Comparison of pyrogen assays by testing products exhibiting low endotoxin recovery.

The use of pyrogen tests to assess the risk of endotoxin in biological products has increased recently due to concerns of some regulatory authorities about products exhibiting low endotoxin recovery (LER). Manufacturers increasingly seek to reduce the use of animals unless essential to assure patient safety. The current study compares the ability of the monocyte activation test (MAT) and the bacterial endotoxin test (BET) to the rabbit pyrogen test (RPT) to detect endotoxin spikes in samples of products shown to exhibit LER. Product samples or water were spiked with endotoxin and held for three days or tested immediately in the BET, the RPT, and two variations of the MAT at the same time. Results show high sensitivity to endotoxin of both the BET and MAT, and much lower sensitivity of the RPT, indicating that much higher levels of reference standard endotoxin are required to induce pyrogenicity in the RPT than the 5 endotoxin units (EU) per kg common threshold. The results of the BET and MAT correlated well for the detection of endotoxin spike in water. We also show that LER (masking of endotoxin) found in the BET is also seen in the MAT and RPT, suggesting that the products themselves elicit a biological inactivation of spiked endotoxin over time, thereby rendering it less or non-pyrogenic. We conclude that the non-animal MAT option is a suitable replacement for the RPT to measure spiked endotoxin in biopharmaceuticals.

Validation of a NAT-based Mycoplasma assay according European Pharmacopoiea.

Eucaryotic expression systems are widely used to produce biologicals for human use, e.g. vaccines, recombinant proteins and monoclonal antibodies. As part of the safety testing the current U.S. Food and Drug Administration (FDA) regulatory guidelines as well as several European Pharmacopoiea monographs requests the demonstration of the absence of Mycoplasma in the cell culture in the bioreactors prior to harvest and further downstream processing. In recent years progress has been made in the development of a sensitive NAT-based method for the detection of Mycoplasma species in CHO cells, e.g. Eldering et al. This method is based on a nucleic acid amplification technique using a very sensitive touch-down PCR-profile. The presence of mollicutes DNA in the test specimens is determined by an approx. 450 bp target sequence which is amplified and this amplicon is finally detected by polyacrylamide gel electrophoresis. Based on this method a ready-to-use test kit was developed. In this report the validation of both method variants according the European Pharmacopoiea monograph 2.6.7 "Mycoplasmas" is described. The validation demonstrated the robustness and precision as well as a sufficient specificity of both assay formats. The validated sensitivity fulfills the requirements of the European Pharmacopoiea for a PCR-based method proposed as an alternative to the time consuming indicator cell culture and the culture method for the detection of Mollicutes (requested sensitivity of at least 10 colony-forming-units/mL).