Sterility is not a percentage, which is the whole difficulty

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.

Bioburden, and why the number before the filter matters

A sterilising filter is not an unlimited barrier. Its qualification is expressed as a retention capability under a defined challenge — conventionally a high titre of a small bacterial species per square centimetre of membrane — and its performance in use depends on the load presented to it. A bulk solution carrying a heavy microbial burden presents a filter with a harder problem than one carrying a light burden, and it presents a second problem the filter cannot address at all: the endotoxin released by organisms that die upstream passes through the membrane unimpeded.

Regulated manufacture therefore specifies a pre-filtration bioburden limit, tests against it on every batch, and treats an excursion as an investigation rather than a curiosity. The specification is usually expressed in colony-forming units per hundred millilitres, and a well-controlled process runs far below it.

Of the twenty companies the Journal wrote to, four stated that pre-filtration bioburden is determined on every batch, three stated that it is determined periodically, and the remainder did not answer the question. We regard that distribution as the single most informative result of the correspondence, because bioburden testing is inexpensive, is performed on the bulk rather than the finished container, and is the earliest point at which a problem is cheap to fix.

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.

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.¹

Depyrogenation, and the step that gets skipped

Because endotoxin survives sterilisation, glass components require a separate treatment. The standard is dry heat: a tunnel or oven cycle at two hundred and fifty degrees or above, validated to achieve at least a three-log reduction in a deliberately applied endotoxin challenge. Vials emerging from a qualified depyrogenation tunnel are both sterile and pyrogen-free, and they remain so only if the subsequent handling maintains it.

Elastomeric closures cannot take that treatment and are instead washed, siliconised and steam-sterilised by a validated cycle, with endotoxin control achieved by the washing step and specified as a limit per stopper. Water for injection is controlled at source, with a compendial endotoxin specification, and a water system is the commonest origin of a pyrogen problem in an otherwise competent facility.

Depyrogenation is also, in the Journal’s experience of asking, the step most frequently absent from descriptions of small fill operations. Autoclaved vials are sterile. Autoclaved vials are not depyrogenated, and an operation that describes its glass preparation solely in terms of autoclaving has told you something specific about what it has not done. We put this point to eleven correspondents; two said their glass is depyrogenated by dry heat with a validated cycle, and one asked us what depyrogenation was, which we took as a straight answer and a useful one.

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.²³

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.⁴

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.

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.