Volume 23, Issue 16 2300172
RESEARCH ARTICLE

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

Corentin Beaumal

Corentin Beaumal

Laboratoire de Spectrométrie de Masse BioOrganique, IPHC UMR 7178, CNRS, Université de Strasbourg, Strasbourg, France

Infrastructure Nationale de Protéomique ProFI - FR2048, Strasbourg, France

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Alain Beck

Alain Beck

IRPF, Centre d'Immunologie Pierre-Fabre (CIPF), Saint-Julien-en-Genevois, France

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Oscar Hernandez-Alba

Oscar Hernandez-Alba

Laboratoire de Spectrométrie de Masse BioOrganique, IPHC UMR 7178, CNRS, Université de Strasbourg, Strasbourg, France

Infrastructure Nationale de Protéomique ProFI - FR2048, Strasbourg, France

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Christine Carapito

Corresponding Author

Christine Carapito

Laboratoire de Spectrométrie de Masse BioOrganique, IPHC UMR 7178, CNRS, Université de Strasbourg, Strasbourg, France

Infrastructure Nationale de Protéomique ProFI - FR2048, Strasbourg, France

Correspondence

Christine Carapito, Laboratoire de Spectrométrie de Masse BioOrganique, IPHC UMR 7178, CNRS, Université de Strasbourg, 25 rue Becquerel, F-67087 Strasbourg, France.

Email: [email protected]

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First published: 06 May 2023

Abstract

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.

CONFLICT OF INTEREST STATEMENT

The authors have declared no conflict of interest.

DATA AVAILABILITY STATEMENT

Additional supporting information may be found online in the Supporting Information section at the end of the article. The dataset was deposited with the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD039582 (for DIA data) and PXD039585 (for DDA data) [68].