Sterility Testing

Question 1

What is sterility testing?

Answer 1

A Quality Control (QC) test to ensure the sterility of the end product

Question 2

List 4 reasons why sterility testing is important

Answer 2

1) Ensures sterility of product
2) Maintain therapeutic efficiency
3) Control microbial instability
4) GMP requirement

Question 3

What are the 8 steps that Sterility Testing involves

Answer 3

1) Sampling from a batch
2) Select culture media
3) Test culture media
4) Select test method
5) Validate test method
6) Perform sterility testing
7) Validate test results
8) Decision on batch release

Question 4

How do you choose a sampling batch in Aseptically prepared products?

Answer 4

Select the first and last items at regular intervals during manufacturing (i.e. during filtration process)

Question 5

How do you choose a sampling batch in Terminally sterilised products?

Answer 5

Select from the sites least accessible to sterilising agent

Question 6

How many samples do you choose from a batch according to CGMP?

Answer 6

Minimum 10% for a small batch (20-300)

At least 30 containers for larger lots of >300

You can reduce this number of samples if the final container is sterilised and has a biological indicator.

Question 7

How much volume do you sample for 20-300mL?

Answer 7

sample all
10-20 mL -> sample 10mL
>20-300mL -> sample 20mL

Question 8

What are two types of culture media used for microbial growth detection?

Answer 8

1) Anaerobic culture medium

2) Aerobic culture medium
detects aerobic bacteria @ 36degrees & detects fungi @ 23degrees incubation

Question 9

What 4 factors do we need to test for on a Culture media to ensure its suitable to detect microbial growth?

Answer 9

1) Sterility
2) Fertility
3) pH
4) Particulates

Question 10

List 3 sterility test methods

Answer 10

1) Direct Transfer
2) Addition of concentrated media
3) Membrane Filtration

Question 11

Explain the Direct Transfer method

Answer 11

1) Aseptic transfer of pooled sample directly into the culture media
2) Media then incubated at appropriate Temperature
3) 14 days incubation period
4) Inspect media every 2 days

Question 12

What are some limitations of the Direct Transfer method?

Answer 12

• Immiscibility (don’t mix enough to see results)
• Incompatibility (precipitation)
• Anti-microbial activity (of the sample)

Question 13

Explain the ‘Addition of Concentrated Media’ method

Answer 13

1) Concentrated media is aseptically added to the test product (opposite of direct transfer)
2) Incubation for the growth detection

Limitations are the same as direct transfer method

Question 14

Explain the ‘Membrane Filtration’ method

Answer 14

1) Pooled sample is filtered through a membrane filter (0.45micron) in a sterilised filtration unit in LFC
2) Filter will trap or retain any contaminants present in the sample
3) After filtration, aseptically divide the filter and incubate filter pieces in the culture media for 14 days

Question 15

What advantage does the ‘Membrane Filtration’ method have over the over 2 methods of sterility test methods?

Answer 15

Able to test samples that have anti-microbial activity.

Filter may retain this sample, but can be easily rinsed off with a sterile diluent.

Question 16

Explain how the sterility test methods are validated?

Answer 16

Before the test is performed (initial validation) media are incubated with product samples deliberately contaminated with organisms (anaerobic, aerobic & fungi). The methods are validated if there is growth shown in 48 hours.

Question 17

Explain the test used to perform sterility tests

Answer 17

NEGATIVE CONTROL TEST

• Mimics sterility testing procedure (i.e. direct transfer, addition of concentrated media or membrane filtration)
• But uses undoubtedly sterile articles (e.g. WFI)
• Must show no growth
• Monitor background contamination

Question 18

How do you validate sterility testing results?

Answer 18

STASIS TEST

• Perform at the end of sterility testing period (14 days)
• Select media with negative growth
• Inoculate 10-20 viable oraganisms
• Incubate for 48 hours –> +ve growth

Question 19

What is the purpose of the Stasis test

Answer 19

1) To confirm negative growth is not due to media inactivation
2) Validate that primary negative result is due to the sterility of the product

Question 20

What results are needed for the sample and the negative control tests for the batch to pass the sterility tests?

Answer 20

Samples (sterility testing) = negative growth

Negative controls = negative growth

Question 21

When does a batch fail sterility tests

Answer 21

When the primary samples show positive growth and the negative controls show negative growth.

Question 22

How do you increase the probability of detecting contamination?

Answer 22

Increase the sample size (repeating test) may increase the chance of detection