Journal of Pharmaceutical Sciences 2014-07-01

Development and application of a robust N-glycan profiling method for heightened characterization of monoclonal antibodies and related glycoproteins.

Tanya Q Shang, Andrew Saati, Kelly N Toler, Jianming Mo, Heyi Li, Tonya Matlosz, Xi Lin, Jennifer Schenk, Chee-Keng Ng, Toni Duffy, Thomas J Porter, Jason C Rouse

文献索引：J. Pharm. Sci. 103(7) , 1967-78, (2014)

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摘要

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(®), is described.© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.