On 01 July 2020, the EDQM published European Pharmacopoeia (EP) Supplement 10.3 which now contains the new chapter 2.6.32 Test for bacterial endotoxins using recombinant factor C (rFC). This is a milestone for the EP, as it now introduces the recombinant Factor C assay as a compendial assay for endotoxin detection.
Chapter 2.6.32 closely follows the existing chapter 2.6.14 Bacterial Endotoxins, covering equipment, reagents and general calculations before describing the technique and detailing the requirements for preparatory and routine testing.
The rFC test is described as a quantitative, endpoint fluorescent assay, employing recombinant Factor C based on the genetic sequence from the horseshoe crab (all species). Lonza’s PyroGeneTM Assay is a prime example of this and has been well established as such for over 15 years.
Like the classic LAL assays, users have to first demonstrate the linearity of the standard curve in an initial qualification (IQ) assay. This is followed by the typical product-specific validation, where inhibition/enhancement (I/E) testing is carried out to determine the best sample preparation and ensure the sample doesn’t interfere with the test. For interference testing, the EP specifically points out that due to the absence of Factor G in the rFC reagent, reactivity to glucans will not occur.
The setup and requirements for preparatory and routine assays are identical to the quantitative photometric LAL assays:
- Standard curve: at least three standards, additional standards for each additional log range, at least triplicates (IQ) / duplicates (I/E and routine); correlation coefficient ≥ |0.980|
- Negative control: water for BET, at least triplicates (IQ) / duplicates (I/E and routine); no detectable endotoxin
- Positive Product Control (PPC): concentration at or near the middle of the standard curve, at least duplicates; recovery 50-200%
With so much in common, labs are able to easily transfer from their current LAL method to an rFC assay and are now backed up in this decision by a major regulatory agency. Moreover, users of Lonza’s WinKQCLTM Software will find that the PyroGeneTM rFC assay is already available as an option, making the switch on the software side as easy as clicking a button.
Written by Saskia
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
