A vial can be 99.4 per cent pure and not sterile in the same afternoon

We began looking at this seriously after a reader sent us a certificate with a query attached. The certificate reported 99.2 per cent purity for a lyophilised peptide, and the reader wanted to know whether that meant the remaining 0.8 per cent could be bacterial. It is a reasonable question and the answer is that the figure has nothing to do with bacteria in either direction. A vial containing a substantial microbial burden would very probably return a purity figure indistinguishable from a clean one, because the mass involved is far below the integration threshold of any method in commercial use.

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.

Why nothing in this market is terminally sterilised

Where a product will tolerate it, moist-heat terminal sterilisation is the preferred route by a wide margin, for the simple reason that the object subjected to the lethal process is the sealed, filled, finished container. There is no subsequent opportunity for contamination, and the lethality delivered can be measured directly through the load.

Peptides make poor candidates. Autoclave conditions accelerate hydrolysis of the amide backbone, deamidation of asparagine and glutamine residues, and aggregation, and a cycle sufficient to guarantee microbial lethality will typically destroy a measurable fraction of the active substance. Lyophilised presentations do not escape the problem, because the solution is filled before it is dried, and the sterility of the finished cake is inherited from the sterility of that solution and the environment of the fill.

The consequence is that every product in this market is aseptically processed. That places the entire burden of sterility assurance on filtration, environmental control, component preparation and operator technique — precisely the four things about which the trade publishes nothing. The Journal states this as a structural observation, not as an accusation: the same is true of many legitimate aseptically filled products, and the difference lies in whether an inspectorate has looked.¹

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.²

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 in general terms on request. QST and BCH answered our five questions in full. WXT and FGP declined on the grounds that research-use products are not represented as sterile injectables, which is a legally sound answer that concedes the point 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.

Five lines that would change what a certificate is worth

Nothing the Journal asks for here requires a regulator, an inspectorate or a change in the law, and none of it is commercially sensitive. All five items are already known to whoever released the batch.

First, state the fill route: aseptic or terminal, and if aseptic, in what class of environment. Second, state the pre-filtration bioburden result against its specification, or state that bioburden is not determined. Third, state whether the sterilising filter was integrity-tested after use. Fourth, report bacterial endotoxin per milligram of peptide, with the method and the inhibition-enhancement result, or state that it was not determined. Fifth, where a sterility test has been performed, state the batch size, the number of containers tested and the method, so that a reader can do the arithmetic in the section above. Every one of the five is an ordinary element of a release specification for a biological product, and none of them is an invention of this publication.³

A certificate carrying those five lines would remain a one-page document and would be worth several times what the current one is worth, principally because four of the five lines are permitted to say no. A stated negative is a fact a reader can use. An omission is a space a reader fills with an assumption, and the assumption is always more favourable than the truth would have been.

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.

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.