The fourteen-day problem, and what it does to a commercial testing model

The explanation is structural rather than any failure of ambition. A purity run occupies an instrument for between twelve and forty minutes and produces a document the same week. A compendial sterility test requires aseptic transfer of the entire container contents into two growth media, incubation at two temperatures for fourteen days, and a facility qualified for that transfer, because a false positive from the analyst’s own sleeve is worse than no result. The test destroys the sample. Nothing about that sequence fits a service priced for private buyers submitting single vials.

The compendial sterility test, described plainly

The compendial test proceeds by one of two routes. In membrane filtration, the entire contents of the sampled containers are passed through a retentive membrane which is then divided between two growth media. In direct inoculation, the contents are transferred into the media directly. The media are a fluid thioglycollate medium incubated at thirty to thirty-five degrees for anaerobes and aerobes, and a soybean-casein digest medium incubated at twenty to twenty-five degrees for fungi and aerobes. Incubation runs for fourteen days with periodic examination for visible growth.

The number of containers sampled depends on batch size, and for a parenteral batch above five hundred containers the requirement is twenty. Every one of those twenty is destroyed. Method suitability must be demonstrated separately, because a preserved formulation or an antimicrobial residue can inhibit the very growth the test is looking for, and the bacteriostatic and fungistatic properties of the article have to be neutralised or shown absent before a negative result means anything.¹

A reader who takes nothing else from this section should take the sample size. Twenty containers, fourteen days, destroyed. That is the entire empirical basis of the finished-product sterility claim, and it is why the process argument carries the weight.

The statistics, worked in one paragraph

Suppose a batch of ten thousand vials in which one vial in a thousand is contaminated — a rate that would be a serious finding in any regulated operation and is invisible to any buyer. The probability that a single randomly chosen vial is clean is 0.999. The probability that all twenty sampled vials are clean is 0.999 raised to the twentieth power, which is approximately 0.980. The sterility test therefore passes this batch about ninety-eight times in a hundred.

Push the contamination rate up tenfold, to one vial in a hundred, and the test still passes the batch roughly eighty-two times in a hundred. To reach an even chance of detection at a one per cent contamination rate you would need to sample about seventy containers; to have a reasonable prospect of catching a one-in-a-thousand rate you would need to sample several hundred, which for most batches means testing a substantial fraction of the product.

This is not a criticism of the compendial test, which is designed as a final check against gross failure and performs that function. It is the reason no serious manufacturer treats a passed sterility test as the basis of the sterility claim, and the reason that a research supplier offering to have a vial sterility-tested on request is offering something considerably weaker than it sounds.

What endotoxin is, and why sterility does not address it

Endotoxin is a structural component of the outer membrane of Gram-negative bacteria: a lipopolysaccharide with a lipid A anchor that is the pyrogenic moiety, a core oligosaccharide, and a variable O-antigen chain. It is shed during growth and released in quantity on cell lysis, which means that killing a bacterial population does not remove its endotoxin and may increase the free concentration.

Three physical properties make it a separate discipline. It is thermally robust, surviving autoclave conditions with little loss of pyrogenicity, so terminal sterilisation is not a depyrogenation step. It is small and amphipathic, forming aggregates that pass a 0.22 micron membrane without difficulty, so sterilising filtration is not a depyrogenation step either. And it is active in humans at very low mass — the threshold pyrogenic dose corresponds to something in the region of a nanogram per kilogram of body weight.

The practical consequence is stark. A vial can pass a sterility test, contain no viable organism of any kind, and carry an endotoxin burden many times a defensible parenteral limit, because the organisms responsible died somewhere upstream in a water system, a holding tank or a poorly stored component.²

The four services, and what they will sell you

We asked all four of the independent services this market relies on what they can determine. Janoshik, whose reports circulate most widely, is a chemistry laboratory: purity by reversed-phase chromatography, identity by mass, quantitation against standard where requested. Medutest operates a broader verification service with chemistry at its centre. PeptideMeter is likewise a chemistry and verification operation. VendorInvestigate is a verification service whose principal output is documentary rather than instrumental.

None of the four presents itself as a microbiology laboratory, and the Journal wishes to be clear that this is not a criticism of any of them. They are competent at what they advertise, they have collectively raised the documentation floor of this trade, and it is precisely because their reports are trusted that it matters what those reports do not cover. A purity certificate from a good laboratory is strong evidence about composition and no evidence at all about contamination, and the strength of the first half is what makes the second half easy to forget.

Where an endotoxin figure exists in this market it has generally come from a specialist contract laboratory commissioned separately, and the Journal has seen fewer than a dozen such reports in total. Two of them accompanied vials we submitted ourselves.

What we submitted, and what came back

Between the second and fourth quarters the Journal purchased fourteen vials of lyophilised research peptide from nine suppliers, at catalogue prices, through ordinary channels and without identifying ourselves. Each was photographed sealed, logged, and stored at two to eight degrees on arrival. Twelve were submitted to a contract laboratory accredited to the general competence standard for testing laboratories for bacterial endotoxin determination by kinetic chromogenic assay, with method suitability established for each matrix. Two were submitted for a compendial sterility test by membrane filtration, which destroyed both.

We disclose the following limitations without being asked. Fourteen vials from nine suppliers is not a survey. Single determinations carry the uncertainty of single determinations. A negative sterility result on one vial says nothing about the batch it came from, for exactly the statistical reasons set out above. And a vial that has crossed a border in a padded envelope has a thermal and mechanical history we cannot reconstruct.

What the exercise establishes is narrower than a survey and, we think, worth publishing: that the tests exist, that they are commercially available to a private purchaser at a known price, that the numbers they return are interpretable against a calculable limit, and that nothing prevented any of the nine suppliers from commissioning them first.³

A note on method and sourcing

The compendial material in this article is drawn from the current general chapters of the United States Pharmacopeia and the European Pharmacopoeia, read in the original rather than in summary, and from the international standards on aseptic processing and on laboratory competence. Where a chapter has changed status recently — as the recombinant reagent chapters have — we say so, because a reader consulting an older edition will find a different framing.

Where the Journal reports a number it obtained itself, it states the laboratory’s accreditation status, the method family, whether method suitability was established, and the number of determinations. Where we report what a company told us, we distinguish an answer from a refusal and a refusal from a non-response, because those three things are routinely collapsed in coverage of this trade and they are not the same.

Corrections to this department are handled by the standards desk, which reads every letter and records the outcome in the log. Readers who believe a paragraph here overstates its evidence are asked to write to standards@compoundjournal.com; readers with documents to send, including certificates they would like read, should write to letters@compoundjournal.com. We do not publish correspondents’ names without permission and we do not identify the source of a certificate.

We will keep asking the five questions, printing the answers, and recording the refusals as refusals. Two companies changed their certificates after the first round of this correspondence, which is a small result for a year of letters and rather better than none. Documents to letters@compoundjournal.com; disputes about anything above to standards@compoundjournal.com.