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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.
Anal Chem; 90(1): 866-871, 2018 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-29206445
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.
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