Host Cell Protein Quantification Workflow Using Optimized Standards combined with Data-Independent Acquisition Mass Spectrometry

ABSTRACT: Monitoring of host cell proteins (HCPs) during the manufacture of monoclonal antibodies (mAb) has become a critical requirement to provide effective and safe drug product. ELISA assays are still the current gold standard for the quantification of protein impurities. However, this technique has several limitations and does, among others, not enable the precise identification of proteins. In this context, mass spectrometry (MS) has emerged as an alternative and orthogonal method that delivers qualitative and quantitative information on all identified HCPs. However, in order to be routinely implemented in biopharmaceutical companies, liquid chromatography (LC)-MS based methods still need to be standardized to provide highest sensitivity and robust and accurate quantification. In this study, we developed 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 performance of the HCP Profiler solution was compared to more conventional standard protein spikes and the DIA approach was benchmarked against classical data-dependent acquisition (DDA) using samples collected at various stages of the manufacturing process. Finally, we further explored the possibility to use spectral library-free DIA. As a result, our method showed accurate and reproducible (coefficients of variation (CVs) < 10%) quantification of HCPs while reaching a sensitivity down to the sub-ng/mg mAb level.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Cricetulus Griseus (chinese Hamster) (cricetulus Barabensis Griseus)

TISSUE(S): Cell Culture

SUBMITTER: Steve Hessmann  

LAB HEAD: Christine Carapito

PROVIDER: PXD029305 | Pride | 2023-07-20

REPOSITORIES: pride