期刊
JOURNAL OF PHARMACEUTICAL SCIENCES
卷 103, 期 7, 页码 1967-1978出版社
WILEY-BLACKWELL
DOI: 10.1002/jps.24004
关键词
biopharmaceuticals characterization; HPLC; protein structure; LC-MS; liquid chromatography; mass spectrometry; monoclonal antibody; glycosylation
资金
- Pfizer BioTherapeutics Pharmaceutical Sciences organization
A highly robust hydrophilic interaction liquid chromatography (HILIC) method that involves both fluorescence and mass spectrometric detection was developed for profiling and characterizing enzymatically released and 2-aminobenzamide (2-AB)-derivatized mAb N-glycans. Online HILIC/mass spectrometry (MS) with a quadrupole time-of-flight mass spectrometer provides accurate mass identifications of the separated, 2-AB-labeled N-glycans. The method features a high-resolution, low-shedding HILIC column with acetonitrile and water-based mobile phases containing trifluoroacetic acid (TFA) as a modifier. This column and solvent system ensures the combination of robust chromatographic performance and full compatibility and sensitivity with online MS in addition to the baseline separation of all typical mAb N-glycans. The use of TFA provided distinct advantages over conventional ammonium formate as a mobile phase additive, such as, optimal elution order for sialylated N-glycans, reproducible chromatographic profiles, and matching total ion current chromatograms, as well as minimal signal splitting, analyte adduction, and fragmentation during HILIC/MS, maximizing sensitivity for trace-level species. The robustness and selectivity of HILIC for N-glycan analyses allowed for method qualification. The method is suitable for bioprocess development activities, heightened characterization, and clinical drug substance release. Application of this HILIC/MS method to the detailed characterization of a marketed therapeutic mAb, Rituxan (R), is described. (c) 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:1967-1978, 2014
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