A single liquid chromatography–quadrupole time‐of‐flight mass spectrometric method for the quantification of total antibody, antibody‐conjugated drug and free payload of antibody–drug conjugates

Abstract

A single hybrid affinity-captured-LC-TOF-MS/MS method was developed and applied for the quantification of total antibody, antibody conjugated drug and free payload of antibody drug conjugate (ADC). Adcetris®, a valine-citrulline monomethyl auristatin E conjugated ADC, was used as a model ADC compound. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 30.65-613.00 ng/mL with an equation y = ax + bx + c for the antibody-conjugated drug of Adcetris®. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control samples. For the analysis of total antibody, a signature peptide (TTPPVLDSDGSFFLYSK, molecular weight 1874) was used after affinity capture using magnetic beads and on-bead trypsin digestion. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 5.00-100.00 μg/mL with an equation y = ax + bx + c for total antibody. For free payload analysis of monomethyl auristatin E, a protein precipitation method followed by LC-TOF-MS/MS analysis was used. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 1.01-2200 ng/mL with an equation y = ax + bx + c for free payload. Pharmacokinetic study samples and in vitro stability samples in rat were successfully analyzed by this a hybrid affinity-captured-LC-TOF-MS/MS method. This single platform method is a useful complementary method for the pharmacokinetics study of ADC with valine-citrulline linker at the early drug discovery stage.

Quantification for Antibody-Conjugated Drug in Trastuzumab Emtansine and Application to In Vitro Linker Stability and In Vivo Pharmacokinetic Study in Rat Using an Immuno-Affinity Capture Liquid Chromatography-Mass Spectrometric Method

“…Each 24 µL of STD and QC plasma sample was mixed with 350 µL of 0.1% tween 20 in PBS and 30 µL of protein A magnetic bead. In addition, 20 µL of all study samples were also mixed with 354 µL of 0.1% tween 20 in PBS and 30 µL protein A magnetic bead (https://www.merckmillipore.com/KR/ko/product/PureProteome-Protein-A-Magnetic-Bead-System,MM_NF-LSKMAGA10#anchor_BRO (accessed on 1 September 2021)) [8]. After vortexing, the mixture samples were incubated gently for about 2 h at room temperature and then the samples were fixed on a magnetic rack and washed with 200 µL of 0.1% tween 20 treated PBS and 200 µL of PBS.…”

“…Antibody-drug conjugate (ADC) is a promising biopharmaceutical consisting of three components: a monoclonal antibody, a linker, and cytotoxic drugs (payloads) [1][2][3][4][5][6][7][8][9]. ADCs undergo receptor-mediated endocytosis after reaching target cells through a specific antigen-antibody response.…”

Cutting-edge multi-level analytical and structural characterization of antibody-drug conjugates: present and future

“…Bioanalytical methods are rapidly being developed to quantitatively monitor the transformation of ADCs in various in vitro or in vivo biological matrices such as serum/ plasma [190] and tumor tissues [191]. Indeed, a crucial property of conjugates is their stability in biofluids as the release over time of cytotoxic drugs into the bloodstream constitutes a considerable health threat [192].…”

Assessments of the In Vitro and In Vivo Linker Stability and Catabolic Fate for the Ortho Hydroxy-Protected Aryl Sulfate Linker by Immuno-Affinity Capture Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometric Assay

“…The antibody-drug conjugate (ADC) is a targeted anti-cancer agent consisting of a monoclonal antibody, linkers, and cytotoxic drugs (payload) [1][2][3][4][5][6]. The major characteristics of ADCs for anti-cancer therapy are that it can improve the therapeutic window by delivering cytotoxic drugs selectively to target cells [7][8][9].…”

“…Using this approach, it is possible to quantify total antibody (tAb) through the determination of a signature peptide and also to perform peptide mapping analysis. In the case of ADC with cleavable linkers, it is also possible to quantify antibody-conjugated drug (acDrug) by the usage of linker-specific enzyme such as cathepsin B, papain and beta-galactosidase [4,5].…”