Why FGP can be entirely straightforward and still tell you nothing about bioburden

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.

Sterile is a claim about a process

In ordinary speech, calling something sterile describes a state. In pharmaceutical manufacture it describes a provenance. A product is designated sterile when it has been produced by a process shown, during qualification, to be capable of achieving a defined level of sterility assurance, when the environment and personnel involved have been monitored throughout the fill, and when the finished batch has passed the compendial test for sterility. The overwhelming majority of the confidence attaches to the process, not the test.

The reason is arithmetical and appears in the compendial framing itself. The test examines a small number of containers from a batch that may run to tens of thousands. Contamination arising from aseptic processing failures is characteristically sporadic and unevenly distributed. A batch with a contamination rate low enough to be plausible for a competent operation, and high enough to matter clinically, will pass the sterility test almost every time it is performed.¹

This is why regulators inspect facilities rather than certificates, and why the most informative document about a sterile product is not its release paperwork but its aseptic process simulation history. Neither is available to anybody buying research peptides.

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 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 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.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.

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.