It is time to reconsider the use of naturally-occurring endotoxins in endotoxin recovery studies: Part 2 of a BioPhorum harmonized endotoxin recovery study

Abstract

The evaluation of Naturally Occurring Endotoxins (NOEs) for Low Endotoxin Recovery (LER) studies has been a topic in the industry and regulatory agencies have been hesitant to endorse NOE use in LER studies over purified Lipopolysaccharide (LPS) standards such as Control Standard Endotoxin (CSE) or Reference Standard Endotoxin (RSE). In a recent study involving 11 BioPhorum member companies across 13 sites, NOEs prepared in high and low nutrient conditions were evaluated in two common monoclonal antibody buffer formulations: 10 mM Sodium Citrate, 0.05 % Polysorbate 80, pH 6.0 and 20 mM Histidine, 0.05 % Polysorbate 80, pH 6.0. 12 g-negative bacterial isolates were used to prepare NOE analytes, which were spiked into the formulation buffers. Additionally, the NOEs were spiked into Limulus Amebocyte Lysate (LAL) reagent water as controls and purified LPS into the citrate/polysorbate buffer as the LER control. Results showed the average of three runs per organism was >50 % recovery, at the conclusion of the 7-day period, regardless of nutrient culture preparation conditions. Furthermore, purified LPS controls became undetectable (<50 % recovery) in the citrate/polysorbate buffer, highlighting the presence of LER. These findings highlight the potential value of using NOEs from relevant manufacturing facilities to assess overall risk when purified LPS recovery is insufficient.