The question no chromatogram has ever answered

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.

The filter, and the test that proves it survived

Sterilising-grade filtration through a membrane of 0.22 micron nominal rating is the operation on which aseptic processing rests. The membrane is qualified by challenge with a small bacterium at high concentration under the process conditions, and a filter that permits no passage under that challenge is accepted as sterilising for that fluid.

The critical practice is not the filtration but the integrity test that follows it. A membrane can be damaged during installation, during sterilisation, or by pressure excursions in use, and a damaged membrane looks exactly like an intact one. Bubble point, diffusive flow and pressure hold tests each detect a breach by measuring gas behaviour across a wetted membrane, and a post-use test is the only evidence that the filter was intact while the product was passing through it. A pre-use test alone establishes nothing about the state of the membrane at the end of the run.

This is the question the Journal has found most useful when assessing whether a fill operation is a real one. It is specific, it has a yes or no answer, the answer is recorded in the batch documentation as a matter of course, and it cannot be answered plausibly by anybody who is not actually running the process.

Particulate matter, visible and subvisible

Injectable products are required to be essentially free of visible particulates, and every container in a regulated batch is inspected against dark and light backgrounds under defined illumination. The detection threshold for a trained inspector is somewhere near fifty microns for a contrasting particle, and the inspection is a hundred per cent operation rather than a sample-based one, which makes it unusual among quality tests.

Subvisible particles are counted instrumentally. Light obscuration is the primary compendial method, with microscopic membrane counting as the alternative when the sample defeats it. For a small-volume injection the limits are six thousand particles at or above ten microns and six hundred at or above twenty-five microns per container. Products that are themselves proteinaceous get a distinct chapter, because inherent aggregates complicate the counting and the method has to distinguish them from extrinsic contamination.

For a private buyer, visible inspection is the one quality operation available without instrumentation, and it is worth doing carefully: a lyophilised cake that has collapsed to a glassy disc, a reconstituted solution showing a faint tyndall haze on rotation, a fibre against a dark card. None of those is a sterility finding. All of them are evidence about how the product was made and how it has travelled.¹²

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.

What this article is not asserting

Precision about the scope of a criticism is part of the job, so it is worth spending a paragraph on what is not being said. This piece does not allege that any company named in it has sold contaminated material. It does not allege that any of them has concealed a result, falsified a document or misrepresented a test. It does not claim that the products discussed are dangerous, and it does not claim that they are safe, because neither claim is supportable from the evidence we have.

What the piece asserts is documentary. A certificate describing chemistry is being read as a release package covering microbiology. That mismatch is created by the format of the document rather than by anybody’s intent, and it is closed by adding lines rather than by changing behaviour.

There is also a legal point the Journal has no wish to elide. Research-use-only material is not approved for human use in any jurisdiction, is not required to meet parenteral standards, and is not represented by its sellers as meeting them. Everything in this article about endotoxin limits and sterility assurance describes the framework that would apply to a parenteral medicine. Applying that framework to a research chemical is a comparison, not a compliance requirement, and readers should hold both halves of that sentence at once.

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.