High-Temperature Reversed-Phase LC Separation of Heavy and Light Chain Fragments of Therapeutic Monoclonal Antibodies and Antibody-Drug Conjugate Produced by Chemical Reduction

Abstract

To construct liquid chromatography (LC)-based bioanalytical method for therapeutic monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs), twelve commercially available therapeutic mAbs and one ADC were chemically reduced, and the generated fragments were analyzed by high-temperature reversed-phase LC. For most therapeutic mAbs, single peaks of light and heavy chains were detected, indicating a possibility of homogeneous LC analysis using light chains. However, characteristic fragmentations were observed in infliximab, pembrolizumab, ramucirumab, and trastuzumab emtansine. We also performed a simple validation using the fragmented light chains for the bioanalysis of bevacizumab. The limit of detection (LOD) and limit of quantification (LOQ) of bevacizumab were 0.63 and 2.10 µg/mL, respectively, with dithiothreitol reduction, and 0.74 and 2.48 µg/mL, respectively, with tris (2-carboxyethyl) phosphine reduction. These results indicate that both the reductants confer sufficient linearity, LOQ, and LOD for the light chain analysis of bevacizumab. Thus, this method, combined with affinity purification, can be used for the bioanalysis of bevacizumab.