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.

Immunoaffinity LC–MS/MS for Quantitative Determination of A Free and Total Protein Target as A Target Engagement Biomarker

“…Therefore, a highly sensitive method with a LLOQ of 1 pM was required to enable measurement of the magnitude and duration of free IP-10 suppression and to differentiate anti-IP-10 mAb drug candidates. In this work, IA-LC-MS/MS assays, which combine the advantages of both immunocapture and LC-MS/MS platforms for specifically isolating and detecting proteins of interest [15][16][17][18][19][20][21][22][23], have been developed. The strategies for maximizing sensitivity by selecting appropriate digestion routes and surrogate peptides, and optimizing the immunocapture procedure to achieve high recovery and minimal matrix effect will be detailed in this paper.…”

Current Mass Spectrometric Tools for the Bioanalyses of Therapeutic Monoclonal Antibodies and Antibody-Drug Conjugates

“…42 However, to maintain the efficiency of tryptic digestion, either an extended peptide (flanking peptide, winged peptide), to which a digestion site sequence is attached, may be used, at both ends of the SIL-peptide, 43 or a combination with SIL-peptide 44 may be used. In pharmaceutical companies and others, it is becoming a common practice to use SIL-protein, prepared from cells grown in a medium containing SIL-labeled amino acids, as IS.…”

Internal Standards for Absolute Quantification of Large Molecules (Proteins) from Biological Matrices by LC-MS/MS

“…Variation in digestion efficiency can be accounted for with the use of an extended SIL-peptide which has cleavable groups flanking either side of side of a SIL-peptide [29,64,65]. Generally, the cleavable groups consist of three to six amino acids residues from the original protein sequence at both the N-and C-terminus [29,65,66].…”