Advanced mass spectrometry workflows for accurate quantification of trace‐level host cell proteins in drug products: Benefits of FAIMS separation and gas‐phase fractionation DIA

Therapeutic monoclonal antibodies (mAb) production relies on multiple purification steps before release as a drug product (DP). A few host cell proteins (HCPs) may co‐purify with the mAb. Their monitoring is crucial due to the considerable risk they represent for mAb stability, integrity, and effica...

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Bibliographic Details
Published in:Proteomics (Weinheim) 2023-08, Vol.23 (16), p.e2300172-n/a
Main Authors: Beaumal, Corentin, Beck, Alain, Hernandez‐Alba, Oscar, Carapito, Christine
Format: Article
Language:English
Subjects:
Antibodies, Monoclonal - chemistry
Antibodies, Monoclonal - metabolism
Chemical Sciences
Chromatography, Liquid - methods
data‐independent acquisition mass spectrometry (dia‐ms)
drug product (dp)
Enzyme-linked immunosorbent assay
Fractionation
high‐field asymmetric ion mobility spectrometry (faims)
host cell protein (hcp) impurities
Immunoassays
Immunogenicity
Ion Mobility Spectrometry
Ionic mobility
Ions
Life Sciences
Liquid chromatography
Mass spectrometry
Mass spectroscopy
Monitoring
Monoclonal antibodies
Proteins
Scientific imaging
Separation
Tandem Mass Spectrometry
Vapor phases
Workflow
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Summary:Therapeutic monoclonal antibodies (mAb) production relies on multiple purification steps before release as a drug product (DP). A few host cell proteins (HCPs) may co‐purify with the mAb. Their monitoring is crucial due to the considerable risk they represent for mAb stability, integrity, and efficacy and their potential immunogenicity. Enzyme‐linked immunosorbent assays (ELISA) commonly used for global HCP monitoring present limitations in terms of identification and quantification of individual HCPs. Therefore, liquid chromatography tandem mass spectrometry (LC‐MS/MS) has emerged as a promising alternative. Challenging DP samples show an extreme dynamic range requiring high performing methods to detect and reliably quantify trace‐level HCPs. Here, we investigated the benefits of adding high‐field asymmetric ion mobility spectrometry (FAIMS) separation and gas phase fractionation (GPF) prior to data independent acquisition (DIA). FAIMS LC‐MS/MS analysis allowed the identification of 221 HCPs among which 158 were reliably quantified for a global amount of 880 ng/mg of NIST mAb Reference Material. Our methods have also been successfully applied to two FDA/EMA approved DPs and allowed digging deeper into the HCP landscape with the identification and quantification of a few tens of HCPs with sensitivity down to the sub‐ng/mg of mAb level.
ISSN:1615-9853
1615-9861
DOI:10.1002/pmic.202300172