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 utilised 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 significant improvements in protein identifications in complex sample matrices while simultaneously reducing analysis times. Here, we utilise the Orbitrap Astral MS to perform HCP analysis on 37 protein biotherapeutics. The use of a short 60 samples-per-day separation method, dramatically reduced analysis time without sacrificing HCP identifications. 544 HCPs were detected across the cohort and 63.60% of those found to be quantifiable in at least one product using Hi3 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). Over 80% of quantifiable HCPs were detected at concentrations below 10 ng/mg, ~9% below concentrations of 1 ng/mg. These included HCPs considered as “high-risk” by the Biophorum Development Group. This study shows how the Orbitrap Astral MS can improve detection of low-level HCPs while significantly reducing analysis times, allowing for rapid and more informed understanding of a products HCP content.