Quantitative Host Cell Protein Analysis across Innovator Monoclonal Antibody-Based Protein Therapeutics using the Orbitrap Astral Mass Spectrometer

Protein biotherapeutics such as monoclonal antibodies (mAbs) are generally produced using cell-based production systems that generate endogenous proteins, commonly referred to as host cell proteins (HCPs), along with the desired drug products. A critical challenge is the removal of HCPs from the final drug product as they can be detrimental to product efficacy and patient safety, even at low levels. HCP clearance is monitored during process optimisation and assessed before product release. Tandem liquid chromatography-mass spectrometry (LC-MS/MS) is increasingly utilized as an orthogonal method to enzyme-linked immunosorbent assays (ELISA) for HCP monitoring during downstream processing due to its untargeted nature and ability to determine total HCP concentrations. The dynamic range of the recombinant proteins makes detection of low-level HCPs difficult. However, the recently developed Orbitrap Astral MS has the potential to overcome such challenges, having previously demonstrated 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 a cohort of 37 protein biotherapeutics. Its speed and sensitivity enable the use of a short 60 samples-per-day (SPD; 24-minute injection to injection) separation method, dramatically reducing analysis time compared to standard LC-MS methods 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. Over 80% of quantifiable HCPs were detected at concentrations below 10 ppm, including approximately 9% below concentrations of 1 ppm. These included HCPs considered as “high-risk” by the Biophorum Development Group. Excluding an outlier, on average 20 HCPs were identified per product. Overall, this study shows how the Orbitrap Astral MS can improve detection of low-level HCPs while significantly reducing analysis times, allowing for a rapid and more informed understanding of a products HCP content.