PXD054948

PXD054948 is an original dataset announced via ProteomeXchange.

Title	Multi-level – Intact, Subunits, and Peptides – Characterization of Antibody-Based Therapeutics by a Single-Column LC-MS setup
Description	A comprehensive characterization of biotherapeutics, mandated by regulatory authorities, require analyses of a protein drug at multiple structure levels. Such multi-level characterization can be performed by mass spectrometry (MS), with established conventional MS-based assays of product quality attributes (PQAs) comprising intact protein and subunit middle-up MS with analytes resolved on a C4 column, and bottom-up peptide mapping with analytes resolved on C18 column. Recent advances in MS have facilitated the increasing use of middle-down analysis, expanding the qualitative analytical capability of MS for protein characterization. Recent studies using less retentive reversed-phase LC in bottom-up MS also offer an opportunity for streamlining equipment configuration to a single-column LC-MS setup for multi-level characterization of therapeutic pro-teins. In this study, we developed a robust middle-down LC-MS method on a ZenoTOF 7600, and evaluated a C4 LC-MS setup for the characterization of NISTmAb, RG7221 bispecific antibody (bsAb) and Fc-fusion etanercept by intact protein, subunit middle-up/down and bottom-up analyses. Successful multi-level characterization of the analytes using C4 LC-MS was demonstrated; notably, high sequence coverage and comprehensive PTMs profiling, including the mapping of all 13 O- and 3 N-glycosylation sites on etanercept in a single run, were obtained by bottom-up C4 LC-MS. This is also the first report on middle-down analysis of the major etanercept TNFR and Fc subunit glycoforms. A streamlined single-column LC-MS setup will enable more robust and efficient MS workflows for PQAs assessment and simplify the integration of LC-MS analyzer as a Process Analytical Technology (PAT) instrument for biopharma applications.
HostingRepository	PRIDE
AnnounceDate	2025-02-26
AnnouncementXML	Submission_2025-02-25_18:00:52.787.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Kia Ngee Low
SpeciesList	scientific name: Cricetulus griseus (Chinese hamster) (Cricetulus barabensis griseus); NCBI TaxID: 10029;
ModificationList	iodoacetamide derivatized residue
Instrument	ZenoTOF 7600

Revision	Datetime	Status	ChangeLog Entry
0	2024-08-15 22:41:02	ID requested
⏵ 1	2025-02-25 18:00:53	announced

10.1021/ACS.ANALCHEM.4C06350;

submitter keyword: subunits,Intact, LC-MS, EAD, C4-column, peptides

Xuezhi Bi
contact affiliation	Senior Principal Scientist I,Bioprocessing Technology Institute, A*Star, Singapore Adjunct Associate Professor, Duke-NUS Medical School, Singapore Adjunct Associate Professor,Singapore Institute of Technology, Singapore
contact email	bi_xuezhi@bti.a-star.edu.sg
lab head
Kia Ngee Low
contact affiliation	Bioprocessing Technology Institute research entity of Agency for Science, Technology and Research (A*STAR)
contact email	low_kia_ngee@bti.a-star.edu.sg
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/02/PXD054948

PRIDE project URI

• PRIDE
  1. PXD054948
     1. Label: PRIDE project
     2. Name: Multi-level – Intact, Subunits, and Peptides – Characterization of Antibody-Based Therapeutics by a Single-Column LC-MS setup