Heavily Glycosylated Proteins

Heavily Glycosylated Proteins

Heavily glycosylated proteins

Glycosylations are one of the most frequent post-translational modifications. For example, at least 50% of human proteins are glycosylated with some estimates being as high as 70%. These glycoproteins contain complex carbohydrates whose presence or absence has a great impact on the efficacy, stability and activity of proteins.

There are two main types of glycosylations: N-glycosylation and O-glycosylation. In N-glycosylation, the carbohydrate residue is linked by an amine bond to an asparagine (N), except when is followed by proline. O-glycosylations occur on serine (S) or Threonine (T) residues. Of these two forms of glycosylation, N-glycosylation of proteins is the most commonly found.

When proteins have a high amount of glycosylation, they are themed heavily glycosylated proteins and as required in the ICH Q6B guidelines, these glycoproteins require specific characterization: 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 glycosilation site or also known as “glycosilation site occupancy”, and 4) The N-linked or O-linked glycans profile.

Taking advantage of the extensive knowledge of our scientist in mass spectrometry, AMSbiopharma offers a strategy for a complete structural characterization of your pharmaceutical at three different levels: Intact protein, glycopeptides and released glycans.

Analysis
Methodology
Level
Intact glycoprotein profiling
(glycoforms profile)
High performance liquid chromatography coupled with high resolution mass spectrometry (HPLC-TOFMS)
Intact glycoprotein
Glycosilation site analysis
Glycosilation site occupancy
High performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) of specific glycopeptides
Glycopeptides
N-linked and O-linked Glycans profile
High performance liquid chromatography coupled with high resolution mass spectrometry (HPLC-TOFMS) of the procainamide derivatized released glycans
Released glycans

Moreover, like the rest of biotherapeutics and with the aim of ensure a complete characterization of the heavily glycosylated proteins as is included in the ICH Q6B guidelines, AMSbiopharma offers the following analytical services: