Isotyping and Semi-Quantitation of Monkey Anti-Drug Antibodies by Immunocapture Liquid Chromatography-Mass Spectrometry

Abstract

There is an urgent demand to develop new technologies to characterize immunogenicity to biotherapeutics. Here, we developed an immunocapture LC-MS assay to isotype and semi-quantify monkey anti-drug antibodies (ADAs) to fully human monoclonal antibody (mAb) drugs. ADAs were isolated from serum samples using an immunocapture step with the Fab of the full-length mAb cross-linked to magnetic beads to minimize matrix interference. A positive monoclonal antibody control against the human immunoglobulin kappa light chain was used as a calibration standard for ADA quantitation. The final LC-MS method contains 17 multiple reaction monitoring (MRM) transitions and an optimized 15-min LC method. The results suggested that IgG1 was the most abundant isotype in ADA-positive samples. IgG2 and IgG4 were identified at lower levels, whereas IgG3 and IgA levels were only observed at very minor levels. In addition, levels of total ADA measured by the LC-MS assay were comparable to results obtained using a traditional ligand binding assay (LBA). The LC-MS ADA assay enabled rapid immunogenicity assessment with additional isotype information that LBAs cannot provide.

Approaches for the detection and analysis of antidrug antibodies to biopharmaceuticals: A review

“…A similar scheme has been evaluated for use in the analysis of antibodies against biotherapeutics [117]. A related strategy for direct ADA analysis has used an immunocapture step that is based on the F ab region of a mAb that is coated on a magnetic bead [118]. This technique has been used with LC‐MS for the isotyping and semiquantitation of ADAs against fully‐humanized mAb‐based drugs in monkey serum [118].…”

Low pKa of Lys promotes glycation at one complementarity-determining region of a bispecific antibody

“…mAb-1 was purified from the collected monkey serum samples by affinity purification (24,25). In brief, a biotinylated anti-human antibody was conjugated to Dynabeads MyOne Streptavidin T1 magnetic beads (Invitrogen, Carlsbad, CA) at room temperature for 10 min.…”

Current Analytical Strategies for Antibody–Drug Conjugates in Biomatrices

“…Meanwhile, an LBA-LC–MS/MS approach for immunogenicity testing has been explored in the past few years [ 9 , 19 , 20 ]. The LBA-LC–MS/MS assay is capable of providing relative concentrations of ADA isotypes in the samples without use of a tired assay strategy and setup of cut-points.…”