Host cell protein quantification workflow using optimized standards combined with data-independent acquisition mass spectrometry

Monitoring of host cell proteins (HCPs) during the manufacturing of monoclonal antibodies (mAb) has become a critical requirement to provide effective and safe drug products. Enzyme-linked immunosor-bent assays are still the gold standard methods for the quantification of protein impurities. However, this technique has several limitations and does, among others, not enable the precise identification of pro-teins. In this context, mass spectrometry (MS) became an alternative and orthogonal method that de-livers qualitative and quantitative information on all identified HCPs. However, in order to be routinely implemented in biopharmaceutical companies, liquid chromatography-MS based methods still need to be standardized to provide highest sensitivity and robust and accurate quantification. Here, we present a promising MS-based analytical workflow coupling the use of an innovative quantification standard, the HCP Profiler solution, with a spectral library-based data-independent acquisition (DIA) method and strict data validation criteria. The performances of the HCP Profiler solution were compared to more con-ventional standard protein spikes and the DIA approach was benchmarked against a classical data -dependent acquisition on a series of samples produced at various stages of the manufacturing process. While we also explored spectral library-free DIA interpretation, the spectral library-based approach still showed highest accuracy and reproducibility (coefficients of variation < 10%) with a sensitivity down to the sub-ng/mg mAb level. Thus, this workflow is today mature to be used as a robust and straightforward method to support mAb manufacturing process developments and drug products quality control.(c) 2023 The Author(s). Published by Elsevier B.V. on behalf of Xi'an Jiaotong University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Automated summary

Monitoring host cell proteins (HCPs) during monoclonal antibody (mAb) manufacturing is crucial for ensuring the safety and efficacy of drug products. Mass spectrometry (MS) has emerged as an alternative technique for both qualitative and quantitative analysis of HCPs. Liquid chromatography-MS based methods still need to be standardized to provide accurate and sensitive quantification.