Comparative evaluation of chelators in the context of analysis of antibody oxidative stability

Oxidation is a critical post-translational modification for antibodies that naturally possess many solvent-exposed amino acid residues. Presence of oxidized methionine and tryptophan residues may potentially impact safety, efficacy, clearance, and immunogenicity of antibodies, emphasizing the importance of including oxidation assays as a part of the antibody control strategy. Sub-unit Reversed Phase HPLC is a commonly employed method to measure oxidation, wherein enzymatic digestion of antibodies followed by disulfide reduction is essential for sample preparation. Peaks resulting from intact sub-units i.e. light chain (LC), single-chain crystallizable fragment (Fc), and N-terminal half of heavy chain (Fd) and additional peaks arising due to subunit oxidation can be easily resolved to quantify oxidative degradation. Our data suggest that the performance of the method can be compromised due to sample oxidation during the analysis window imposing restrictions on sample throughput and increasing method variability. Drug substance batches of six monoclonal antibodies with variable subclasses and isoelectric points showed an increase of 5-50 % in subunit oxidation levels when tested over 48 hours. With an aim to minimize oxidation during analysis we modified the sample preparation technique using a variety of metal chelators including Ethylenediaminetetraacetic acid (EDTA), Ethylene Glycol-bis (β-aminoethyl ether)-N,N,N',N'-Tetraacetic Acid (EGTA), Diethylene Triamine Pentaacetic Acid (DTPA), Nitrilotriacetic acid (NTA) and Hydroxyethyl Ethylenediamine Triacetic Acid (HEDTA). EGTA showed the most prevalent stabilizing influence on subunit oxidation for all drug substance batches screened. Overall, our results demonstrate that careful optimization of sample preparation during oxidation analysis can increase sample throughput significantly and highlight the feasibility of using EGTA as a more suitable alternative to EDTA, the most employed trace-metal chelator in the biopharmaceutical space.