Discovery and reduction of tryptophan oxidation-induced IgG1 fragmentation in a polysorbate 80-dependent manner

Abstract

The fragmentation of therapeutic antibodies, particularly in the complementarity determining region (CDR), is considered a critical quality attribute. However, our understanding of CDR fragmentation remains limited. Here, we report the mechanism behind CDR fragmentation of a therapeutic IgG1 formulation, which is prone to fragmentation in heavy-chain CDR during storage, and screened excipients to control fragmentation based on the mechanism. In the degraded samples, fragments were generated from the cleavage around Tyr102, and a high level of oxidation was observed. Trp100 correlated most strongly with fragments among all the oxidized sites in multiple batches of mAb1 drug products. Oxidant-induced selective upregulation of mAb1 Trp100 oxidation level increased fragment level. This trend was rescued by the addition of Trp, indicating that Trp100 oxidation induced mAb1 fragmentation. A minor change in the mAb1 structure explored the potential mechanism of the regulatory relationship between Trp100 oxidation and fragmentation. Moreover, there was no obvious Trp100 oxidation or fragments in the mAb1 formulation without polysorbate 80 (PS80). Accordingly, substitution of PS80 with new surfactants, a combination of antioxidants, or EDTA with PS80, maintained mAb1 stability. This study demonstrated that mAb1 CDR fragmentation results from Trp100 oxidation and provides new excipients to resolve mAb fragmentation during formulation development.