Application of Recombinant Factor C Reagent for the Detection of Bacterial Endotoxins in Pharmaceutical Products

Abstract

Recombinant Factor C (rFC) is non-animal-derived reagent used to detect bacterial endotoxins in pharmaceutical products. Despite the fact that the reagent was first commercially available nearly 15 years ago, the broad use of rFC in pharmaceutical industry has long been lagging, presumably due to historical single-source supplier concerns and the lack of inclusion in worldwide pharmacopeias. Commercial rFC reagents are now available from multiple manufacturers, thus single sourcing is no longer an issue. We report here the successful validation of several pharmaceutical products by an end-point florescence-based endotoxin method using the rFC reagent. The method is equivalent or superior to the compendia bacterial endotoxins test method. Based on the comparability data and extenuating circumstances, the incorporation of the end point fluorescence technique and rFC reagent in global compendia bacterial endotoxins test chapters is desired and warranted. Public health has been protected for over 30 years with the use of a purified blood product of the horseshoe crab, limulus amebocyte lysate. More recently, this blood product can be produced in biotech manufacturing processes, which reduces potential impacts to the horseshoe crab and related species dependent upon the crab, for example, migrating shorebirds. The pharmaceutical industry has been slow to adopt the use of this reagent, Recombinant Factor C (rFC), for various reasons. We evaluated the use of rFC across many pharmaceutical products, and in other feasibility demonstration experiments, and found rFC to be a suitable alternative to the animal-derived limulus amebocyte lysate. Incorporation of rFC and its analytical method into national testing standards would provide an equivalent or better test while continuing to maintain patient safety for those who depend on medicines and while securing pharmaceutical supply chains. In addition, widespread use of this method would benefit existing animal conservation efforts.

Application of a Recombinant Three-Factor Chromogenic Reagent, PyroSmart, for Bacterial Endotoxins Test Filed in the Pharmacopeias

“…To address these issues, some products have been marketed, including PyroGene and EndoZyme, which use recombinant factor C alone and a fluorogenic substrate. 19,20) PyroSmart is characterized by three recombinant factors including factor C, factor B and the proclotting enzyme and a chromogenic substrate. 21) Several reports regarding evaluations of PyroGene have been published.…”

When Would Immunologists Consider a Nanomaterial to be Safe? Recommendations for Planning Studies on Nanosafety

“…These assays are based on the activation of a recombinant form of Factor C, which is one of the first elements involved in the Limulus coagulation cascade and involve a fluorogenic detection method. [36,37] Endozyme is a homogenous test for soluble samples while EndoLISA uses a recombinant bacteriophage protein to immobilize endotoxins and detect them in a principle similar to an enzyme-linked immunosorbent assay (ELISA). [38] Both methods are highly sensitive and quantitative, however, they also can be affected by NMs due to interferences with the fluorescent light detection (e.g., quenching), inhibition of the protein function, or protein immobilization (EndoLISA).…”

Metode Monocyte Activation Test (MAT) dan Recombinant Factor C (rFC) sebagai Alternatif Metode Pengujian Pirogen bagi Perusahaan Farmasi di Indonesia

“…Kelebihan dari pengujian dengan metode rFC antara lain: rFC lebih spesifik terhadap endotok sin, proses produksi rekombinan protein telah divalidasi sehingga reprodusibilitas dari metode ini lebih baik dibandingkan dengan reagen LAL alami. Metode rFC merupakan metode analisa yang modern dan sederhana, cepat, spesifik, sen sitif, serta lebih hemat biaya untuk endotoksin tes [20]. Perbandingan lainnya adalah metode analisa yang dilakukan, pada metode rFC dilaku kan pe ngukuran berdasarkan deteksi fluore sens, sedangkan pada metode LAL pengukuran de ngan deteksi absorbansi.…”

“…Meskipun hingga saat ini belum diketahui efeknya pada manusia, namun diharapkan metode MAT dapat mengatasi gang guan LER karena metode ini meniru reaksi piro gen pada manusia [24]. [20]. Hasil efektifitas dari rFC menunjukkan dapat men deteksi kandungan endotoksin pada 100 ng protein rekombinan [30].…”