Stress Temperature Studies in Small Scale Hastelloy® Drug Substance Containers Lead to Increased Extent of and Increased Variability in Antibody-Drug Conjugate and Monoclonal Antibody Aggregation: Evidence for Novel Oxidation-Induced Crosslinking in Monoclonal Antibodies.

Health authorities require that suitable stability of the drug substance be shown in relevant materials of construction. ICH Q1A(R2) explicitly states that "stability studies should be conducted on drug substance packaged in a container closure system that is the same as or simulates the packaging proposed for storage and distribution". Stainless steel containers are commonly used for the long-term storage of frozen bulk drug substances (DSs). Hastelloy®-based metal containers are sometimes used due to their higher corrosion resistance and significantly lower iron content to mitigate the potential corrosion-related risks associated with high salt formulations. Despite their benefits, we have found that elevated temperature stability studies in small scale Hastelloy® containers can lead to degradation that is not representative of degradation under typical storage conditions relevant to the manufacturing process. We provide evidence for an oxidation-induced aggregation mechanism that is based on Fenton chemistry with peroxide being supplied by the autoxidation of polysorbate at stress temperatures. Further, variation in the rates of iron leaching between individual small scale containers is shown to be the cause of the variable rates of degradation through strong correlations between leached iron levels and the extents of oxidation and aggregation. The addition of a metal chelator or the removal of polysorbate from the formulation mitigates the oxidation and the non-representative behavior. Extended characterization by LC-MS and O labeled peptide mapping shows that a significant portion of the aggregate formed under these conditions is covalently crosslinked and that the predominant covalent species is either a dityrosine or tyrosine-tryptophan crosslink between an Fc peptide and a Fab peptide. This report is the first time either of these two crosslinks have been reported for antibodies with detailed analytical characterization. Because the behavior observed in these studies is not representative of degradation under typical storage conditions relevant to the manufacturing process, this study demonstrates that small scale stress studies in metal containers should be performed with caution and that extended incubation times can lead to non-representative degradation mechanisms.