Targeted mass spectrometry for absolute quantification of total human β‑nerve growth factor in the development of a bispecific therapeutic antibody.

Nerve growth factor (NGF) functions not only as a neurotrophic factor that supports neural survival and differentiation but also as a mediator and modulator of pain. Therapeutic agents that target circulating NGF have been developed for the treatment of pain. MEDI7352 is a bispecific antibody that simultaneously targets NGF and tumor necrosis factor. To support its clinical development and characterize target engagement, quantification of total NGF in human serum after administration of MEDI7352 is essential. Here we describe an ultrasensitive, consistent, and reliable high-throughput assay for the quantitation of total NGF in human serum, with a linear assay range of 10-1000 pg/mL. We used a non-competing polyclonal antibody for immuno-affinity (IA) capture to enrich NGF, followed by trypsin digestion and solid-phase extraction cleanup. The resulting tryptic peptide was quantified by microflow liquid chromatography-mass spectrometry (LC-MS). The assay was analytically validated and subsequently applied across multiple MEDI7352 clinical studies. Measurement of baseline serum NGF concentrations in patients with painful diabetic neuropathy (PDN) and osteoarthritis (OA) revealed statistically significant differences between the two cohorts. After MEDI7352 administration, the total NGF concentrations increased in a dose-dependent manner, concordant with pharmacokinetic (PK) exposure. Notably, the increase in total NGF and PK exposure was more pronounced in antidrug antibody (ADA) negative patients compared to ADA positive patients, indicating that total NGF biomarker measurements not only inform on target engagement of the therapeutic drug but also provide insights into the impact of immunogenicity. Overall, this novel IA-LC-MS hybrid approach enabled a sensitive bioanalytical assay for the characterization of soluble protein target and is readily generalizable to support other therapeutic programs in drug development or applications in other research areas.