The certificate is silent on contamination, and it never claimed otherwise

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.

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.

Container closure integrity, method by method

The compendial guidance divides leak test methods into probabilistic and deterministic families and is explicit about preferring the second. Probabilistic methods — dye ingress under vacuum, microbial immersion challenge, bubble emission — rely on a sequence of stochastic events, and their sensitivity is poor and hard to quantify. A dye ingress test can pass a container with a defect large enough to admit an organism, because the dye happened not to travel.

Deterministic methods measure a physical quantity with a continuous response. Vacuum decay monitors pressure rise in an evacuated test chamber. High-voltage leak detection measures current through the container wall and is well suited to liquid-filled units. Laser-based headspace analysis interrogates the gas inside a sealed container non-destructively, which permits repeated measurement of the same unit across a stability programme. Helium mass spectrometry resolves the smallest defects of any method in routine use.

The threshold that matters is the maximum allowable leakage limit — the defect size below which microbial ingress does not occur under the conditions the product will see. Establishing it for a given package is a piece of work, and once established it converts an argument about seals into a measurement.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.

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.