Category: Chemical Biology
The glycans or polysaccharides attached to proteins after protein post-translation modification play critical roles in eukaryotic cell protein functions, such as protein assembling and folding stability, signal transduction, ligand binding, protein interaction, etc. In the therapeutic immunoglobulin, the N-glycosylation on amide nitrogen of asparagine is a critical quality attribute in the pharmacology, affecting immunogenicity, pharmacokinetics and pharmacodynamics. Glycan sample preparation (deglycosylation and dye labeling) is a key step for glycan analysis.
We have developed a fully automated platform and workflow for N-glycan sample preparation using Thermo Fisher GlycanAssure reagents. The magnetic beads-based, automated procedure provides streamline workflow for glycoprotein denaturation and deglycosylation, APTS labeling of the released glycans and cleaning of the excess free dyes. The hand-on time for 13 samples is as short as ~5 minutes because the operators only need to add their glycoprotein to micro-tubes before they start the automated run. The setup of the instrument is simple and easy as all of the reagents are prefilled in the prefilled cartridges, which are stable at -20C for at least one year. The total sample preparation time on the instrument is 1 hour and 45 min and the APTS labeled glycans are ready to be analyzed on UPLC and CE instruments. The fully automated platform is demonstrated to result in high quality of glycan profiles analyzed on both UPLC and CE. The glycan profiles of NIST mAb, human serum IgG, RNase B and Fetuin are comparable to the GlycanAssure manual kits, as well as comparable to other glycan sample preparation kits in the market.
Nan Liu– R&D scientist, Thermo Fisher, Livermore, California
I am an R&D scientist at Pharma Analytics group, BioProduction Division of Thermo Fisher Scientific. I obtained Ph.D in Genetics and Molecular Biology at University of St. Andrews, UK. I worked at Lawrence Livermore National Laboratory for 13 years and have publications in PNAS, Molecular Cell and Science, etc. I joined Applied Biosystems (now part of Thermo Fisher) in 2007 and have been leading the product development for Residual DNA quantitation kit, Residual host cell protein quantitation kit, and the fully automated glycan sample preparation kit, etc.