Physicochemical and biological impact of metal-catalyzed oxidation of IgG1 monoclonal antibodies and antibody-drug conjugates via reactive oxygen species

Abstract

Biotherapeutics are exposed to common transition metal ions such as Cu(II) and Fe(II) during manufacturing processes and storage. IgG1 biotherapeutics are vulnerable to reactive oxygen species (ROS) generated via the metal-catalyzed oxidation reactions. Exposure to these metal ions can lead to potential changes to structure and function, ultimately influencing efficacy, potency, and potential immunogenicity of the molecules. Here, we stress four biotherapeutics of the IgG1 subclass (trastuzumab, trastuzumab emtansine, anti-NaPi2b, and anti-NaPi2b-vc-MMAE) with two common pharmaceutically relevant metal-induced oxidizing systems, Cu(II)/ ascorbic acid and Fe(II)/ H 2 O 2 , and evaluated oxidation, size distribution, carbonylation, Fc effector functions, antibody-dependent cellular cytotoxicity (ADCC) activity, cell anti-proliferation and autophaghic flux. Our study demonstrates that the extent of oxidation was metal ion-dependent and site-specific, leading to decreased FcγRIIIa and FcRn receptor binding and subsequently potentially reduced bioactivity, though antigen binding was not affected to a great extent. In general, the monoclonal antibody (mAb) and corresponding antibody-drug conjugate (ADC) showed similar impacts to product quality when exposed to the same metal ion, either Cu(II) or Fe(II). Our study clearly demonstrates that transition metal ion binding to therapeutic IgG1 mAbs and ADCs is not random and that oxidation products show unique structural and functional ramifications. A critical outcome from this study is our highlighting of key process parameters, route of degradation, especially oxidation (metal catalyzed or via ROS), on the CH1 and Fc region of full-length mAbs and ADCs. Abbreviations : DNPH 2,4-dinitrophenylhydrazine; ADC Antibody drug conjugate; ADCC Antibody-dependent cellular cytotoxicity; CDR Complementary determining region; DTT Dithiothreitol; HMWF high molecular weight form; LC-MS Liquid chromatography–mass spectrometry; LMWF low molecular weight forms; MOA Mechanism of action; MCO Metal-catalyzed oxidation; MetO Methionine sulfoxide; mAbs Monoclonal antibodies; MyBPC Myosin binding protein C; ROS Reactive oxygen species; SEC Size exclusion chromatography

Visible Light Induces Site-Specific Oxidative Heavy Chain Fragmentation of a Monoclonal Antibody (IgG1) Mediated by an Iron(III)-Containing Histidine Buffer

“…We note that Glover et al recently showed a less pronounced effect of Fe­(II) compared to Cu­(II) during metal-catalyzed oxidation of several mAbs and mAb-derived conjugates . They did not identify specific binding sites for Fe­(II), but this observation does not exclude the binding of Fe­(III).…”

“…29,48 Figure 11B shows the crystal structure of a mononuclear citrate/Fe(III) complex determined by Matzapetakis et al 29 We note that Glover et al recently showed a less pronounced effect of Fe(II) compared to Cu(II) during metal-catalyzed oxidation of several mAbs and mAb-derived conjugates. 49 They did not identify specific binding sites for Fe(II), but this observation does not exclude the binding of Fe(III). The experimental test of Glover et al relied on the site-specific formation of reactive oxygen species, especially hydroxyl radicals, via the Fenton reaction of Fe(II) with hydrogen peroxide.…”

A Review of Protein- and Peptide-Based Chemical Conjugates: Past, Present, and Future

“…In addition, polymerization conditions must be carefully optimized to be compatible with proteins, which require the use of aqueous solvents and low temperatures; as a result, achieving a balance between mild polymerization conditions and low polymer dispersity is often challenging. Finally, ATRP uses transition metal catalysts such as copper, which is a potential concern as metal ions can bind to proteins and trigger chemical degradation, including oxidation and fragmentation [ 264 , 265 ].…”

Correlated analytical and functional evaluation of higher order structure perturbations from oxidation of NISTmAb

“… 16 As the level of MetO increases, the resulting SPR data can provide a readout in terms of reduced binding affinity. 16 , 17 Recently, a rapid SPR assay was developed to quickly detect Met oxidation in both the antigen-binding fragment (Fab) and Fc domains. 18 Using a model therapeutic IgG1κ mAb, NISTmAb, 19 , 20 specific molecular probes were used to assess the binding to one region in the Fc (Protein A) and two regions in the Fab (Protein L, F peptide).…”