Allen Burgenson, associate director, Global Subject Matter (SME) Testing, Lonza, discusses the a more sustainable route to future-proof testing and quality control in pharma.
Key insights:
- The industry is increasingly looking to more sustainable BET methods — to minimise supply chain risk and future-proof pharmaceutical quality control (QC) testing operations.
- Research has shown that the rFC method is a suitable BET assay for the products tested, providing a sustainable alternative BET method, and that rFC assays can be comparable to LAL-based methods across various product matrices.
- Eli Lilly’s Emgality was the first FDA-approved drug released using rFC, back in 2018. Since then, regulatory bodies around the world have approved many other drugs tested using rFC.
Traditional LAL-based testing — risks for the modern QC lab
Testing injectables and parenteral pharmaceuticals for bacterial endotoxins is critical for their safe release to the market. Traditionally, the test of choice for bacterial endotoxin testing (BET) has been the limulus amebocyte lysate (LAL) test, which requires the blood of the horseshoe crab (HSC).
However, rapid expansion of the pharmaceutical market is driving an increased need for BET, and the finite resources critical for LAL. Estimates suggest the global LAL testing market will grow by 9% per year between 2022 and 2029.
As such, the industry is increasingly looking to more sustainable BET methods — methods that reduce dependence on natural resources to minimise supply chain risk and future-proof pharmaceutical quality control (QC) testing operations.
Recombinant factor C: the route to more sustainable QC operations
Figure 1: Illustration of the rFC assay reaction. rFC assays work via a single enzymatic step.
Recombinant Factor C (rFC) is one sustainable method. First developed and commercialised in 2003, rFC was a critical development in the biomedical industry’s continued commitment to reduce reliance on HSCs in the face of growing BET demand. The rFC assay uses a recombinant form of Factor C, the first protein of the HSC blood coagulation cascade that reacts with endotoxins. In the rFC assay, endotoxins activate the recombinant factor C protein, which then cleaves a fluorogenic substrate to produce a fluorescent signal (figure 1).
The rFC assay offers several advantages over traditional LAL-based assays. As recombinant Factor C is manufactured in a lab and not derived from HSCs, it is more sustainable. rFC assays can therefore better accommodate growing testing demand, imparting greater supply chain security. Additionally, because the rFC assay works via a single enzymatic step, it is less susceptible to interference. Conversely, the traditional multi-step LAL cascade is activated by glucans as well as endotoxins, which can lead to false positive BET results (figure 2).
Figure 2: Illustration of the LAL assay reaction. Unlike the rFC assay, the traditional multi-step LAL clotting cascade is susceptible to interference by glucans.
Finally, while HSC blood contains myriad factors that vary between animals, the rFC assay contains only Factor C. rFC assays therefore retain all the endotoxin reactivity of LAL-based tests, but none of the biological variability, leading to greater lot-to-lot consistency.
It is important to note that, like LAL tests, rFC must be tested with each pharmaceutical product to confirm its suitability, as no single BET method works for all products.
An established, equivalent method for BET
Despite the benefits, some organisations have hesitated to adopt rFC methods, owing to concerns around the method being too new or lacking equivalency to LAL tests. However, the rFC assay is already a well-established alternative. The test has been commercially available for almost two decades, and, in that time, a wealth of peer-reviewed studies have demonstrated the assay’s feasibility, specificity, and comparability to traditional LAL-based tests.
Perhaps the most important of these studies is that published in Pharmacopeial Forum in early 2010. This multi-site study was the first comparison of the rFC assay with traditional LAL tests and followed the requirements of the United States Pharmacopeia (USP) chapter <1225>. Researchers compared the rFC assay to Kinetic-QCL Kinetic Chromogenic LAL (KQCL) across 10 different pharmaceutical products. As per USP chapter <1225>, researchers evaluated specificity, precision, accuracy, linearity, quantitation limit, and range. In all cases, results showed rFC to be comparable to the LAL-based method when using reference standard endotoxin (figure 3). The study thus concluded that the rFC method is a suitable BET assay for the products tested, providing a sustainable alternative BET method.
In 2021, researchers conducted another comparative study (expected to be published in Pharmacopeial Forum in Q1 2023), this time looking at natural endotoxin contamination. Researchers spiked four parenteral products with natural water to simulate a purification system breach, and then tested the products using LAL and rFC methods. Results demonstrated that rFC assays can be comparable to LAL-based methods across various product matrices.
Global regulations also support rFC assays, with the rFC assay being either a compendial or alternative method as per several pharmacopeias (Table 1).
Table 1: rFC assay status according to the European, Chinese, United States, and Japanese pharmacopeias.
Besides being supporting by numerous studies and regulations, rFC tests have been — and continue to be — widely used. Eli Lilly’s Emgality was the first FDA-approved drug released using rFC, back in 2018. Since then, regulatory bodies around the world have approved many other drugs tested using rFC. Our latest market data indicates hundreds of organisations, spanning more than thirty countries and an array of pharmaceutical industry segments, currently use rFC for BET.
Straightforward validation
The rFC assay’s status as a non-pharmacopeial assay in the US and Japan means laboratories must validate it for use with products destined for global distribution. The added validation process has prevented some manufacturers from using rFC. However, the validation process is significantly easier than laboratories presume.
Assuming a laboratory has already validated a quantitative LAL-based method for its product, QC personnel can validate rFC in as little as five days. The validation process is identical to that used for LAL-based methods, with just one added step — “validation of alternative method” (figure 3).
While the validation procedure is straightforward enough, BET product vendors offer solutions that further streamline the process. For example, some vendors provide a full, easy-to-follow validation protocol with rFC products, and plate readers are available that can accommodate multiple testing types, including rFC, in a single instrument. The latter means lab personnel can directly compare results when evaluating traditional and alternative methods, saving time and space during validation. What’s more, endotoxin detection and analysis software is available that can accommodate multiple BET methods, including rFC, regardless of hardware platform, with the most advanced solutions also supporting rFC assay automation. Lab personnel don’t therefore require any additional software training for adopting rFC assays, and rFC testing post-validation can be as efficient as automated LAL-based testing.
Future-proofed QC operations
BET is critical for the safe release of injectables and parenteral pharmaceuticals. But demand for LAL-based assays is projected to grow rapidly in coming years. The rFC assay is a sustainable alternative option, with proven equivalence to LAL-based methods, and boasting many advantages for QC labs. Most importantly, making the transition from LAL-based methods to rFC is easier than QC personnel commonly perceive, and BET vendors are stepping up to help make the process even simpler.