Measurement of free thiols in antibody therapeutics is important for product development and assessment of critical quality attributes. Earlier studies demonstrated fast separation of free thiol variants of IgG1 using reversed-phase high performance liquid chromatography (RP-HPLC) with diphenyl resin. Here, we report using N-tert-butylmaleimide (NtBM) alkylation followed by RP-HPLC and online mass spectrometry for rapid total and domain-specific free thiol characterization of IgG1. By increasing hydrophobicity, NtBM alkylation improves separation of free thiol variants from disulfide-linked main peak species. The unique mass shift by NtBM alkylation offers unambiguous characterization of free thiol variants by online mass spectrometry. Variant peaks separated by RP-HPLC were antibody molecules containing two NtBM-alkylated cysteines, corresponding to IgG1 containing two free thiols before alkylation. Further characterization of the collected fractions of variants by peptide mapping revealed that each variant contained unpaired cysteines located in specific IgG1 domains (CH1, CH3, VH and VL domains). Total molecular-level and domain-specific free thiol content measured by this method correlate well with orthogonal differential alkylation peptide mapping analysis, which measures free thiol level at individual cysteine residues. This method provides high throughput quantitation of total and domain-specific free thiol content in IgG1 molecules, facilitating rapid, comprehensive product and manufacturing process characterization.
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