title: Generic Hybrid Ligand Binding Assay Liquid Chromatography High-Resolution Mass Spectrometry-Based Workflow for Multiplexed Human Immunoglobulin G1 Quantification at the Intact Protein Level: Application to Preclinical Pharmacokinetic Studies.
abstract: The quantitative analysis of human immunoglobulin G1 (hIgG1) by mass spectrometry is commonly performed using surrogate peptides after enzymatic digestion. Since some limitations are associated with this approach, a novel workflow is presented by hybridizing ligand binding assay (LBA) with liquid chromatography-high-resolution mass spectrometry (LC-HRMS) for hIgG1 quantification directly at the intact protein level. Different hIgG1s, including a [13C]-labeled version used as internal standard, were immuno-enriched from rat serum with a fully automated platform based on streptavidin coated tips and a biotinylated mouse anti-hIgG capture antibody targeting the fragment crystallizable region followed by overnight deglycosylation prior to LC-HRMS analysis. The proposed quantitative workflow utilized extracted ion chromatograms (XICs) from the nondeconvoluted full-scan MS spectrum. The assay was validated in terms of selectivity, sensitivity, accuracy/precision, carry-over, dilution linearity, and reproducibility. Consistent data between the conventional approach based on surrogate peptide analysis and our proposed workflow were obtained in vitro and in vivo with the advantage of a less extensive sample pretreatment. Multiplexing capabilities for simultaneous quantification of different hIgG1s within the same spiked sample were also exemplified. Altogether our results pave the way not only for the thorough application of intact hIgG1 quantification by LBA-LC-HRMS but also as a generic quantitative analytical method for other hIgG isotypes or next generation biotherapeutics.
authors.affiliation: Novartis Institutes for Biomedical Research , Drug Metabolism and Pharmacokinetics, Novartis Campus, 4056 Basel, Switzerland.|Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg , CNRS, IPHC UMR 7178, 67000 Strasbourg, France.
authors.forename: Christian
authors.identifier: 0000-0002-2405-0227
authors.initials: C
authors.lastname: Lanshoeft
authors.affiliation: Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg , CNRS, IPHC UMR 7178, 67000 Strasbourg, France.
authors.forename: Sarah
authors.initials: S
authors.lastname: Cianférani
authors.affiliation: Novartis Institutes for Biomedical Research , Drug Metabolism and Pharmacokinetics, Novartis Campus, 4056 Basel, Switzerland.
authors.forename: Olivier
authors.initials: O
authors.lastname: Heudi
mesh_terms: D000818:Animals; D019921:Biotinylation; D002851:Chromatography, High Pressure Liquid; D004353:Drug Evaluation, Preclinical; D005260:Female; D006207:Half-Life; D006801:Humans; D007118:Immunoassay; D007074:Immunoglobulin G / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D008024:Ligands; D010455:Peptides / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
chemical_list: D007074:Immunoglobulin G; D008024:Ligands; D010455:Peptides