Dataset Information

Targeted Quantitation of Host Cell Proteins by Capillary LC/IMS/MS/MS in Combination with Rapid Digestion on Immobilized Trypsin Column Under Native Conditions

ABSTRACT: Host cell protein (HCP) impurities are considered a critical quality attribute of biopharmaceuticals because of their potential to compromise safety and efficacy, and LC/MS-based analytical methods have been developed to identify and quantify individual proteins instead of employing enzyme-linked immunosorbent assay to assess total HCP levels. Native digestion enablesenables highly sensitive detection of HCPs, but requires overnight incubation to generate peptides, limiting the throughput of sample preparation. In this study, we developed an approach employing native digestion on a trypsin-immobilized column to improve the sensitivity and throughput. We examined suitable databases for the identification of HCPs derived from Chinese hamster ovary (CHO) cells and selected RefSeq's Chinese Hamster as the optimal database. Then, we investigated methods to identify HCPs with greater efficiency than that of denatured in-solution digestion. Native in-column digestion not only reduced the digestion time from overnight to 10 minutes, but also increased the number of quantified HCPs from 154 to 226. In addition to this rapid digestion methodology, we developed high-throughput LC/MS/MS with a monolithic silica column and　parallel reaction monitoring-parallel accumulation-serial fragmentation. The optimized system was validated with synthetic peptides derived from high-risk HCPs, confirming excellent linearity, precision, accuracy, and low LOD and LOQ (1-3 ppm). The optimized digestion and analysis method enabled high-throughput quantification of HCPs, and is expected to be useful for quality control and characterization of HCPs in biopharmaceuticals.

ORGANISM(S): Cricetulus Griseus

SUBMITTER: Yasushi Ishihama

PROVIDER: PXD053135 | JPOST Repository | Fri May 02 00:00:00 BST 2025

REPOSITORIES: jPOST

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Dataset's files

Source:

			Action	DRS
	AMED%20MAB%20denatured%20in%20column%20digestion%20(dimethyl-heavy),%20synthetic%20peptides%20(dimethyl-light).zip	Other
	AMED%20MAB%20native%20in%20column%20digestion%20(dimethyl-heavy),%20synthetic%20peptides%20(dimethyl-light).zip	Other
	AMED%20MAB,%20denatured%20in%20column%20digestion.zip	Other
	AMED%20MAB,%20denatured%20in%20solution%20digestion.zip	Other
	AMED%20MAB,%20native%20in%20column%20digestion%20trypsin%20poor.zip	Other

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Publications

Quantitation of Host Cell Proteins by Capillary LC/IMS/MS/MS in Combination with Rapid Digestion on Immobilized Trypsin Column Under Native Conditions.

Tomioka Ryota R Ogata Kosuke K Ishihama Yasushi Y

Mass spectrometry (Tokyo, Japan) 20240914 1

Host cell protein (HCP) impurities are considered a critical quality attribute of biopharmaceuticals because of their potential to compromise safety and efficacy, and LC/MS-based analytical methods have been developed to identify and quantify individual proteins instead of employing enzyme-linked immunosorbent assay to assess total HCP levels. Native digestion enables highly sensitive detection of HCPs but requires overnight incubation to generate peptides, limiting the throughput of sample prep ...[more]

PMID: 39296308

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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.