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Development of an automated, interference-free, immunoaffinity-based mass spectrometric assay for quantification a therapeutic monoclonal antibody in human sera

Krantz, Carsten and Warren, Andrew Paul and Miess, Christian (2018) Development of an automated, interference-free, immunoaffinity-based mass spectrometric assay for quantification a therapeutic monoclonal antibody in human sera. Bioanalysis, 10 (13). pp. 1023-1037. ISSN 1757-6180

Abstract

Hybrid ligand binding-mass spectrometry assays are increasingly being applied to quantitative measurement of proteins in biological matrices. These assays combine the high specificity and enrichment capabilities of selective affinity reagents with the unmatched specificity and unequivocal analyte identification provided by mass spectrometry. Often these assays involve initial analyte enrichment at the protein level. Although these assays can work well, they are limited by the lack of appropriate well-characterized affinity reagents and, as with some immunoassays, may suffer from interferences caused by off-target protein binding, autoantibodies and anti-immunoglobulin antibodies. To address such problems, the method known as stable isotope standards and capture by anti-peptide antibodies (SISCAPA) was developed, which involves the use of well characterized, high affinity antibodies to enrich proteotypic peptide surrogates of the target protein followed by their identification and quantitation by mass spectrometry. The SISCAPA method involves tryptic digestion of the proteins in the sample matrix which destroys unwanted antibodies and other interfering proteins. Here we report the development of an automated, SISCAPA-based two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) assay for measurement of total levels a monoclonal antibody currently in clinical development. An affinity purified polyclonal antibody specific for a peptide in the complementarity determining region (CDR) of the antibody was used to enrich the surrogate peptide from trypsin-digested human plasma, followed by 2D-LC-MS/MS quantitation. The assay was compared to a standard ligand binding assay currently being used for the antibody quantitation. The data indicate that the peptide-based SISCAPA-2D-LC-MS/MS assay is a valuable alternative to specific protein quantification assays devoid of target or binding protein interference.

Item Type: Article
Date Deposited: 21 Aug 2018 00:45
Last Modified: 21 Aug 2018 00:45
URI: https://oak.novartis.com/id/eprint/34726

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