Impact of Multiple Freeze Thaw Cycles on Degradation of Trastuzumab and Rituximab in Different Formulations

Freeze-thaw operations during manufacturing, storage, and distribution are known to significantly impact the physical and chemical stability of mAbs. Most of the investigations thus far have focused on aggregation arising from freeze-thaw, while other possible chemical degradation pathways like oxidation have not been explored much. The present study aims to evaluate the impact of repeated freeze-thaw on the oxidation of two mAbs (trastuzumab and rituximab) in different buffer systems (formulation buffer, phosphate-buffer saline and histidine buffer). The two mAbs were selected as model mAbs in histidine-containing and histidine-free formulations. Aggregation and charge variants were also estimated in this study to understand the trend in agreement with current literature on freeze-thaw stress. The two mAbs were exposed to repeated freezing (-20 ᵒC) and thawing (5 ᵒC) cycles daily (28 days) and weekly (4 weeks). An increase in oxidation up to 3X is observed for trastuzumab samples in the formulation buffer, while the increase is less (1X oxidation) for rituximab samples. Oxidation of the two mAbs in histidine buffer alone was also assessed, and a significant increase of 7X and 8X oxidation was observed in trastuzumab and rituximab, respectively. The presence of oxidized and charged species was also confirmed with intact mass and peptide mapping analysis. The results highlight the increased mAb oxidations in histidine-polysorbate 20 buffer compared to the citrate-polysorbate 80 formulation buffer upon repeated freeze-thaw. Maximum formation of high molecular weight species (15.94%) was observed in the phosphate-buffer saline samples of trastuzumab. An increase in acidic (19.54–23.22%) and basic variants (5.23–9.44%) was observed for rituximab, whereas a decrease in acidic variants (32.60–26.63%) was observed for trastuzumab samples. In view of the results, we can surmise that manufacturers need to investigate and accordingly design freeze-thaw procedures for storage of their in-process samples, drug substance, or drug product.