What is Protein Glycosylation?
Glycosylation is one of the most important post-translational modifications to consider during protein characterization and it is a regulatory requirement to characterize the glycans on your biopharmaceutical. Protein glycosylation can be significantly affected by even simple changes in the manufacturing processes, such as a pH change or a change of growth media, and these alterations can impact the functionality of your biopharmaceutical.
Glycosylation is defined as the enzymatic attachment of carbohydrates, or sugars, to the protein backbone. A protein that is glycosylated is known as a glycoprotein. The two most common types of protein glycosylation are known as N-glycosylation and O-glycosylation. Of these two forms of glycosylation, N-glycosylation of proteins is the most commonly found.
|Attached to asparagine residues||Attached to serine/threonine residues|
|Linear protein consensus sequence||No linear protein consensus sequence|
|10-20 monosaccharide residues in size on average but some variability around this||2-5 monosaccharide residues in size on average but some variability around this|
|Attached as a large precursor glycan||Attached as a single N-acetylgalactosamine monosaccharide|
|Conserved pentasaccharide core||No conserved core – several core structures are possible|
|Precursor is broken down and remodelled||Monosaccharides are added individually to the core N-acetylgalactosamine residue|
|Contains the monosaccharide mannose||Does not contain the monosaccharide mannose|
Analyzing Carbohydrate Structure
It is vital to generate structural information on glycosylation since a detailed knowledge of product structure is imperative for any drug to be brought to market. This is true for both novel products and also biosimilars. Furthermore, comparability of the glycan profiles across various batches will prove that your manufacturing process is under control and for this reason, the regulatory authorities will request monosaccharide composition analysis data (e.g. levels of fucose, mannose, galactose and sialic acid) and N-linked (and O-linked if applicable) structural data on glycosylation from various batches.
Monosaccharide composition analysis
Obtaining data on monosaccharide composition is the first step in glycan characterization. BioPharmaSpec uses Gas Chromatography with Mass Spectrometric detection (GC-MS) to identify and quantitate of the levels of the neutral sugars (most commonly Fucose, Mannose and Galactose), amino sugars (N-Acetylglucosamine, N-Acetylgalactosamine) and Liquid Chromatography with Fluorescence for the detection of sialic acids (N-Acetylneuraminic acid and N-Glycolylneuraminic acid) following DMB labeling.
Oligosaccharide analysis at BioPharmaSpec involves the isolation and subsequent analysis of N-glycans and O-glycans from a glycoprotein using mass spectrometry and chromatographic analytical processes. These are very powerful tools for the investigation of the complex set of glycan structures present on a glycoprotein and are applicable to the analysis of both protein N-glycosylation and protein O-glycosylation. The use of chemical and enzymic procedures for sample work up generates released glycans in a suitable condition for analysis.
BioPharmaSpec has many years of experience in the field of protein glycosylation analysis of both mammalian and non-mammalian cell lines and can design and execute a set of analyses appropriate for your needs. This covers not only analysis of the total population of glycans on a glycoprotein but also the structure of glycans at individual protein glycosylation sites in the molecule.
Methodology for N-linked Glycan Analysis
An example of the methodology used for releasing, purifying and analyzing the N-linked glycans from a monoclonal antibody is shown below.
The N-linked glycan fraction can then be analyzed using mass spectrometry and/or liquid chromatography based techniques or on line LC-MS. An example of the data obtained from liquid chromatographic analysis of 2-AB labeled N-linked oligosaccharides is shown below.
Released N-glycans can also be analyzed by MALDI-MS, usually following permethylation. An example of the data obtained from analysis of the N-glycans released from a Monoclonal Antibody is shown below.
MS/MS data can also be obtained of individual glycans for antennal fragments. These data sets can be used to provide a relative quantitation of the N-linked oligosaccharides observed and suggest the structures of the N-linked glycans present.
To provide a further layer of structural detail, BioPharmaSpec can also employ chemical derivatization procedures in conjunction with GC-MS analysis to determine the nature of the linkages present between monosaccharides in the population of glycans. This is very important since certain monosaccharides, such as sialic acid, can be linked in different ways in glycoproteins and this can have an effect on function. Furthermore, for the production of Biosimilars, the Biosimilar must structurally match the Innovator drug.
The ICH Q6B guidelines require full characterization of the glycosylation of a glycoprotein. BioPharmaSpec can generate all of the information required for this full structural characterization. This applies to both the total glycan population but also the structures of the glycans at individual glycosylation sites if more than one is present.
Glycosylation analysis: bespoke for your biopharmaceutical
BioPharmaSpec is highly experienced in glycosylation analysis and can suggest methods that are appropriate for your molecule and will give you the data you need. Talk to our scientists today or request a proposal.