Quantitative Host Cell Protein Analysis of Antibody-Based Protein Therapeutics Using the Orbitrap Astral Mass Spectrometer.

Host cell proteins (HCPs) are endogenous proteins generated in cellular production systems alongside the biotherapeutic of interest. Removal of HCPs is crucial as they can be detrimental to product efficacy and patient safety. Due to its ability to determine individual HCP concentrations, liquid chromatography tandem mass spectrometry is increasingly utilized as an orthogonal method to ELISA for HCP monitoring. For protein biotherapeutics like monoclonal antibodies, their dynamic range makes detection of low-level HCPs difficult. The Orbitrap Astral MS has the potential to overcome such challenges, offering improvements in protein identifications in complex sample matrices while simultaneously reducing analysis times. Here, we utilize the Orbitrap Astral MS to perform HCP analysis on 36 protein biotherapeutics. Our workflow used a short 60 samples-per-day separation method and was initially benchmarked against four previously published studies, demonstrating comparable levels of HCP identifications. 236 HCPs were detected across the cohort and 55% of those found to be quantifiable in at least one product using label free quantitation. Functional analysis revealed that most detected HCPs had functions related to catalysis or binding, predominately catalytic activity (46%, 97 gene IDs) or protein binding (44%, 91 gene IDs). Nearly 80% of quantifiable HCPs were detected at concentrations below 10 ng/mg, with 8% detected below concentrations of 1 ng/mg. These included HCPs considered as "high-risk" by the Biophorum Development Group. This study shows how new generation mass spectrometry instruments can enable detection of low-level HCPs while allowing for a rapid and more informed understanding of a product's HCP content.