High-concentration L-methionine as a potent antioxidant for oxidation resistance and stability enhancement in high-concentration antibody therapeutics

Abstract

The development of high-concentration monoclonal antibody (mAb) formulations for subcutaneous administration is faces critical stability challenges, particularly oxidation and aggregation, which compromise efficacy and safety. While antioxidants are commonly employed, existing studies predominantly focus on low concentrations, leaving the potential of high-concentration antioxidants underexplored. Here, we present the first systematic evaluation of high-concentration L-methionine (L-Met,>20 mM) as a novel antioxidant to address these limitations. Through accelerated stability testing coupled with multi-dimensional analytical techniques, we demonstrated that L-Met at concentrations exceeding 20 mM surpasses conventional antioxidants in mitigating oxidation and aggregation. Synergy with 200 mM trehalose further enhanced stability by reducing oxidative degradation and inhibiting protein aggregation. Comprehensive biophysical analyses confirmed no adverse effects, with some aspects showing improved outcomes in structural integrity, colloidal stability, and thermal behavior. The optimized formulation (25 mM L-Met +200 mM trehalose) also exhibited robust protection against light-induced degradation and broad applicability across therapeutic antibodies. This work pioneers a high-concentration antioxidant strategy, addressing a critical gap in mAb formulation science and offering a translatable solution for stabilizing next-generation high-concentration biologics.