ABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/chemistry , Chromatography, High Pressure Liquid/methods , Chromatography, Reverse-Phase/methods , Recombinant Proteins/chemistry , Sulfhydryl Compounds/analysis , Antibodies, Monoclonal/analysis , Dithionitrobenzoic Acid/chemistry , Hydrophobic and Hydrophilic Interactions , Linear Models , Maleimides/chemistry , Recombinant Proteins/analysis , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
AIM:To set up a determination of tanshinone Ⅱ_A and cryptotanshinone in Compound Danshen Tablet(Radix Salviae Miltiorrhizae, Radix Notoginseng, Borneolum Syntheticum) by RP-HPLC. METHODS:Two components could be separated through YWG-C_~18 column with methanol-water (75∶25 by volume) as a mobile phase. The flow rate was 1.0 mL?min~-1 , and the detection wavelength was at 270 nm. RESULTS: The linear range of tanshinone Ⅱ_A and cryptotanshinone were 2.5-50.0 ?g?mL~-1 and 0.625-12.5 ?g?mL~-1 , respectively. The average recovery was 99.76% with RSD 0.92% for tanshinone Ⅱ_A and 98.34% with RSD 0.71% for cryptotanshinone, respectively. CONCLUSION: The method is simple, accurate and rapid with good reproducibility. It can be used for the quality control of Compound Danshen Tablet.
