Dataset Information

A Sensitive, Rapid, Robust and Reproducible Workflow for Host Cell Protein Profiling in Biopharmaceutical Process Development

ABSTRACT: There is a growing industry and regulatory need to detect host cell protein (HCP) impurities in the production of protein biopharmaceuticals, as certain HCPs can impact product stability, safety, and efficacy, even at low levels.1-6 In some cases, regulatory agencies require the identification as well as the quantification of HCPs in drug products for risk assessment, and this is an active and growing topic of conversation in industry and amongst regulators. In this study, we developed a sensitive, robust, and reproducible workflow for HCP detection and quantification in the significantly shorter turnaround time than previously reported using an Evosep ONE LC system coupled to an Orbitrap Fusion Lumos mass spectrometer. Due to its fast turnaround time, this HCP workflow can be integrated into process development for the high-throughput (60 samples analyzed per day) identification of HCPs. The ability to rapidly measure HCPs and follow their clearance throughout the downstream process can be used to pinpoint sources of HCP contamination, which can be used to optimize biopharmaceutical production to minimize HCP levels. Analysis of the NIST monoclonal antibody (mAb) reference material using the rapid HCP profiling workflow detected the largest number of HCPs reported to date, underscoring an improvement in performance along with an increased throughput. The HCP workflow can be readily implemented and adapted for different purposes to guide biopharmaceutical process development and enable better risk assessment of HCPs in drug substances (DS) and drug products (DP).

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Jiao Ma

LAB HEAD: Greg Kilby

PROVIDER: PXD018427 | Pride | 2020-06-02

REPOSITORIES: Pride

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Publications

Sensitive, Rapid, Robust, and Reproducible Workflow for Host Cell Protein Profiling in Biopharmaceutical Process Development.

Ma Jiao J Kilby Greg W GW

Journal of proteome research 20200605 8

There is a growing industry and regulatory need to detect host cell protein (HCP) impurities in the production of protein biopharmaceuticals, as certain HCPs can impact product stability, safety, and efficacy, even at low levels. In some cases, regulatory agencies require the identification and the quantification of HCPs in drug products (DPs) for risk assessment, and this is an active and growing topic of conversation in the industry and amongst regulators. In this study, we developed a sensiti ...[more]

PMID: 32449362

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Project description:Host cell proteins (HCPs) are process-related impurities that are generated by the host organism and are typically present at low levels in recombinant biopharmaceutical products, such as therapeutic antibodies. While overall HCP levels are usually monitored by enzyme-linked immunosorbent assay (ELISA), liquid chromatography coupled to mass spectrometry (LC-MS) is emerging as a powerful tool that can provide both qualitative and quantitative information about HCP levels during purification process development. However, a major challenge for LC-MS-based methods is that there can be a more than 5 orders of magnitude difference in the concentration between HCPs and therapeutic antibody in solution, which precludes the effective identification of low abundance HCPs in antibody product. This work reports a simple and powerful strategy to identify HCPs in antibody drug substance by applying molecular weight cut-off (MWCO) filtration step followed by shotgun proteomic analysis. After dissociating the interaction between HCPs and antibody with an anionic detergent, the depletion of antibody from HCPs can be easily achieved with the MWCO filtration step. Using this method, we observed that the dynamic range across proteins in the HCP samples was significantly decreased by 1,000-fold. In addition, by spiking in known amounts of HCPs to purified antibody drug substance with low levels of HCPs, we demonstrated that our method could detect HCP at a concentration as low as 1 ppm. When applying this methodology to the study of HCPs in NIST monoclonal antibody (NISTmAb), more than 150 HCPs were confidently identified, which doubles the number of identified HCPs that have been previously reported. Parallel reaction monitoring (PRM) results confirmed that the novel HCPs found using this method were present in very low abundance (0.01-8 ppm), highlighting that our method reduces the dynamic range by removing antibody interference and improving the sensitivity of HCP identification and quantification.

Dataset Information

A Sensitive, Rapid, Robust and Reproducible Workflow for Host Cell Protein Profiling in Biopharmaceutical Process Development

Publications

Sensitive, Rapid, Robust, and Reproducible Workflow for Host Cell Protein Profiling in Biopharmaceutical Process Development.

Similar Datasets

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