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.

When proteins have a high amount of glycosylation, they are termed heavily glycosylated proteins. Examples of biopharmaceuticals in this category include Erythropoietin (EPO), Darbepoetin (DPO) and Follicle Stimulating Hormone (FSH). These glycoproteins bring specific and additional protein characterization challenges, because it is a regulatory requirement to characterize the glycosylation profile of your biopharmaceutical to the extent possible.

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.

Aspect of glycosylation Methodology/ instrumentation used for data provision Detected carbohydrates
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 sites 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.

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:

Analytical requirement Methodology used for data provision Instrumentation requirements
Amino acid sequence N-terminal sequencing (Edman chemistry)

Mass-spectrometric sequencing

N-terminal sequencer

On line LC-MS (with MSe and/or MS/MS)

MALDI-MS and MS/MS

Amino acid composition 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 MSe and/or MS/MS)
Sulphydryl group(s) and disulphide bridges Non-reduced peptide mapping On line LC-MS (with MSe 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 MSe and/or MS/MS)

MALDI-MS

SEC

CE-SDS

Analysis of other modified forms Intact molecular weight

Peptide mapping

Oligosaccharide profiling

Isoform profiling

On line LC-MS (with MSe and/or MS/MS)

MALDI-MS

icIEF

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