Glycosylation involves enzymatic addition of a precursor oligosaccharide (or glycan) to certain sites in an expressed protein with known peptide motifs within a protein sequence. It is a complex process and can give rise to a wide range of different oligosaccharide structures that are unique to each protein.

The final structures are also dependent on, but not limited to, the cell line used to express the protein, expression time, cell culture conditions and post-harvest purification. The changes that can occur during these events can potentially affect the bioactivity, safety and efficacy of the protein therapeutic.

The oligosaccharide structures are classed as either high-mannose, hybrid or complex oligosaccharides, and when attached to the protein they form a product with a known peptide composition but multiple oligosaccharide structures. These differing final glycosylated molecules are termed glycoforms.

It is important to have an understanding of the structure of the glycans to reduce risks associated with patient safety (due to the formation of potentially allergenic structures), and loss of biological activity of the glycoprotein therapeutic (due to non-optimal glycosylation). Due to the complex nature of glycosylation and structural characterisation, different analytical approaches are required to fully assess the glycan or glycoprotein structure.

Glycosylation analytical strategies require multiple technologies, including selective enzymatic cleavage, mass spectrometry and a range of HPLC-based chromatographic methods (SEC, HILIC, HIAX and IEX). Typically the following analyses are recommended:

  • Identification of glycosylation sites through peptide mapping
  • Confirmation of peptide sequence
  • Determination of Glucose Unit (GU) values (HILIC)
  • Determination of glycan structure (neutral versus charged, percentage bi- tri and tetra-antennary)
  • Determination of glycan structure (enzyme arrays, identification of galactose residues, ESI-MS and ESI-MS/MS)
  • Total sugar (neutral monosaccharides: galactose, mannose, fucose, glucose, N-acetyl glucosamine and, N-acetyl galactosamine)
  • Determination of sialic acid residues, total and specific (N-acetyl neuraminic acid and N-glycolyl neuraminic acid)

RSSL can provide detailed glycosylation analysis in accordance with the relevant guidelines (EMA, FDA and ICH), and to GMP standards. Our analytical programs are designed to support early stage development, through to product characterisation and regulatory submission. The design of an analytical package is based on our expertise and experience, and to ensure the precise information required for different stages of product development, regulatory submission and final drug substance/product is obtained.

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