Factors Affecting Reduction of Reference Endotoxin Standard Activity Caused by Chelating Agent/Detergent Matrices:...

Reduction of reference standard endotoxin activity was kinetically analyzed under low endotoxin recovery conditions and was considered as an apparent first-order reaction. Temperature, pH, and salt concentrations affected the rates of reduction of reference standard endotoxin activity. Temperature appeared to be the most important factor affecting low endotoxin recovery. Components of low endotoxin recovery matrices, such as citrate and polysorbate 20, showed similar low endotoxin recovery effect at concentrations commonly used. Phosphate concentrations showed negative correlation against the half-life of reference standard endotoxin activity in solutions containing phosphate buffer and polysorbate 20. Activation energy for low endotoxin recovery with naturally occurring endotoxin was higher than that with reference standard endotoxin, and this explained one of the reasons for naturally occurring endotoxin resistance to low endotoxin recovery. Lower temperature, lower pH, and a higher salt concentration are preferable to avoid low endotoxin recovery in a hold-time study. This study provides useful data for anticipation of the severity of the low endotoxin recovery effect and future hold-time studies in the biopharmaceutical field.LAY ABSTRACT: Endotoxin derived from Gram-negative bacteria is potentially harmful when it is parenterally administrated. Therefore, injectables and medical devices are tested by the bacterial endotoxins test to detect contamination by endotoxin of those products. Low endotoxin recovery is a phenomenon of reduction of detectable standard endotoxin activity by certain matrices of biopharmaceutical products containing a chelating agent and a detergent, and it is a controversial topic because its mechanism and clinical risks are unknown. The author analyzed the kinetics of low endotoxin recovery to elucidate the mechanism of low endotoxin recovery and to propose conditions to avoid low endotoxin recovery.