High Sensitivity and Precision High-Temperature Reversed-Phase LC Analysis of Bevacizumab for Intact Bioanalysis of Therapeutic Monoclonal Antibodies

Abstract

We optimized several analytical conditions for more sensitive and precise HT-RPLC analysis of the therapeutic monoclonal antibody (mAb), bevacizumab. Specifically, we (1) optimized the sample preparation process to reduce adsorption and aggregation of bevacizumab, (2) introduced a sample concentration process using a centrifugal ultrafiltration unit to increase detection sensitivity, and (3) used another therapeutic mAb as an internal standard to improve analytical precision. The optimized method for bevacizumab analysis was shown to have low detection and quantification limits of 0.010 and 0.032 µg/mL, respectively, good correlation coefficients (r 2 > 0.9997), and good intra-and inter-day precisions within < 12.0 %. This study provides an important methodology for the intact bioanalysis of therapeutic mAbs, not merely their LC measurement.