Polysorbates 20 and 80 Degradation by Group XV Lysosomal Phospholipase A2 Isomer X1 in Monoclonal Antibody Formulations

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 A(2) isomer X1 (LPLA(2)) by shotgun proteomics. Recombinant LPLA(2) was over expressed, purified, and functional integrity confirmed against a diheptanoyl phosphatidylcholine substrate. Incubation of recombinantly produced LPLA(2) with PS20 and PS80 resulted in hydrolysis of PS20 and PS80 monoester but a much slower rate was observed for higher-order PS80. Endogenous LPLA(2) was detected and quantitated at less than 1 ppm in 3 formulated antibodies while LPLA(2) was not detected (or less than 0.1 ppm) in a fourth formulated antibody. Furthermore, antibodies with detectable quantities of endogenous LPLA(2) demonstrated polysorbate hydrolysis while in contrast the antibody without detectable LPLA(2) did not show polysorbate hydrolysis. Comparison of polysorbate degradation products generated from the formulated antibody and samples of polysorbate incubated with recombinant LPLA(2) resulted in similar elution profiles by liquid chromatographyemass spectrometry. These results suggest that LPLA(2) may play a key role in polysorbate degradation in some antibody preparations. (C) 2016 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved.