Glycosylation is the attachment of carbohydrates to the backbone of a protein through an enzymatic reaction. Once attached, the carbohydrate chain goes through a series of enzymic reactions that result in remodelling of the glycan unit which can lead to considerable heterogeneity of the glycan structure depending on the extent of these enzymic activities. 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.
Glycan Characterization for Heavily Glycosylated Proteins
As required in the ICH guidelines (Topic Q6B), the carbohydrate content (neutral sugars, amino sugars and sialic acids) should be determined. In addition, glycan structures should be characterized both in terms of size, monosaccharide linkages and relative abundance to the extent possible, and particular attention should be paid to their degree of sialylation, the glycans present at each of the sites (glycosylation site analysis) and the degree of glycosylation at each site (glycosylation site occupancy).
BioPharmaSpec scientists have been characterizing heavily N- and O-glycosylated proteins for over three decades and can work with you to devise the best structural and physicochemical characterization strategies for your development plans.
There are a variety of characterization methods used in order to identify the monosaccharides and oligosaccharides (N- and O-glycans) present on your biopharmaceutical.
| Monosaccharides – identification and quantitation | Gas Chromatography with Mass Spectrometric detection (GC-MS) | Neutral sugars (most commonly Fucose, Mannose and Galactose) Amino sugars (N-Acetylglucosamine, N-Acetylgalactosamine) |
| Sialic acids – identification and quantitation | Liquid Chromatography with Fluorescence following DMB labelling | N-Acetylneuraminic acid and N-Glycolylneuraminic acid |
| Oligosaccharides – total population analysis | Liquid chromatography with Mass Spectrometric detection (LC-MS) of fluorescently tagged glycans | N-glycans |
| Matrix Assisted Lased Desorption Ionization Mass Spectrometry (MALDI-MS) of permethylated glycans | N and O -glycans | |
| Oligosaccharides – linkage analysis of the monosaccharides in the glycan chains | Gas Chromatography with Mass Spectrometric detection (GC-MS) | All linked neutral and amino sugars |
| Site occupancy – determination of relative percentage occupancy | Liquid chromatography with Mass Spectrometric detection (LC-MS) of specific proteolytic and deglycosylated digests | N-glycans |
| Site analysis – analysis of the glycan populations at individual glycosylation site | Liquid chromatography with Mass Spectrometric detection (LC-MS) of specific proteolytic digests. Separated glycopeptides are then collected for analysis of the glycans at each site | N- and O-glycans |
Immunogenic epitopes
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 specific for that cell type. Because many biopharmaceuticals are produced in non-human cell lines, there is a risk that, due to the presence of non-human glycan structures, the glycoproteins produced could be immunogenic to humans. An example of this is the Galα1-3Gal epitope which is produced in murine cell lines but not naturally found in humans. Bacterial protein glycosylation has also been observed to produce immunogenic glycan epitopes.
![Alpha_gal-Heavily_glycosylated_proteins[1]](https://biopharmaspec.com/wp-content/uploads/2020/11/Alpha_gal-Heavily_glycosylated_proteins1.svg "Alpha_gal-Heavily_glycosylated_proteins[1]")
BioPharmaSpec scientists have considerable experience in the analysis of unusual or uncommon glycans structures and can design specific analyses for the investigation of these potentially immunogenic epitopes, such as the Galα1-3Gal structure.
Other Structural and Physicochemical Protein Characterization for Heavily Glycosylated Proteins
The expectations of the EMA and US FDA, in terms of structural characterization, are outlined in ICH Topic Q6B.
ICH Topic Q6B provides examples of technical approaches to be considered for structural characterization and confirmation, and evaluation of physicochemical properties of the desired product, drug substance and/or drug product. The guideline recognizes that new analytical technology and modifications to existing technology are continuously being developed and should be utilized when appropriate.
The structural and physico-chemical techniques used to provide the necessary data both to determine the structural characteristics of new biologics as well as to support the comparability assessment of biosimilars and the regulatory process are outlined in the table below:
| Amino acid sequence | N-terminal sequencing (Edman chemistry) Mass-spectrometric sequencing | N-terminal sequencer On line LC-MS (with MSθ and/or MS/MS) MALDI-MS and MS/MS |
| Amino acid analysis | Amino acid analysis | Amino acid Analyzer (e.g. RP-HPLC with fluorescence detector) |
| Terminal amino acid sequence | N-terminal sequencing (Edman or mass spectrometry) Mass spectrometry based sequencing for the C-terminus | N-terminal sequencer Mass spectrometer capable of providing N- and C-terminal sequence information |
| Peptide map | Peptide mapping | On line LC-MS (with MSθ and/or MS/MS) |
| Sulphydryl group(s) and disulphide bridges | Non-reduced peptide mapping | On line LC-MS (with MSθ and/or MS/MS) |
| Carbohydrate structure | Monosaccharide composition analysis Oligosaccharide population analysis Glycosylation site analysis | GC-MS LC and on line LC-MS (with MSe and/or MS/MS) MALDI-MS and MS/MS |
| Molecular weight or size | Intact molecular weight analysis | On line LC-MS MALDI-MS |
| Isoform pattern | IsoElectric Focusing (IEF) | Imaged Capillary IEF (icIEF) |
| Extinction coefficient | Optical density measurement and protein concentration determination | UV spectrophotometer Amino acid Analyzer (e.g. RP-HPLC with fluorescence detector) |
| Electrophoretic analysis | Charge and size based electrophoretic analysis | icIEF Capillary Gel Electrophoresis (CE-SDS) |
| Liquid Chromatography | Charge, size and hydrophilicity/hydrophobicity based chromatographic analyses | Ion-exchange chromatography (IEX) Size Exclusion Chromatography (SEC) Reversed Phase Chromatography (RP-HPLC) |
| Spectroscopic profiles | Secondary and tertiary structure analysis | NMR (1D and 2D)1 Circular Dichroism (CD) Fourier Transform-Infra Red (FT-IR) Fluorescence (Intrinsic and Extrinsic) |
| Analysis of truncated forms | Intact molecular weight analysis Peptide mapping Size based chromatographic and electrophoretic analysis | On line LC-MS (with MSθ and/or MS/MS) MALDI-MS SEC CE-SDS |
| Aggregation | Size based chromatography, Analytical UltraCentrifugation (AUC) and Field Flow Fractionation (FFF) | Size Exclusion Chromatography with Multi Angle Laserlight Scattering (SEC-MALS) Sedimentation Velocity-Analytical UltraCentrifugation (SV-AUC) Field Flow Fractionation (FFF; AF4) |
1 HDX-MS data can also be used if NMR data is not available
Where to start...
Although there is a comprehensive range of glycosylation analysis methods available at BioPharmaSpec, our scientists can quickly suggest the best methods to obtain the glycan data you need.