Polysorbates 20 and 80 Degradation by Group XV Lysosomal Phospholipase A 2 Isomer X1 in Monoclonal Antibody Formulations

Abstract

Decreases in polysorbate (PS80) content were observed while evaluating the long-term storage stability of Chinese hamster ovary–derived, purified monoclonal antibodies. It was determined that polysorbate had been enzymatically degraded; therefore, studies were performed to identify and characterize the protein(s) responsible. Polysorbate degrading activity was enriched from Chinese hamster ovary media leading to the identification of group XV lysosomal phospholipase A₂ isomer X1 (LPLA₂) by shotgun proteomics. Recombinant LPLA₂ was over expressed, purified, and functional integrity confirmed against a diheptanoyl phosphatidylcholine substrate. Incubation of recombinantly produced LPLA₂ with PS20 and PS80 resulted in hydrolysis of PS20 and PS80 monoester but a much slower rate was observed for higher-order PS80. Endogenous LPLA₂ was detected and quantitated at less than 1 ppm in 3 formulated antibodies while LPLA₂ was not detected (or less than 0.1 ppm) in a fourth formulated antibody. Furthermore, antibodies with detectable quantities of endogenous LPLA₂ demonstrated polysorbate hydrolysis while in contrast the antibody without detectable LPLA₂ did not show polysorbate hydrolysis. Comparison of polysorbate degradation products generated from the formulated antibody and samples of polysorbate incubated with recombinant LPLA₂ resulted in similar elution profiles by liquid chromatography–mass spectrometry. These results suggest that LPLA₂ may play a key role in polysorbate degradation in some antibody preparations.