Development and Application of a Robust N-Glycan Profiling Method for Heightened Characterization of Monoclonal Antibodies and Related Glycoproteins.

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 J Pharm Sci 103:1967-1978, 2014 [ABSTRACT FROM AUTHOR]