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

ABSTRACT: 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.

INSTRUMENT(S):

ORGANISM(S): Cricetulus Griseus (chinese Hamster) (cricetulus Barabensis Griseus) Mus Musculus (mouse)

SUBMITTER: Josh Smith

LAB HEAD: Dr. Jonathan Bones

PROVIDER: PXD067004 | Pride | 2026-04-20

REPOSITORIES: Pride

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			Action	DRS
	20230406_OLMP1_NIBRT_HCPs_EyleaB3Mar_500ng_CHO_Uniprot_All.mzML	Mzml
	20230406_OLMP1_NIBRT_HCPs_EyleaB3Mar_500ng_CHO_Uniprot_All.mzid	Mzid
	20230406_OLMP1_NIBRT_HCPs_EyleaB3Mar_Rep1_500ng_01.raw	Raw
	20230406_OLMP1_NIBRT_HCPs_EyleaB3Mar_Rep1_500ng_02.raw	Raw
	20230406_OLMP1_NIBRT_HCPs_EyleaB3Mar_Rep2_500ng_01.raw	Raw

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Project description:Host cell proteins (HCPs) are process-related impurities that are generated by the host organism and are typically present at low levels in recombinant biopharmaceutical products, such as therapeutic antibodies. While overall HCP levels are usually monitored by enzyme-linked immunosorbent assay (ELISA), liquid chromatography coupled to mass spectrometry (LC-MS) is emerging as a powerful tool that can provide both qualitative and quantitative information about HCP levels during purification process development. However, a major challenge for LC-MS-based methods is that there can be a more than 5 orders of magnitude difference in the concentration between HCPs and therapeutic antibody in solution, which precludes the effective identification of low abundance HCPs in antibody product. This work reports a simple and powerful strategy to identify HCPs in antibody drug substance by applying molecular weight cut-off (MWCO) filtration step followed by shotgun proteomic analysis. After dissociating the interaction between HCPs and antibody with an anionic detergent, the depletion of antibody from HCPs can be easily achieved with the MWCO filtration step. Using this method, we observed that the dynamic range across proteins in the HCP samples was significantly decreased by 1,000-fold. In addition, by spiking in known amounts of HCPs to purified antibody drug substance with low levels of HCPs, we demonstrated that our method could detect HCP at a concentration as low as 1 ppm. When applying this methodology to the study of HCPs in NIST monoclonal antibody (NISTmAb), more than 150 HCPs were confidently identified, which doubles the number of identified HCPs that have been previously reported. Parallel reaction monitoring (PRM) results confirmed that the novel HCPs found using this method were present in very low abundance (0.01-8 ppm), highlighting that our method reduces the dynamic range by removing antibody interference and improving the sensitivity of HCP identification and quantification.

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

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