The recombinant Factor C (rFC) assay has been developed to help alleviate the pressures of a growing demand for the Limulus amoebocyte lysate (LAL) assay. Here, given the recent pending recognition by the European Pharmacopoeia of rFC as a compendial test, the advantages of rFC testing will be explored as well as a discussion of rFC as an extension of the LAL paradigms.
In November of 2019, the European Pharmacopoeia (Ph. Eur.) Commission adopted 13 new monographs and four new general chapters. Among the new chapters is Chapter 2.6.32, entitled ‘Test for bacterial endotoxins using recombinant factor C (2.6.32)’. This will be published in the coming weeks in Ph. Eur. Supplement 10.3 and available on the European Directorate for the Quality of Medicines (EDQM) website.
The chapter will be effective at the start of 2021. Thus rFC testing will be a compendial test very soon in Europe. Tests for bacterial endotoxins using recombinant factor C, therefore, will not have to be validated per se, other than in consideration of their use for a specific substance or product in a specific analytical environment which is referred to as a verification in USP terminology. USP <85>, for example, is a verification whereas USP <1225> is a full validation.
Improving the LAL paradigm
The advantages of the rFC test revolve around the most recent paradigm: the difficulty associated with sustaining and improving modern endotoxin test methods in light of diminishing horseshoe crab populations. The rFC test is recognised as possessing several distinct advantages to the LAL assay, here referred to as the “3S’s”:
- Scientific characterisation
- Reproducibility
- Quality
- Sustainability
- Availability
- Specificity
These advantages will be briefly discussed.
Scientific characterisation
The lack of characterisation of LAL has never been an acknowledged problem. However, LAL was used for a decade before an alternative activation mechanism was discovered (the Factor G pathway). It was a shocking revelation at the time that materials other than endotoxin could activate the Limulus cascade to produce a positive reaction when little or no endotoxin was present.
rFC consists of just the recombinant protein and a small fluorescent peptide and thus no Factor G. The protein content of rFC can be measured to perfection whereas the proteins in LAL are at least 8 different ones (Factor C, Factor B, proclotting enzyme, coagulogen, Factor G, and serine protease inhibitors 1, 2, and 3), including some denatured by processing, and likely more proteins exist in LAL that are not specified.
The LAL formulation process that differs LAL manufacturer to manufacturer contributes to the lack of scientific characterisation in that they are all different.
Some LAL formulations are chloroform extracted leaving denatured proteins (inhibitors) whereas some contain zwittergent (the concentration added can have a large effect on endotoxin detection levels) and each LAL is paired with an LPS that is alternatively prepared (relative to RSE) and formulated with various excipients.
Characterisation suggests that one should know exactly what is contained in a product and this for LAL is impossible.
The lack of characterisation of LAL has never been an acknowledged problem
There is some irony in that current attempt to correlate a highly characterised material (rFC) with a less characterised material that is used as the gold standard and that varies widely supplier to supplier is made difficult given the lack of specificity of LAL to endotoxin.
Classical LAL assays are sourced from horseshoe crabs and thus subject to underlying natural variability from lot to lot. Production lot to lot, rFC has been shown to maintain its quality attributes in terms of standard curve values and reaction parameters obtained. The biotechnological production of rFC allows users to source a consistent product that is not subject to the variables of naturally harvested proteins. This is important for reproducible and reliable testing.
Product variability that occurs with pooled batches of harvested animals includes age, gender, size, environment, etc. Global companies prefer uniformity and standardised platforms as applied across global supply chains. Thus, a recombinant produced product can better meet these criteria.
rFC is a product of biotechnology; thus, as opposed to being harvested from a sea creature that is undergoing survival pressures, it can be produced “at-will” in cell culture. The genus Limulus is listed as “vulnerable” in the US and Asia the genus Tachypleus is listed as “endangered” on the IUCN Red List.
The global availability of LAL has been met to date in a supply chain that depends upon geographically isolated production and subsequent transatlantic or transpacific export from the US east coast. On a smaller scale, the use of Tachypleus amebocyte lysate (TAL) produced locally in Asia has also been used. The “at-will” production of a recombinant reagent can help meet the logistical demands to support global supply chains.
rFC is not licensed by FDA as LAL has historically been because it is not a blood-derived product, therefore, more localised production of rFC may provide advantages in terms of pharma auditing and oversight of production quality.
Specificity
If LAL was to be approved today it would have to contend with USP specificity requirements. In this regard, it has been accepted, yet this should not be confused with an optimum situation. In contrast to rFC, LAL contains Factor G, which is the beta-glucan zymogen (biosensor for beta-glucan).
Beta-glucan has been found commonly as a breakdown product of cellulosic filters in drug manufacturing processes, as well as a ubiquitous contaminant of natural waters (lakes, rivers and sewers) as a by-product of plant, algae and fungal growth. The activation of LAL by beta-glucans is referred to as a “false positive” as there is no beta-glucan standard in the endotoxin test. Even when masked with beta-glucan blocking buffer (a high concentrated solution of beta-glucans), it has been shown to affect the variability of LAL assay results.
Activity of LAL may also be affected by the synergistic effects of small amounts of endotoxin exaggerated by the presence of trace levels of beta-glucans. The lack of false-positive results from rFC testing removes a sometimes confounding element of LAL detection.
In contrast to rFC, LAL contains Factor G, which is the beta-glucan zymogen (biosensor for beta-glucan)
In recent years, many scientific studies have been published showing reliable detection of a wide variety of Gram-negative bacterial endotoxin using rFC-based assays. In comparison, the performance of equivalency of the rFC and LAL methods has been demonstrated by many pharmaceutical users. Multiple pharmacopoeias are today beginning to include the rFC in their official texts.
Naturally sourced waters such as those from lakes, rivers or sewage should not be used for comparison purposes in testing rFC versus LAL. For pharmaceutical water testing, purified water spiked with endotoxin (natural or standard) is used for equivalence testing (rFC vs. LAL). The potential contaminants from purified water systems have been shown to come from biofilm rather than pass-through from natural sources which are uncharacterised and expected to contain beta-glucans.
In terms of validation requirements for pre-compendial comparison testing (though regulators have expressed the policy of accepting the pending compendial status that now applies to rFC), water could be used from various natural sources including The Sea of Cortez, Japan’s lake Biwa, or Bay of Bengal (for example). However, each natural water may contain specific impurities from local flora that interfere with either rFC or LAL and thus should not be used for equivalence testing.
Users should use purified water as neither USP <85> or USP <1225> requires the testing of any water but that actually used as purified for drug manufacturing purposes. It would be exceedingly difficult to reproduce testing from uncharacterised water sources.
rFC as a continuation of LAL
Any envisioning of “the future” of testing contains elements extrapolated from the past. There are three main elements that predict a positive global outcome for recombinant Factor C (rFC) that require very little extrapolation:
- The advent of biotechnology and the replacement of animal proteins via the cloning of the necessary animal genes (insulin, growth hormone, etc) and production in bioreactors via single-celled organisms has given us advanced medicines and cures for many diseases and thus serves as a successful paradigm for replacement.
- The specificity, sensitivity and expanded utility of using rFC methods has been repeatedly demonstrated. The paradigm change of testing a coagulation output (Limulus) versus an original fever output (Rabbit Pyrogen) was a much bigger change than changing from a natural to recombinant protein version where both are inherent coagulation measures. Note that the horseshoe crab has no fever reaction and the assumption that fever reaction equals coagulation reaction is seldom discussed. For practical purposes of contaminant detection, the two are accepted as synonymous.
- The continued use of horseshoe crabs as harvested from limited geographical locations is not sustainable, and pharmaceutical manufacturers will want to prepare for such a change.
Since the initial exploratory use of LAL for radiopharmaceutical testing the past 50 years have established several widespread paradigms that will be enhanced by rFC testing including:
- The use of the LAL test to supplant the rabbit pyrogen test (RPT) as a method of precluding fever reactions in patients subject to injectable drug treatments. The detection of “all pyrogens” was not a particularly sensitive or relevant target for injectable drugs as drugs are a product of water-based manufacturing where GNB are the predominant contaminant.
- Initially, quality control consisted of the end-product testing of finished drugs only. With the advent of a convenient in vitro test, LAL has rapidly taken over the task of testing; over the years testing has expanded from end-point only to raw material, in-process, purified waters as well as end-point release testing. The increased test coverage of the entire manufacturing process has brought about a vast increase in the microbiological safety of drug products.
- The rise of test volume (both domestically and globally) has been paralleled by the demise in the reagent source. Horseshoe crabs on the US eastern seaboard (Limulus) are listed as “vulnerable” and those in Asia (Tachypleus) are now listed as officially “endangered”. While most agree that biomedical bleeding has not been the cause of such a demise, the industry must still contend with the fact that sustainable methods are needed to replace LAL testing.
- More recently the advent of “ease of use” testing has greatly benefited users. The LAL test began as a fairly user-intensive effort that included manually inverting reaction tubes for gel clot testing and has transitioned to first, semi-automated kinetic testing where absorbance is monitored over time without further user intervention, and finally, to configurations in which test standards and reagents are combined in a single prepackaged unit (cartridge and precoated plate testing). Already there are “ease of use” improvements in the rFC assay including pre-spiked plates.
Each paradigm change has aided the pharmaceutical industry by helping make product contamination events very rare. 3 Today, there is an expectation that, for sustainability reasons, global pharmaceutical companies should explore the use of rFC in routine testing of purified water, raw materials, and components as well as finished drug products.
The US Food and Drug Administration (FDA) has been receptive to such a change, as seen in the recent approval (2019) of the first drug to be released using rFC testing. Many global companies also continue to pursue raw material, water and component testing as well as a finished product using rFC.
There are some in industry that worry about the legacy of LAL; however, the uptake of recombinant methods should be viewed as a fulfilment of that legacy rather than an affront to it, just as the change from animal-sourced medicinal proteins, such as recombinant human insulin, was not an affront to early efforts (animal-derived insulin) to treat disease. Indeed, the biotechnological revolution is a direct response to the recognised utility of natural proteins.
There are also some that would delay and defer the development of approved rFC methods for commercial purposes with the intent of continuing the regulated monopoly that now exists on Limulus harvest. This is imprudent as it puts at risk the entire supply chain of medicinal products.
When the final horseshoe crab is bled and there is no more supply from which to make LAL, what will the detractors of rFC propose in that day?