120th General Meeting of the KCS

Type Oral Presentation
Area Oral Presentation of Young Analytical Chemists I
Room No. Room C311+C312
Time THU 10:47-:
Code ANAL1.O-44
Subject Analytical Platforms Employing LC-MS for Glycosylation Assessment of Therapeutic Glycoprotein
Authors Nayoung Yun, Myung Jin Oh, Hyun Joo An*
Graduate School of Analytical Science and Technology, Chungnam National University, Korea
Abstract Glycosylation of a therapeutic protein can significantly affect drug’s stability, immunogenicity, PK/PD, and bioactivity. Therefore, glycomic analyses are necessary to assess biotherapeutic quality and biosimilar similarity. Current glycome analyses involve the use of complementary methods for assessing specific glycosylation attributes including glycan composition and structure, abundance, and glycosylation site with microheterogeneity. Here, we demonstrated a stepwise approach using MS platform for glycomic characterization, namely glycan analysis by glycomic approach, site-specific analysis using glycoproteomic approach, and intact protein analysis. Two representative therapeutic glycoproteins, interferon-beta-1a (IFN-β1a) and trastuzumab, were used to compare the performance (results) of different analytical platforms. Interferon-β-1a (IFN-β1a) possessing single N-glycosylation site was used for this study. IFN-β1a and trastuzumab were prepared and analyzed using three independent analytical platforms. First, in intact protein analysis, the standards were desalted using 10K MWCO filters. Without further purification or enrichment IFN-β1a was directly injected onto the nano LC C8 chip/Q-TOF MS. For glycopeptide approach, the standards were digested with pronase E and enriched and fractionated according to size and polarity by graphitized carbon solid phase extraction (GCC-SPE). In parallel, N-glycans were released from therapeutic proteins by PNGase F and then selectively enriched by GCC-SPE. The enriched glycopeptides and glycans were chromatographically separated and analyzed by nano LC PGC chip/Q-TOF MS, respectively. Regardless of analytical platforms, glycosylation profiling in both qualitative and quantitative analysis showed high correlation (R=0.91~0.99). Each paltform showed different strengths and weaknesses, providing different levels of information on glycosylation. The results would be used as the reference for stepwise glycomic characterization during the development & production stage of biotherapeutics.
E-mail yny9339@gmail.com