Study on the Method Validation of Recombinant Factor C Endotoxin Testing Assay
Keywords:
bacterial endotoxin, recombinant factor C, vaccine, monoclonal antibodyAbstract
Background: Bacterial endotoxins, the pyrogens derived from gram-negative bacteria, are heat-stable and can contaminate vaccine, injectable drug, and medical device products for human and veterinary use during manufacturing. The Limulus Amebocyte Lysate (LAL) method for analyzing the endotoxin determination uses a lysate of horseshoe crab blood, which has a high post-bleeding mortality rate. In accordance with the 3Rs principle of animal welfare, there has been a global effort to find a replacement for the use of animals. A recombinant factor C (rFC) assay has been developed as a replacement for the horseshoe crab lysate assay. Currently, the European and United State Pharmacopoeias have recognized it as a standard method.
Objectives: To study the validation of the recombinant factor C method for analyzing the endotoxin determination for use as a standard method in quality control of biological products to cover all analytical methods in European and United Stated pharmacopoeias.
Methods: This experimental research was conducted from October 2022 to December 2023 for validation of the endotoxin determination using recombinant factor C method in the parameters such as linearity, accuracy and precision (repeatability and reproducibility by different days and analyst).
Results: The study found that the method demonstrated a linear relationship between the measured values and the concentration of the standard with a correlation coefficient of ≥ 0.980. The accuracy was assessed by spiking known concentrations of endotoxin into samples, the % recovery was within the acceptance criteria of 50-200%. The precision was evaluated by repeatability and intermediate precision studies. The coefficient of variation (CV) of % recovery was less than 25%. It could be used as a standard method for conducting the endotoxin determination in human vaccine and monoclonal antibody products. However, plasma-derived or blood products could not be analyzed due to interference from the samples.
Conclusions: The recombinant factor C method, which has been studied for its accuracy, is suitable and reliable. It can be used as a standard method for analyzing the quality of vaccines and monoclonal antibodies sold in Thailand.
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