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.

Development of Novel Separation and Analytical Methods for Optically Active Substances and Therapeutic Monoclonal Antibodies Based on Liquid Chromatography

“…Other mAbs (bevacizumab, rituximab, and nivolumab) exhibited good linearity with R 2 ≥0.996, and the mean concentration factors were similar to those obtained for trastuzumab. Additionally, we reported (1) the sensitive detection of bevacizumab by HT-RPLC separation followed by postcolumn fluorescence derivatization using o-phathalaldehyde and 2-mercaptoethanol [53] and (2) HT-RPLC separation of heavy and light chain fragments of therapeutic mAbs and antibody-drug conjugate produced by chemical reduction [54].…”

Sensitive Method for LC Analysis of Therapeutic Monoclonal Antibodies Using a Centrifugal Filtration Device with Adsorption Suppression Treatment

“…However, the column was too short to separate therapeutic mAbs or human endogenous IgG. High-temperature reversed-phase LC [10,11,15,16], which we have already reported, can be applied here as it allows simultaneous analysis of multiple antibody drugs and bioanalysis. Table 1 shows the peak heights and concentration factors before and after centrifugal concentration of the trastuzumab solutions.…”

Fluorescence Bioanalysis of Bevacizumab Using Pre-Column and Post-Column Derivatization – Liquid Chromatography After Immunoaffinity Magnetic Purification

“…In this method, target drugs in blood samples are purified using immunoaffinity magnetic beads immobilized with anti-idiotype mAbs. The purified drugs are separated further using HT-RPLC [17][18][19][20], which is suitable for excellent separation among IgGs, using a large pore-size octyl column. The separated drugs are detected sensitively with their own fluorescence.…”