One column, one wavelength, one very large blind spot

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.

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.

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.

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.