119th General Meeting of the KCS

Type Oral Presentation
Area Oral Presentation of Young Analytical Chemists Ⅰ
Room No. 303호
Time THU 10:12-:
Code ANAL1.O-29
Subject High-throughput Screening of Native Glycans on Therapeutic Glycoproteins using Liquid Handling System
Authors 박경미, 안현주*
충남대학교 분석과학기술대학원, Korea
Abstract Glycosylation of biotherapeutics is critically associated with the stability, biological activity, half-life, and safety of drug. The glycosylation could be changed by production environment such as host cell system and culture conditions. Therefore, monitoring the glycosylation at each stage of manufacture process is a suitable approach to assess production quality for drugs. Automative high-throughput tools are currently developed and innovated to treat a large number of samples in a short time span and get high quality data by minimization of hands-on time. Here we have developed the automated analytical platform to enrich and fractionate native glycans using liquid handling system combined with 96-well microplate for a high-throughput manner. Human IgG used as a motif of therapeutic mAbs, a well known glycoprotein, was chosen to develop sample preparation procedure. Overall processes including protein denaturation by heating block, N-glycan release by PNGase F, and glycan purification and enrichment by solid phase extraction have been streamlined and optimized. Glycans were profiled by nanoLC chip/Q-TOF MS. We found total 24 glycans consisting of complex/hybrid type N-glycans w/wo sialic acid residues in both automated and manual treatment. The CVs of normalized peak intensities of top 10 N-glycans were less than 8.2 %, which indicates high comparability between two methods. Moreover, we validated the quantitative reproducibility for 5 different well positions in the 96-well plate. Two independent replicates experiments were performed per day. The Pearson correlation coefficient R between all samples was determined to be above 0.9. Glycan preparation using the automated platform showed to be rapid, reproducible, and reliable and it can be applied for real therapeutic glycoproteins.
E-mail pkm2432@gmail.com