We asked four laboratories for a sterility test. Here is what came back.

The explanation is structural rather than any failure of ambition. A purity run occupies an instrument for between twelve and forty minutes and produces a document the same week. A compendial sterility test requires aseptic transfer of the entire container contents into two growth media, incubation at two temperatures for fourteen days, and a facility qualified for that transfer, because a false positive from the analyst’s own sleeve is worse than no result. The test destroys the sample. Nothing about that sequence fits a service priced for private buyers submitting single vials.

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

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 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.

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.³⁴

The endotoxin limit, calculated

The limit is derived, not looked up. For a parenteral product other than an intrathecal one, the threshold pyrogenic dose is taken as five endotoxin units per kilogram of body weight per hour. The endotoxin limit for the product is that figure divided by the maximum dose per kilogram administered within an hour. For an intrathecal route the threshold falls to 0.2 endotoxin units per kilogram, a twenty-five-fold reduction that reflects the absence of the systemic buffering the bloodstream provides.

Work it for a concrete case. A seventy-kilogram subject has an hourly allowance of three hundred and fifty endotoxin units. A vial nominally containing five milligrams of peptide, reconstituted to two millilitres, from which a fifth of a millilitre is drawn, delivers a tenth of the vial contents. If the whole vial carried three hundred and fifty endotoxin units, that draw would deliver thirty-five — a tenth of the allowance. The limit expressed per milligram of peptide is what a certificate should carry, because it is the only form of the number that survives a change in reconstitution volume.

None of the arithmetic is difficult. What is missing from this trade is not the calculation but the measured numerator.⁵

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.⁶⁷

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.⁸

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.⁹

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.