Bioanalysis of bevacizumab and infliximab by high-temperature reversed-phase liquid chromatography with fluorescence detection after immunoaffinity magnetic purification

Abstract

This study presents two simple and rapid methods for the quantification of therapeutic mAbs based on LC. Two mAbs (bevacizumab and infliximab) in plasma samples were purified using magnetic beads immobilized with a commercially-available idiotype antibody for each mAb. Purified mAbs were separated with HT-RPLC and detected with their native fluorescence. Using immunoaffinity beads, each mAb was selectively purified and detected as a single peak in the chromatogram. The HT-RPLC achieved good separation for the mAbs with sharp peaks within 20 min. The calibration curves of the two mAbs ranged from 1 to 20 μg mL−1 (bevacizumab) and 1–10 μg mL−1 (infliximab), and they had strong correlation coefficients (r2 > 0.998). The LOD of bevacizumab and infliximab was 0.07 and 0.15 μg mL−1, and the LLOQ of bevacizumab and infliximab was 0.12 and 0.25 μg mL−1, respectively. Thus, the sensitivities were sufficient for clinical analysis. Immunoaffinity purification with HT-RPLC produced a selective and accurate bioanalysis without an LC-MS/MS instrument. Both methods could become general-purpose analytical methods and complement the results obtained with conventional LBA.