Multi-level – Intact, Subunits, and Peptides – Characterization of Antibody-Based Therapeutics by a Single-Column LC-MS setup

ABSTRACT: 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.

INSTRUMENT(S): ZenoTOF 7600

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

SUBMITTER: Kia Ngee Low  

LAB HEAD: Xuezhi Bi

PROVIDER: PXD054948 | Pride | 2025-02-26

REPOSITORIES: Pride