The addition of a carbohydrate moiety to a protein molecule is referred to as protein glycosylation. 

Protein glycosylation helps in proper folding of proteins, stability and in cell to cell adhesion commonly needed by cells of the immune system. There are two main types of glycosilations: N-glycosilation and O-glycosilation. In N-glycosilation the glycan residue is linked by an amine bond to an asparagine (N), except when is followed by proline. O-glycosilations occur on serine (S) or Threonine (T) residues. Of these two forms of glycosylation, N-glycosilation of proteins is the most commonly found.

Based on ICH Q6B guidelines, a complete study of the glycosylated protein or monoclonal antibody is required, where is it essential a specific characterization including:

1) A glycoforms profile.

2) The different glycans that can occupy the same glycosylation site in different copies of a protein, a phenomenon themed “microheterogeneity or glycosylation site analysis”.

3) The degree of glycosylation at each glycosylation site or also known as “glycosylation site occupancy”, and 4) The N-linked or O-linked glycans profile.

AMSbiopharma scientists have implemented a complete strategy for a complete structural characterization of your glycosylated biopharmaceutical at three different levels: Intact protein, glycopeptides and released glycans.

Intact glycoprotein profiling (glycoforms profile): Intact glycoproteins can be directly analyzed for general glycan patterns. Electrospray ionization and high-resolution mass spectrometry (ESI-TOFMS) coupled with reversed phase HPLC is the most common method for intact glycoprotein analysis which enables the precise mass determination and characterization of intact glycoproteins. The ability of ESI-TOFMS to place multiple charges enables accurate determination of highly complex glycoprofile at the intact protein level.

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Glycosylation site profiling:  Glycopeptides analysis links the glycosilation information to exact location (glycosilation site) and the degree of glycosilation (glycosilation occupancy). 

Photos courtesy of SCIEX (

Glycan analysis: N-glycans are enzymatically released and the derivatized to be analyzed by HPLC-MS. There are a number of derivatization methods available for HPLC-MS analysis of glycans, each of which imparts different properties that affect both glycan retention on HPLC columns and MS analyses. Derivatizing reagents as 2-aminobenzamide, procainamide, reduction with permethylation for N-glycan analysis and rapid commercial kits. Released glycan identification is carried out by comparison against available database.

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