title: Sequential protein and peptide immunoaffinity capture for mass spectrometry-based quantification of total human β-nerve growth factor.
abstract: Nerve growth factor (NGF) is a neurotrophin that is implicated in the modulation of pain perception. Tanezumab, a humanized monoclonal antibody (mAb) specific for NGF, is highly potent in sequestering NGF and has demonstrated efficacy for treatment of chronic pain in clinical trials. We describe a novel, sensitive immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for quantitative determination of human serum NGF levels at baseline and after tanezumab treatment. The assay combines magnetic bead-based NGF immunoaffinity enrichment using a non-neutralizing polyclonal antibody followed by digestion and quantitation of a NGF-derived tryptic peptide via high-flow peptide immunoaffinity enrichment and nanoflow LC-MS/MS. Following validation, the assay was employed to measure total NGF concentrations in samples from clinical studies. The assay had a <10% interassay relative error and <15% interassay coefficient of variation across a range from 7.03 to 450 pg/mL human NGF. Generally, human basal serum NGF concentrations were between 20 and 30 pg/mL which, upon treatment with tanezumab, elevated in a dose-dependent manner into the high pg/mL to low ng/mL range. This is the first report of clinical trial implementation of a MS-based assay that uses sequential protein and peptide immunoaffinity capture for protein target quantitation. The use of robotic sample preparation and a robust chromatography configuration enabled this technology to advance into the routine clinical analysis and now provides a bioanalytical platform for the development of similar assays for other protein targets.
authors.affiliation: Pfizer Inc., 1 Burtt Road, Andover, Massachusetts 01810, United States. hendrik.neubert@pfizer.com
authors.forename: Hendrik
authors.initials: H
authors.lastname: Neubert
authors.forename: David
authors.initials: D
authors.lastname: Muirhead
authors.forename: Musarat
authors.initials: M
authors.lastname: Kabir
authors.forename: Chris
authors.initials: C
authors.lastname: Grace
authors.forename: Adriaan
authors.initials: A
authors.lastname: Cleton
authors.forename: Rosalin
authors.initials: R
authors.lastname: Arends
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D004285:Dogs; D006801:Humans; D007118:Immunoassay / Q000379:methods; D008252:Macaca fascicularis; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D020932:Nerve Growth Factor / Q000032:analysis* / Q000235:genetics; D010446:Peptide Fragments / Q000032:analysis* / Q000235:genetics; D051381:Rats; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: C502240:NGF protein, human; D010446:Peptide Fragments; D020932:Nerve Growth Factor