Sterility is not a percentage, which is the whole difficulty

A certificate of analysis for a research peptide is, in the overwhelming majority of cases, a document about chemistry. It reports a chromatographic purity figure, sometimes a mass, occasionally a water content, and it is generated from a few milligrams of powder dissolved in a vial of mobile phase. Nothing in that procedure has any bearing on whether the sealed container it came from holds viable microorganisms, whether the powder carries pyrogenic material from an upstream water system, or whether the stopper has maintained a seal since the day it was crimped. The document is accurate and it is answering a different question.

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

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 statistics, worked in one paragraph

Suppose a batch of ten thousand vials in which one vial in a thousand is contaminated — a rate that would be a serious finding in any regulated operation and is invisible to any buyer. The probability that a single randomly chosen vial is clean is 0.999. The probability that all twenty sampled vials are clean is 0.999 raised to the twentieth power, which is approximately 0.980. The sterility test therefore passes this batch about ninety-eight times in a hundred.

Push the contamination rate up tenfold, to one vial in a hundred, and the test still passes the batch roughly eighty-two times in a hundred. To reach an even chance of detection at a one per cent contamination rate you would need to sample about seventy containers; to have a reasonable prospect of catching a one-in-a-thousand rate you would need to sample several hundred, which for most batches means testing a substantial fraction of the product.

This is not a criticism of the compendial test, which is designed as a final check against gross failure and performs that function. It is the reason no serious manufacturer treats a passed sterility test as the basis of the sterility claim, and the reason that a research supplier offering to have a vial sterility-tested on request is offering something considerably weaker than it sounds.

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.³

Recombinant factor C, and the end of an awkward supply chain

The lysate on which conventional endotoxin testing depends is harvested from horseshoe crabs, which are bled and returned to the sea with a mortality that is disputed and not negligible. The assay also inherits the biological variability of a natural product: lysate lots differ, and the cascade includes a branch responsive to beta-glucans, which is a common source of false positives in the presence of cellulose filter residues.

Recombinant factor C reagents replace the harvested cascade with an expressed enzyme, activated by lipopolysaccharide and read chromogenically or fluorimetrically. The response is specific to endotoxin and insensitive to the glucan branch, lot-to-lot consistency is a manufacturing rather than an ecological question, and comparative studies across a wide range of matrices have found agreement with conventional methods well within the variability of the conventional methods themselves.

The reagent has been available for well over a decade and its slow adoption was a regulatory rather than a scientific matter: for years it sat in an alternative-method chapter, obliging users to validate it as a departure. That has now changed, with dedicated chapters in both the United States and European compendia treating recombinant reagents as methods in their own right, and the Journal expects the harvested lysate to become the historical option within this decade.⁴⁵

What the cake tells you

A lyophilised plug is a physical record of the cycle that produced it. A well-formed cake occupies close to the volume of the solution that was frozen, has a matte, uniform appearance, retains a defined edge where it met the glass, and dissolves rapidly and completely on addition of diluent. That appearance indicates that the product was frozen below its critical formulation temperature and dried without the structure collapsing.

Departures are informative. A shrunken, translucent or glassy plug suggests collapse during primary drying — the ice sublimed faster than the amorphous matrix could hold its structure — which is associated with higher residual moisture and a shorter shelf life. A cake that has partly detached and moves freely suggests mechanical handling after drying, which is cosmetic. A cake showing melt-back at the base, or a visible meniscus line, suggests a shelf temperature excursion. Slow or incomplete reconstitution suggests either collapse or an unfavourable surface.

None of these observations is a purity finding, a sterility finding or an endotoxin finding, and it is important not to overclaim. What they are is the only direct evidence about process control that arrives in the box, and reading them costs nothing. The Journal photographs every cake it receives before opening the vial, for exactly that reason.⁶

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.