Category: Formulation and Quality
Purpose: Protein oxidation is a critical quality attribute for many therapeutic proteins, including monoclonal antibodies. Oxidative stress can occur during routine manufacturing, processing, storage or use. Multiple methods are currently employed as stress tests to induce oxidative damage. In this study, we compared molecular modifications induced by two different oxidizers: Copper (II)/ascorbate (MCO) and 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH) in therapeutic monoclonal antibodies.
Methods: Therapeutic monoclonal antibodies rituximab, trastuzumab, obinutuzumab and ofatumumab were treated with either MCO or AAPH. Their stability and potency were tested at different time point during the treatment using biochemical assays. At the molecular level, we investigated methionine oxidation by LC/MS, carbonyl formation by ELISA and dihydroxyphenylalanine (DOPA) modifications. In case of higher-order structures, circular dichroism (CD), MFI, hydrophobicity, SDS-PAGE, and amyloid-like structure formations were analyzed. Tertiary structure was assessed using 4,4’-dianilino-1,1’-binaphthyl-5,5’-disulfonic acid (bis-ANS) to identify exposed and potentially solvent-accessible hydrophobic regions. We also tested ADCC activity in these mAbs.
Results: The results of LC/MS indicated that in trastuzumab and obinutuzumab, AAPH induced more methionine in Fc subunits than MCO did. Oxidative carbonyl formation which occurs on lysine, arginine, threonine and proline residues, showed that rituximab, trastuzumab and obinutuzumab were more sensitive towards MCO then AAPH. Preliminary analysis by SDS-PAGE revealed cross-linked species as well as fragmentation, resulting in substantially decreased native protein content after MCO treatment. AAPH decreased native protein levels after 6 hours. Analysis of secondary structure by CD suggested that MCO treatment for 24 hours substantially disrupted the alpha-helices and beta-sheets in IgG folds, whereas AAPH treatment showed minimal impact on secondary structural elements. Ofatumumab and rituximab suffered changes to secondary structure from MCO, while trastuzumab and obinutuzumab retained much of their folded state. Bis-ANS fluorescence increased continually during MCO treatment, with AAPH treatment again showing little structural impact even after 24 hours. The ADCC result indicated that MCO decreased ADCC activity in Trastuzumab as soon as 30 min after treatment, but not in the other mAbs.
Conclusion: These data demonstrate that different mAb respond uniquely to different oxidizing conditions in the presence or absence of trace metals. Orthogonal oxidative stress conditions might be informative when characterizing the stability and degradation profile of proteins.