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.

N- Glycosylation

N-glycosylation occurs on the side chain of the amino acid asparagine (asparagine is described with the letter “N” using the single letter amino acid code, hence the name N-glycosylation). The asparagine residue must be present in a well-defined specific three amino acid consensus sequence for N-glycosylation to take place. This consensus sequence is defined as N-X-S/T where “N” is the asparagine residue to be glycosylated, “X” can be any amino acid except proline and “S/T” indicates that a serine or threonine residue must be at this position. Therefore, the prediction of N-glycosylation sites in proteins is possible from a knowledge of the amino acid sequence of that protein. The presence of this sequence in a protein chain does not mean that it will definitely be N-glycosylated, rather it indicates that it is possible for N-glycosylation to occur here. Other factors, such as the overall folding of the molecule, will also play a role in determining if a potential N-glycosylation site is actually glycosylated or not.

O- Glycosylation

O-glycosylation is found on the side chains of the hydroxylamino acids Serine and Threonine (the “O” indicating attachment of sugar residues through the side chain hydroxyl group). Unlike N-glycosylation, O-glycosylation is not controlled be the presence of a linear consensus sequence in the amino acid chain, thus O-glycosylation prediction is not possible from the linear amino acid sequence. This is just one of several differences that exists between N-glycosylation and O-glycosylation. These differences are summarised in the table below.

N-glycosylation O-glycosylation
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

How Does Glycosylation Happen?

Glycosylation is not controlled through a simple template in the way proteins are via the DNA template. Rather, glycosylation of proteins occurs in the endoplasmic reticulum and Golgi apparatus via the action of specifically localized enzymes on the glycoprotein as it passes through the secretory pathway from site of translation to site of release at the plasma membrane of the cell. This is true for both N and O-linked glycosylation of proteins. This means that the N-glycans and O-glycans produced are complex and can exhibit a high degree of heterogeneity, depending on the glycosylation enzymes that have acted and the extent to which that action has occurred.

There are many factors that can affect the profile of glycans on a glycoprotein. These include types of glycan biosynthesis enzymes present in the secretory pathway (which is a function of cell type used in manufacturing), the overall 3D structure of the protein itself, speed of the protein through the secretory pathway, and bioreactor conditions (such as pH or nature of growth media). Furthermore, different glycosylation sites within a molecule can have their own specific sets of glycans.

Glycosylation in Mammals, Plants and Insects

Protein glycosylation occurs in both plants and animals but the nature of glycosylation varies through the different kingdoms as well as between differ phyla and further down through taxonomic classification. Thus plants, insects and mammals all produce glycans with unique sets of structural features. Likewise, different mammalian cell types are also capable of producing unique glycan profiles that are specific for that cell type. This means that cell lines used to produce glycoproteins must be carefully considered since glycan structures should either match the natural source of the glycoprotein or be engineered in a controlled way to give the designed functionality.

The most important point to consider here is that humans produce a specific range of glycans. If an inappropriate cell line is used in the manufacturing process, glycoproteins could be produced with potentially immunogenic structures, since they would be not found on natural human glycoproteins. An example of this is the Galα1-3Gal epitope which is produced in murine cell lines. Bacterial protein glycosylation has also been observed and again this will not be the same as for human glycosylation and could potentially produce immunogenic glycan epitopes.

The Role of Glycosylation

The role of protein glycosylation has been shown to be very diverse, with different effects observed depending on the nature of the glycans and protein to which they are attached. A wide variety of specific functions such as targeting of a glycoprotein to a site of action (eg inflammatory response, lysosomal targeting), control of half-life of a glycoprotein in the blood stream, control of function (e.g. decrease in fucosylation of a monoclonal antibody leads to an increase in antibody dependent cellular cytotoxicity) and maintenance of glycoprotein structure have all be attributed to glycosylation.

Many proteins are modified with glycans, indeed the majority of the therapeutic proteins on the market today are glycoproteins. These include monoclonal antibodies, Erythropoietin, Darbepoietin and FSH amongst others. The fact that so many biotherapeutics are glycosylated makes glycosylation one of the most important post-translational modifications to consider during protein characterization. This potentially high degree of heterogeneity may appear to make glycan analysis a daunting undertaking. However, BioPharmaSpec scientists are used to working with this high degree of complexity and will use their product knowledge and many years of technical expertise in glycan analysis to suggest the best glycan characterization assessment approach for your biopharmaceutical.

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

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.

Prep pf N-linked

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.

2AB LC-MS

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.

MALDI MS

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.

Contact us to understand how to characterize the glycosylation on your biopharmaceutical of interest.