Scientists at The Hospital for Sick Children are developing a multivalent protein comprised of 24 identical protomers, where each protomer is a 50kDa protein with two sites of N-linked glycosylation.
BioPharmaSpec provided support for the glycan analysis of several batches of the product produced in different cell lines. The glycan characterization included:
BioPharmaSpec scientists successfully characterized the glycans present on the product and provided comparability data between different cell lines.
Technical considerations- Glycan analysis
With a focused interest in the N-linked glycans present on the samples, the Projects consisted of:
Monosaccharide composition analysis
Following hydrolysis of the samples, BioPharmaSpec used Gas Chromatography with Mass Spectrometric detection (GC-MS) to analyze TMS derivatized neutral and amino monosaccharides and Liquid Chromatography of DMB-labelled sugars for sialic acid analysis.
The analyses allow detection and quantitation of the monosaccharides of interest e. Fucose, Mannose, Galactose and N-Acetylglucosamine in one experiment and sialic acids (N-Acetylneuraminic acid and N-Glycolylneuraminic acid) in a second experiment.
Oligosaccharide population analysis
HILIC analysis of PNGase F released and RapiFluor labeled N-linked Glycans with Fluorescence detection was carried out incorporating on-line Mass Spectrometric-peak identification.
Separately, to provide orthogonal data for some data sets, permethylated samples of the PNGase F released oligosaccharides were examined by MALDI TOF analysis on a SCIEX 5800 instrument to produce a Glycomic Map.
“We were extremely impressed with BioPharmaSpec’s capabilities and the quality of the data generated to characterize and enable the development of our complex protein.” – The Hospital for Sick Children