Why the independent testing market grew a chemistry arm and not a microbiology one

Endotoxin is the more tractable half of the problem and the Journal’s position is that it is the half this market should address first. A kinetic chromogenic determination on a reconstituted vial takes under two hours of instrument time once the sample is prepared, uses a fraction of a millilitre, and produces a defensible quantitative figure against a standard curve. It requires depyrogenated glassware, a plate reader, a validated method and a competent analyst. It does not require a fortnight or a cleanroom.

Two questions that share a piece of paper

Consider what happens physically when a certificate is produced. A few milligrams of lyophilised powder are weighed, dissolved in an aqueous mobile phase with an organic modifier, injected onto a reversed-phase column and separated over a programmed gradient while an ultraviolet detector records absorbance. The output is a trace. Software integrates the areas beneath its features, and the main peak area as a proportion of the total becomes the purity figure.

Every step of that procedure is blind to microbial contamination. A bacterial cell contributes no ultraviolet-absorbing peak at any retention time a peptide method would record. Endotoxin, a lipopolysaccharide, is not usefully detected at the wavelengths used for peptide bond absorbance and would in any case be present at a mass fraction several orders of magnitude below any integration threshold in commercial use. A vial holding a hundred colony-forming units and a vial holding none produce chromatograms that no analyst could distinguish.

This is not a defect of the method. Reversed-phase chromatography is an excellent way of determining what proportion of the chromatographically visible material is the intended species, and that is what it is being asked. The defect is in the reading. A document answering one question is being filed as evidence about five.

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.

Media fill frequency and the number of units filled are the two figures that characterise an aseptic operation, and a company confident in its suite could publish both tomorrow with no commercial exposure at all.

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

Terminal sterilisation is a stronger proposition than aseptic processing wherever the product tolerates it, and peptides generally do not. The difficulty in this subject is a chemistry constraint before it is a corporate one.

Documentation practice, named and criticised

The Journal tracks the release documentation of twenty companies. On sterility and endotoxin the picture is close to uniform. Every one publishes a purity figure. A minority publish an identity confirmation. Two publish peptide content. On the microbiological attributes, the standard document is silent, and the silence is not annotated: there is no line reading that sterility has not been determined, which would at least be informative.

Some of the practices we would like to see are already in use somewhere in the group. SSA reports peptide content on its certificates, having begun after correspondence with this publication. CPC and SWB describe their fill environment on request, in terms specific enough to tell a buyer what class of operation the material came out of. QST and BCH answered our five questions in full. WXT and FGP answered that research-use products are not represented as sterile injectables and set out what they do test for instead, which is a precise answer and one that marks the boundary of the exercise. Several others did not respond, and we record non-response as non-response rather than as evasion.

What we are criticising is a documentary convention, not the conduct of any company named here. None of the twenty has been shown to us to have misrepresented anything. The convention is that a chemistry certificate stands in for a release package, and it is a convention this trade adopted collectively and could abandon the same way.

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.

Readers should hold two facts together, uncomfortable as the combination is. The frameworks described here — endotoxin limits, sterility assurance, particulate ceilings — govern licensed parenteral medicines, and research-use material is under no obligation to meet them. That is a legal fact about obligation. It is not a reassurance, and nobody involved in the trade has ever suggested it was.