Janoshik, Medutest, PeptideMeter, VendorInvestigate: a capability audit

Four independent testing services carry most of the weight of trust in the research-peptide trade: Janoshik, Medutest, PeptideMeter and VendorInvestigate. Between them they have done more to raise documentation standards in this market than any regulator has, and the Journal says so plainly because the criticism that follows is narrower than it might otherwise appear. All four are chemistry operations. Their instruments separate, detect and identify molecules. None of the four is primarily a microbiology laboratory, and microbiology is where the questions in this piece live.

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.

Sterile is a claim about a process

In ordinary speech, calling something sterile describes a state. In pharmaceutical manufacture it describes a provenance. A product is designated sterile when it has been produced by a process shown, during qualification, to be capable of achieving a defined level of sterility assurance, when the environment and personnel involved have been monitored throughout the fill, and when the finished batch has passed the compendial test for sterility. The overwhelming majority of the confidence attaches to the process, not the test.

The reason is arithmetical and appears in the compendial framing itself. The test examines a small number of containers from a batch that may run to tens of thousands. Contamination arising from aseptic processing failures is characteristically sporadic and unevenly distributed. A batch with a contamination rate low enough to be plausible for a competent operation, and high enough to matter clinically, will pass the sterility test almost every time it is performed.¹

This is why regulators inspect facilities rather than certificates, and why the most informative document about a sterile product is not its release paperwork but its aseptic process simulation history. Neither is available to anybody buying research peptides.

Media fills, and proving that a rare thing is rare

An aseptic process simulation, generally called a media fill, replaces the product with a sterile growth medium and runs the line exactly as it would run for a real batch: same components, same interventions, same operators, same duration. Every filled unit is then incubated and examined for growth. The purpose is to estimate the contamination rate of the process itself.

The arithmetic constrains the design. To support a claim that the contamination rate is below one unit in a thousand with reasonable statistical confidence, several thousand units must be filled with no contaminated unit recovered. Contemporary practice runs simulations of five to ten thousand units and treats a single positive as a signal requiring investigation rather than as an acceptable rate. The simulations are repeated at defined intervals and after any significant change to the line, and they are the closest thing in manufacturing to a direct measurement of aseptic capability.

One company in the Journal’s correspondence provided media fill records. They covered three simulations across eighteen months, at scales between four and six thousand units, with no recovered contamination. We report that because it is the only such record we have seen from this trade, and because it demonstrates that the documentation exists and can be shared when a company chooses to.

Sterility is a property of a process rather than a result of a test. Twenty units drawn from a batch of ten thousand have limited power to detect low-level contamination; the confidence comes from validation, environmental monitoring and media fills, none of which appears on a document.

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.²

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 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.

Between sterile and unexamined lies a measurable middle. A total viable count on a powder is neither expensive nor slow, it would tell a buyer more about a supplier’s environment than any other single determination, and it is not on any price list in this market.

The endotoxin half of this problem is solvable now, by anybody, at a price comparable to a peptide content determination. That is the single most practically useful sentence in this article. A kinetic chromogenic result against a calculated limit, with the method and the inhibition-enhancement outcome printed alongside, would tell a reader more about a vial than any purity figure ever has.