Glythera Limited is a biotechnology company specializing in the development of next generation biotherapeutics, through the application of their advanced proprietary linker and stable glycan technologies: PermaLink™ and PermaCarb™.
Glythera required glycosylation analysis for their ADC product to provide functionally important post-translational modification information and to ensure that the chemistry used to attach the drug did not impact glycosylation. To perform the carbohydrate population analysis this study, BioPharmaSpec used Mass spectrometric (MS) and Liquid Chromatographic (LC) based methods.
The following analyses were performed by BioPharmaSpec:
- Sample reduction/alkylation/protease digestion
- Release of N-linked glycans with PNGase F
- Reverse-phase purification and separation and collection of the void fraction for:
- 2-AB labelling followed by LC separation and fluorescence detection with UPLC
- Permethylation and Matrix-assisted-laser-desorption (MALDI) mapping using MALDI TOF/TOF mass spectrometry
Sialic acids are usually linked to the 3 or 6 position of the terminal Galactose. Full structural analysis is achieved by confirmation of expected mammalian biosynthetic pathway products using MALDI TOF/TOF MS/MS, followed by linkage analysis of the permethylated samples as the permethyl alditol acetates, using GC-MS. Finally, the stereospecificity of linkages may be confirmed using specific enzymes and MS analysis of the digest products.
“The results from this study were crucial to the plans for our PermaLink technology. We needed to partner with a CRO with specific glycosylation expertise and BioPharmaSpec fulfilled this requirement for us” – Senior Development Scientist, Glythera