Carvalho, Sofia B.Profit, LudivineKrishnan, SushmithaGomes, Ricardo A.Alexandre, Bruno M.Clavier, SeverineHoffman, MichaelBrower, KevinGomes-Alves, Patrícia2025-05-072025-05-072024-03-200168-1656PURE: 90453784PURE UUID: bac6b6ea-af05-4789-9324-c5f4852013f0Scopus: 85185552306PubMed: 38340900http://hdl.handle.net/10362/182734Funding Information: This work was supported by Sanofi. Funding Information: Authors acknowledge the support from the UniMS team, all MS data were generated by the UniMS–Mass Spectrometry Unit, iBET/ITQB, Oeiras, Portugal. The authors also acknowledge Miguel Antunes and Inês Isidro from iBET Data Science team for their contribution to data analysis. The authors acknowledge the experimental work from Emmanuel Fofie, Céline Hemet and Didier Duthé from Sanofi on chromatographic runs. The authors are grateful to Jason Walther for revising the manuscript and English proofreading. Publisher Copyright: © 2024 The AuthorsHost cell proteins (HCPs) are process-related impurities expressed by the host cells during biotherapeutics’ manufacturing, such as monoclonal antibodies (mAbs). Some challenging HCPs evade clearance during the downstream processing and can be co-purified with the molecule of interest, which may impact product stability, efficacy, and safety. Therefore, HCP content is a critical quality attribute to monitor and quantify across the bioprocess. Here we explored a mass spectrometry (MS)-based proteomics tool, the sequential window acquisition of all theoretical fragment-ion spectra (SWATH) strategy, as an orthogonal method to traditional ELISA. The SWATH workflow was applied for high-throughput individual HCP identification and quantification, supporting characterization of a mAb purification platform. The design space of HCP clearance of two polishing resins was evaluated through a design of experiment study. Absolute quantification of high-risk HCPs was achieved (reaching 1.8 and 4.2 ppm limits of quantification, for HCP A and B respectively) using HCP-specific synthetic heavy labeled peptide calibration curves. Profiling of other HCPs was also possible using an average calibration curve (using labeled peptides from different HCPs). The SWATH approach is a powerful tool for HCP assessment during bioprocess development enabling simultaneous monitoring and quantification of different individual HCPs and improving process understanding of their clearance.113568448engBioprocess developmentCHO HCP identificationhigh throughput HCP quantificationmAbsPurification platformSWATH-MSBiotechnologyBioengineeringApplied Microbiology and Biotechnologyjournal article10.1016/j.jbiotec.2024.02.001https://www.scopus.com/pages/publications/85185552306