Development of Immunocapture-LC/MS Assay for Simultaneous ADA Isotyping and Semiquantitation

Therapeutic proteins and peptides have potential to elicit immune responses resulting in anti-drug antibodies that can pose problems for both patient safety and product efficacy. during drug development immunogenicity is usually examined by risk-based approach along with specific strategies for developing fit-for-purpose bioanalytical approaches. enzyme-linked immunosorbent assays and electrochemiluminescence immunoassays are the most widely used platform for ada detection due to their high sensitivity and throughput. during the past decade,lc/ms has emerged as a promising technology for quantitation of biotherapeutics and protein biomarkers in biological matrices,mainly owing to its high specificity,selectivity,multiplexing,and wide dynamic range. in fully taking these advantages,we describe here an immunocapture-lc/ms methodology for simultaneous isotyping and semiquantitation of ada in human plasma. briefly,ada and/or drug-ada complex is captured by biotinylated drug or anti-drug ab,immobilized on streptavidin magnetic beads,and separated from human plasma by a magnet. ada is then released from the beads and subjected to trypsin digestion followed by lc/ms detection of specific universal peptides for each ada isotype. the lc/ms data are analyzed using cut-point and calibration curve. the proof-of-concept of this methodology is demonstrated by detecting preexisting ada in human plasma. © 2016 lin-zhi chen et al.