Facile quantitation of free thiols in a recombinant monoclonal antibody by reversed-phase high performance liquid chromatography with hydrophobicity-tailored thiol derivatization

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 1092; pp. 158 - 167
• Main Authors: Welch, Leslie, Dong, Xiao, Hewitt, Daniel, Irwin, Michelle, McCarty, Luke, Tsai, Christina, Baginski, Tomasz
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15-08-2018
• Subjects: Derivatization; Ellman's reagent; Free thiol; N-tert-butylmaleimide; Recombinant monoclonal antibody; Reversed-phase high performance chromatography; N-tert-butylmaleimide; PV; IgG; NPM; Derivatization; UF/DF; QC; NtBM; Free thiol; Recombinant monoclonal antibody; ESI; UHPLC; R2; UV; RSD; DTNB; TOF; LOQ; mAb; RP-HPLC; LC-MS; NEM; Gdn-HCl; Ellman's reagent; Reversed-phase high performance chromatography
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2018.05.039

Free thiol content, and its consistency, is one of the product quality attributes of interest during technical development of manufactured recombinant monoclonal antibodies (mAbs). We describe a new, mid/high-throughput reversed-phase-high performance liquid chromatography (RP-HPLC) method coupled with derivatization of free thiols, for the determination of total free thiol content in an E. coli-expressed therapeutic monovalent monoclonal antibody mAb1. Initial selection of the derivatization reagent used an hydrophobicity-tailored approach. Maleimide-based thiol-reactive reagents with varying degrees of hydrophobicity were assessed to identify and select one that provided adequate chromatographic resolution and robust quantitation of free thiol-containing mAb1 forms. The method relies on covalent derivatization of free thiols in denatured mAb1 with N-tert-butylmaleimide (NtBM) label, followed by RP-HPLC separation with UV-based quantitation of native (disulfide containing) and labeled (free thiol containing) forms. The method demonstrated good specificity, precision, linearity, accuracy and robustness. Accuracy of the method, for samples with a wide range of free thiol content, was demonstrated using admixtures as well as by comparison to an orthogonal LC-MS peptide mapping method with isotope tagging of free thiols. The developed method has a facile workflow which fits well into both R&D characterization and quality control (QC) testing environments. The hydrophobicity-tailored approach to the selection of free thiol derivatization reagent is easily applied to the rapid development of free thiol quantitation methods for full-length recombinant antibodies.
•Novel and facile total free thiol quantitation method for intact mAbs•RP-HPLC with N-tert-butylmaleimide tagging of free thiols•An hydrophobicity-tailored approach to free thiol derivatization reagent selection•Analytical method performance and robustness suitable for R&D and QC