PXD053135

PXD053135 is an original dataset announced via ProteomeXchange.

Title	Targeted Quantitation of Host Cell Proteins by Capillary LC/IMS/MS/MS in Combination with Rapid Digestion on Immobilized Trypsin Column Under Native Conditions
Description	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.
HostingRepository	jPOST
AnnounceDate	2025-05-02
AnnouncementXML	Submission_2025-05-02_02:22:29.054.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Ryota Tomioka
SpeciesList	scientific name: Cricetulus griseus; NCBI TaxID: 10029;
ModificationList	alpha-amino acetylated residue; L-methionine sulfoxide; S-carboxamidomethyl-L-cysteine
Instrument	timsTOF Pro

Revision	Datetime	Status	ChangeLog Entry
0	2024-06-14 21:21:21	ID requested
⏵ 1	2025-05-02 02:22:29	announced

Tomioka R, Ogata K, Ishihama Y, Quantitation of Host Cell Proteins by Capillary LC/IMS/MS/MS in Combination with Rapid Digestion on Immobilized Trypsin Column Under Native Conditions. Mass Spectrom (Tokyo), 13(1):A0152(2024) [pubmed]

submitter keyword: mAb, HCP, prm-PASEF

Yasushi Ishihama
lab head
Ryota Tomioka
contact affiliation	Kyoto university
dataset submitter

jPOST dataset URI

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