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Quantitation of a Therapeutic Antibody in Serum Using Intact Sequential Affinity Capture, Trypsin Digestion, and LC-MS/MS

Vasicek, Lisa A., Spellman, Daniel S., Hsieh, SuChun, Seghezzi, Wolfgang, Zhang, Shuli, Santostefano, Michael, Bateman, Kevin P.
Analytical chemistry 2018 v.90 no.1 pp. 866-871
blood serum, digestion, drugs, monoclonal antibodies, therapeutics, trypsin
Large molecule quantitation by LC-MS/MS commonly relies on bottom-up or so-called surrogate peptide measurements to infer the whole-molecule concentration. This can lead to questions about what is actually being measured in the assay (intact drug and/or other drug related material). An intact sequential affinity capture (ISAC) assay was developed utilizing two different immunoaffinity (IA) reagents. The reagents were selective for the heavy and light chain of a monoclonal antibody, which when used consecutively, ensures that only the intact form of the antibody is represented by the surrogate peptide. The approach provided comparable results to a traditional sandwich IA assay indicating similar capture populations. The use of an initial ISAC assessment of affinity capture purification, should add a degree of confidence in the use of a single IA-LC-MS/MS quantitation assay.