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Glycosylation analysis of therapeutic glycoproteins

Therapeutic monoclonal antibodies (mAbs) are glycoproteins produced by living cell systems. Glycosylation is one of the most common post-translational modifications (PTMs) of proteins. The glycan moieties attached to the proteins can directly affect protein stability, bioactivity, and immunogenicity. Therefore, glycan variants of a glycoprotein product must be adequately analyzed and controlled to ensure product quality. However, the inherent complexity of protein glycosylation, combining the vast diversity in glycan structure and composition add an additional level of daunting analytical challenge.

In the development of therapeutic glycoproteins (e.g., mAbs), especially biosimilar products, glycan analysis usually involves the use of complementary methods for assessing specific glycosylation attributes, including:

  • glycosylation site
  • glycan structure: the structure of the carbohydrate chains, the oligosaccharide pattern (antennary profile)
  • abundance: the carbohydrate content (neutral sugars, amino sugars, and sialic acids)

Analytical methods:

Methods Application
High-performance liquid chromatography (HPLC) Widely used to quantify the amounts of released oligosaccharides
Mass spectrometry (MS) Coupled with HPLC remains a powerful tool in the characterization of glycosylation site(s) occupancy and carbohydrate structures
Lectin-based microarray Directly measure glycan variations in an intact protein without the need of clipping glycans from the protein backbone
Capillary electrophoresis (CE) Alone or in combination is commonly used to monitor heterogeneity in sialic acids on intact glycoproteins
Isoelectric focusing (IEF)
Ion-exchange (IEX)

The selection of appropriate methods for a specific glycoprotein will depend on the purpose of testing and the ability of individual method in analyzing intact glycoproteins, glycopeptides, released glycans, and monosaccharides.

Glycosylation analysis of therapeutic glycoproteins

We recently provide an update series of services and related products in glycan analysis for determining unique glycan features, including assessment of glycosylation site, glycan structure, and carbohydrate content.

Our experts apply our experience in designing programs to deliver the glycosylation information to meet the requirements of your development stage and strategic packages pertinent to your actual need. Our experience spans tailored services for glycan analysis and a variety of related products. We are ready to support your antibody product development and manufacture, helping you to meet all of your therapeutic glycoproteins characterisation needs.

Service Available:

A panel of services for the characterisation of glycoproteins that Creative Biolabs offers:

  • Identification of glycosylation sites through LC-MS/MS peptide mapping 
  • Capillary electrophoresis methods for glycan profile
  • Confirmation of glycan structural characterization (enzyme array, MALDI-TOF)
  • Determination of glycosylation linkage (GC-MS)
  • Isoform analysis via cIEF
  • Determination of Gu or G0 values (CE or normal phase LC)
  • Glycan occupancy measuring and compositional analysis 
  • Determination and quantitative analysis of sialic acid residues, total and specific
  • LC-MS intact molecular weight for intact molecule heterogeneity analysis
  • Quantitative analysis of total sugar (monosaccharides and oligosaccharide composition neutral and amino sugars) 
  • Glycosylation population profile analysis with glycan ID via LC-MS/MS

Products Available:

Glycan staining or labeling

Sugar Oxidation and Aldehyde Reaction

Cat No. Product Name Associated Applications Price
Glyco-016CL CBGlycoTM Sodium Meta-Periodate Carbonyls (Aldehydes) as Crosslinking Targets Inquiry
Glyco-017CL BMPH (N-β-maleimidopropionic acid hydrazide) Hydrazide Reaction Chemistry-Heterobifunctional Sulfhydryl-to-Carbonyl Hydrazide Crosslinkers

Prepare specific glycoprotein conjugates-Maleimide / Hydrazide-Pyridyldithiol / hydrazide (sulfhydryl-to-carbonyl) Crosslinkers
Inquiry
Glyco-018CL EMCH (N-ε-maleimidocaproic acid hydrazide) Inquiry
Glyco-019CL MPBH (4-(4-N-maleimidophenyl)butyric acid hydrazide) Inquiry
Glyco-020CL KMUH (N-κ-maleimidoundecanoic acid hydrazide) Inquiry
Glyco-021CL PDPH (3-(2-pyridyldithio)propionyl hydrazide) Inquiry
Glyco-022CL CBGlycoTM Alkoxyamine-PEG12-Biotin Hydrazide Reaction Chemistry
Glycoprotein labeling
Applications for Alkoxyamine Crosslinking
-Hydrazide and Alkoxyamine Biotinylation Reagents
Inquiry
Glyco-023CL CBGlycoTM Alkoxyamine-PEG4-Biotin Inquiry
Glyco-024CL CBGlycoTM Hydrazide-Biocytin Inquiry
Glyco-025CL CBGlycoTM Hydrazide-Biotin Inquiry
Glyco-026CL CBGlycoTM Biotin-LC-Hydrazide Inquiry
Glyco-027CL CBGlycoTM Hydrazide-PEG4-Biotin Inquiry
Glyco-028CL CBGlycoTM Coupling Catalyst Aniline Catalysis of Hydrazide Conjugation Inquiry
Glyco-029CL CBGlycoTM Immobilization Kit Glycoprotein immobilization-Coupling Resin Inquiry
Glyco-030CL CBGlycoTM IP Kit Glycoprotein immobilization Inquiry
Glyco-031CL CBGlycoTM Aminolink Coupling Resin Reductive Amination Reaction Chemistry-Sodium Cyanoborohydride (Aminolink Reductant) Inquiry
Glyco-032CL 16% Formaldehyde Solution Formaldehyde and glutaraldehyde crosslinking Inquiry
Glyco-033CL CBGlycoTM Plus Activated Peroxidase HRP Labeling Kit  Protein to glycoprotein conjugation Inquiry
Glyco-034CL CBGlycoTM Activated glutaraldehyde HRP Labeling Kit Protein to glycoprotein conjugation Inquiry
Glyco-035CL CBGlycoTM Maleimide-activated HRP (sulfhydryl-reactive) Protein to glycoprotein conjugation Inquiry
Glyco-036CL CBGlycoTM Aminolink Coupling Resin Protein immobilization through primary amines Inquiry
Glyco-037CL CBGlycoTM Aminolink Plus Coupling Resin Protein immobilization through primary amines Inquiry
Glyco-038CL CBGlycoTM Direct IP Kit Protein immobilization through primary amines Inquiry

Protein Gel Stains

Cat No. Product Name Associated Applications Price
Glyco-039CL CBGlycoTM Blue Stain Reagent  Coomassie dye stains Inquiry
Glyco-040CL CBGlycoTM CBGlycoTM Blue Safe Protein Stain Coomassie dye stains Inquiry
Glyco-041CL CBGlycoTM Protein Stain (sensitive, R-250 coomassie stain) Coomassie dye stains Inquiry
Glyco-042CL CBGlycoTM Silver Stain Kit Silver stains Inquiry
Glyco-043CL CBGlycoTM Silver Stain Kit for Mass Spectrometry Silver stains Inquiry
Glyco-044CL CBGlycoTM Color Silver Stain Kit Silver stains Inquiry
Glyco-045CL CBGlycoTM Zinc Reversible Stain Zinc stains Inquiry
Glyco-046CL CBGlycoTM Krypton Fluorescent Protein Stain Fluorescent dye stains Inquiry
Glyco-047CL CBGlycoTM Glycoprotein Staining Kit Functional group specific stains Inquiry
Glyco-048CL CBGlycoTM 6xHis Protein Tag Stain Kit Functional group specific stains Inquiry
Glyco-049CL CBGlycoTM Pro-Q Emerald 488 Glycoprotein Gel and Blot Stain Kit Functional group specific stains-Specialty Protein Gel Stains Inquiry
Glyco-050CL CBGlycoTM Pro-Q Emerald 300 Glycoprotein Gel and Blot Stain Kit Inquiry
Glyco-051CL CBGlycoTM Pro-Q Diamond Phosphoprotein Gel Stain Inquiry
Glyco-052CL CBGlycoTM Reversible Protein Stain Kit for Nitrocellulose Membranes Inquiry
Glyco-053CL CBGlycoTM Novex Reversible Membrane Protein Stain Kit Inquiry
Glyco-054CL CBGlycoTM Lumio Green Detection Kit Functional group specific stains-Tagged Fusion Protein Gel Stains Inquiry
Glyco-055CL CBGlycoTM InVision His-Tag In-Gel Stain Inquiry
Glyco-056CL CBGlycoTM InVision His-Tag In-Gel Staining Kit Inquiry
Glyco-057CL CBGlycoTM Glycoprotein Staining Kit Glycosylated proteins staining Inquiry
Glyco-058CL CBGlycoTM O-GlcNAc Western Blot Detection Kit O-GlcNAc post-translational modification (PTM) Inquiry
Glyco-059CL CBGlycoTM Glycoprotein Carbohydrate Estimation Kit Protein glycosylation measuring Inquiry

Glycoprotein purification or enrichment

Cat No. Product Name Associated Applications Price
Glyco-060CL CBGlycoTM Glycoprotein Isolation Kit (Concanavilin A) Isolation of glycoproteins Inquiry
Glyco-061CL Jacalin Agarose Immobilized Jacalin (for human IgG purification) Inquiry

Glycoproteome and Glycome Analysis by Mass Spectrometry

Proteases and Protein-Cleaving Reagents

Cat No. Product Name Associated Applications Price
Glyco-062CL Carboxypeptidase Y Purified Inquiry
Glyco-063CL Immobilized Carboxypeptidase Y (Agarose Resin) Immobilized proteases Inquiry
Glyco-064CL Factor Xa (Bovine) Solution Purified Inquiry
Glyco-065CL CBGlycoTM HRV 3C Protease Solution Kit Purified Inquiry
Glyco-066CL Immobilized Ficin (Agarose Resin) Immobilized proteases Inquiry
Glyco-067CL Immobilized Papain (Agarose Resin) Immobilized proteases Inquiry
Glyco-068CL Immobilized Pepsin (Agarose Resin) Immobilized proteases Inquiry
Glyco-069CL Proteinase K Powder Purified Inquiry
Glyco-070CL CBGlycoTM Immobilized V-8 Protease Kit Immobilized proteases Inquiry
Glyco-071CL Staphylococcus aureus V-8 Protease Purified Inquiry
Glyco-072CL Immobilized Trypsin, TPCK Treated (Agarose Resin) Immobilized proteases Inquiry
Glyco-073CL Trypsin, TPCK Treated Purified Inquiry
Glyco-074CL Trypsin Protease, MS Grade MS Grade Inquiry
Glyco-075CL Lys-C Protease, MS Grade MS Grade Inquiry
Glyco-076CL Lys-N Protease, MS Grade MS Grade Inquiry
Glyco-077CL Asp-N Protease, MS Grade MS Grade Inquiry
Glyco-078CL Chymotrypsin Protease (TLCK treated), MS Grade MS Grade Inquiry
Glyco-079CL Glu-C Protease, MS Grade MS Grade Inquiry

SILAC Isotope Labeling Reagents and Kits

Cat No. Product Name Associated Applications Price
Glyco-080CL L-Lysine-2HCl, 13C6 for SILAC Heavy and light amino acids are used to specifically analyze protein expression by mass spectrometry using stable isotope labeling with amino acids in cell culture (SILAC) quantification kits. Inquiry
Glyco-081CL L-Lysine-2HCl for SILAC Inquiry
Glyco-082CL L-Arginine-HCl, 13C6 for SILAC Inquiry
Glyco-083CL L-Arginine-HCl for SILAC Inquiry
Glyco-084CL L-Arginine-HCl, 13C6, 15N4 for SILAC Inquiry
Glyco-085CL L-Lysine-2HCl, 13C6, 15N2 for SILAC Inquiry
Glyco-086CL L-Lysine-2HCl, 4,4,5,5-D4 for SILAC Inquiry
Glyco-087CL L-Leucine for SILAC Inquiry
Glyco-088CL L-Leucine, 13C6 for SILAC Inquiry
Glyco-089CL L-Proline for SILAC Inquiry
Glyco-090CL DMEM Media for SILAC SILAC cell culture media and dialyzed fetal bovine serum (FBS) are optimized for use with stable isotope labeling with amino acids in cell culture (SILAC) to analyze protein expression by mass spectrometry (MS). Inquiry
Glyco-091CL Powdered DMEM Media for SILAC Inquiry
Glyco-092CL RPMI Media for SILAC Inquiry
Glyco-093CL Powdered RPMI Media for SILAC Inquiry
Glyco-094CL DMEM:F12 (1:1) Media for SILAC Inquiry
Glyco-095CL IMDM Media for SILAC Inquiry
Glyco-096CL Ham's F12 Media for SILAC Inquiry
Glyco-097CL McCoy's 5A Media for SILAC Inquiry
Glyco-098CL MEM Media for SILAC Inquiry
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