture media)
3. Select test method (Validate test method)
4. Perform sterility test (Validate test results)
5. Decision on batch release

Question 2

Sampling Plan

Answer 2

• Samples represent the whole batch
• Number of samples and quantity to test from each sample

If aseptically prepared –> take samples at regular intervals during manufacturing, first and last

If terminally sterilised products –> samples form various sites of the steriliser, least accessible site to sterilising agent

Small batch 20-300 take 10%
Large batch >300 take 30 containers
Reduce number of samples if sterilised in final container and using a biological indicator

If sample size is 20 <300ml sample 20 ml

Question 3

Select Culture Media

Answer 3

Anaerobic culture medium using fluid thioglycate broth
- 32 deg for anaerobic bacteria

Aerobic culture media with soybean casein digest broth
- 36 deg for aerobic bacteria
23 deg for fungi

Question 4

Test Culture Media

Answer 4

1. Sterility
   - Check background contamination by sterilising a black culture media at 36 and 20-30 for 3-6 days
   - must show no growth for it to be sterile and used
2. Fertility
   - Confirm media can support growth by deliberately inoculating test organisms and incubate media
   - must show positive growth
3. pH
   - 7.1 / 7.3 +/- 0.2
4. Particulates
   - No visible particulate matters

Question 5

Select Sterility Test Methods

Answer 5

1. Direct transfer
   - aseptic transfer of sample directly into culture media
   - 14 days incubation at temp, inspect every 2 days
   - Limitations: immiscibility, incompatibility, anti-microbial activity
2. Addition of concentrated media
   - Concentrated medium is aseptically added to the sample and incubation for growth
3. Membrane filtration
   - Sample filtered through membrane filter 0.45um in sterilised filtration unit in LFC
   - trap or retain contaminants
   - divide filter and incubate pieces in the culture for 14 days
   Limitations:
   - need to ensure sterility of the filter
   - filter may retain sample i.e. antimicrobial products
   - use sterile diluent to rinse the filter

Question 6

Method Validation

Answer 6

• Need to validate the method before it is performed
• make sure test can actually detect m.o in sample
• use deliberately contaminated test samples to test efficiency of test method
• media incubated with test samples contaminated with CL sphenoides, B subtilise, C albicans and must show growth in 48 hours

Question 7

Perform Sterility test and Validate test results

Answer 7

Validate:

1. Negative Control test
   - Use sterile articles i.e. water for inj to check background contamination
   - mimick sterility testing procedure and must show no growth
2. Stasis test
   - To validate sterility testing results and to confirm that negative growth is not due to media inactivation but due to sterility of product
   - Perform at the end of sterility testing period (14 days)
   - Select the media with negative growth and inoculate organisms, incubate and must show positive growth (similar to fertility test)

Question 8

Decision on batch release

Answer 8

Interpretations of results
Sample growth
Negative control any growth

If batch fails with positive growth, repeat test with double sample size
Increase sample size increase chance of detecting contamination

Question 9

If batch fails test

Answer 9

• identify source
• review test procedures
• review sterility assurance procedures i.e.
  contamination control, water quality, personnel techniques, manufacturing methods, containers packaging, sterilisation methods