Sterility testing

What does the pharmacopoeial sterility test involve?

The sterility test is harmonised across the major pharmacopoeias. ICH Q4B Annex 8(R1) records that Ph. Eur. 2.6.1, JP 4.06 and USP <71> Sterility Tests can be used as interchangeable in the ICH regions, subject to conditions such as the minimum number of containers tested (ICH Q4B Annex 8(R1)). The US FDA's guidance on aseptic processing calls USP <71> the principal source used for sterility testing methods, including procedures and media (US FDA, Sterile Drug Products Produced by Aseptic Processing, 2004).

The harmonised text, published by the European Medicines Agency during the Q4B process, uses two media. Fluid thioglycollate medium is mainly for anaerobic bacteria but also detects aerobic bacteria, and is incubated at 30 to 35 °C. Soya-bean casein digest medium suits fungi and aerobic bacteria, and is incubated at 20 to 25 °C (harmonised Sterility Test text, EMA).

Membrane filtration or direct inoculation: what is the difference?

The harmonised text uses membrane filtration whenever the nature of the product permits, including filterable aqueous preparations and preparations soluble in solvents that have no antimicrobial effect under the test conditions.

Why does a sterility test take at least 14 days?

Organisms present in very small numbers, or damaged by processing, can take days to grow to visible levels. The harmonised text sets incubation at not less than 14 days and requires the media to be examined at intervals and at the end for macroscopic evidence of growth. If the sample itself clouds the medium so that growth cannot be judged, portions are transferred to fresh medium after 14 days and incubated for at least 4 more days.

What happens if something grows?

Growth means the sample does not comply, unless the test can be shown to be invalid for reasons unrelated to the product. The harmonised text limits those reasons to a fault in the facility's microbiological monitoring, a fault in the procedure, growth in the negative controls, or an organism that can be unequivocally traced to the test materials or technique. Only then may the test be repeated, with the same number of units.

US rules for biological products are similar. 21 CFR 610.12 allows one repeat when an investigation finds the initial positive was due to laboratory error or faulty materials, requires the same test method for the repeat, and states that growth in the repeat means the product does not comply (21 CFR 610.12, eCFR).

Why does a passing sterility test say little about a whole batch?

Because only a few units are tested. The FDA aseptic processing guidance states that sterility tests are limited in their ability to detect contamination because of the small sample size, quoting USP that the sampling plan only enables detection of contamination in a lot where 10% of units are contaminated about nine times out of ten. It adds that if a 10,000-unit lot with 0.1% contamination were tested using 20 units, there would be a 98% chance the batch would pass.

This is why sterility is built into manufacturing through validated sterilisation or aseptic processing, with the end test as one check among many. A pass on one vial says that vial grew nothing, not that its batch mates are sterile. The same guidance also warns that poor test facilities can produce false positives, and notes that isolators minimise that risk.

Does Tested.ph offer sterility testing?

No. A meaningful pharmacopoeial result needs several units from one batch and a 14-day incubation, which does not fit single-vial testing. Endotoxin by LAL assay is offered as an option. The lesson on endotoxin and sterility puts the two side by side.