Empowering the Biochemist to Analyze Glycoproteins with Exceptional Reproducibility

BIOshell™ Glycan HPLC Columns

Protein glycosylation is one of the most common and structurally diverse types of protein post translational modifications (PTM). The particular pattern of glycosylation as well as the location of the PTM has been shown to play a major role in numerous physiological and pathological reactions such as cellular development, immunity, blood clotting and apoptosis. Thus, understanding protein glycosylation is essential in elucidating a glycoprotein's cellular function.

Glycoprotein analysis can be challenging, however, because it involves the identification of complex N-linked and O-linked sugar molecules that are often quite structurally similar. Hydrophilic interaction liquid chromatography (HILIC) is a proven technique for the separation and quantitation of isolated glycans under native conditions or after their derivitization with fluorescent labels.

BIOshell Glycan HPLC Columns are specifically engineered to deliver fast, high resolution, reproducible glycan identification using HILIC chromatography. Benefits of using Fused-Core® BIOshell Glycan HPLC Columns for glycoprotein analysis are as follows:

  • Increased resolution, faster separations, and lower back pressure. Based upon Fused-Core Particle Technology, BIOshell HPLC columns offer significant performance benefits over popular columns based on totally porous particles.
  • Excellent reproducibility. Quality Control testing requires tight retention time and peak width specifications ensuring lot to lot reproducibility.
  • Complimentary Sigma-Aldrich products. Sigma-Aldrich carries all of the reagents and consumables needed for each step in glycoprotein analysis: release, labeling, cleanup and analysis as seen in Table 1 below.

 


Applications using BIOshell Glycan HPLC Columns

 


Figure 1: Separation of a Procainamide Labeled Dextran Ladder

The dextran ladder is used as an external standard for the analysis of glycans by normal phase HPLC after fluorescent labeling. When analyzed on the BIOshell Glycan HPLC Column, this standard gives a characteristic ladder profile from monomeric glucose to approximately a 20-mers of glucose oligosaccharide. This ladder provides calibration reference points that can aid in identifying more complex glycans based upon retention characteristics.


Figure 2: Separation of Procainamide Labeled Human IgG Glycans

A sample of human IgG glycans was analyzed on a BIOshell Glycan HPLC Column resulting in the identification of 20 distinct peaks. Excellent separation as well as symmetrical peak shape can be observed in the chromatogram.


Figure 3: Separation of Procainamide Labeled Cetuximab Glycans


Cetuximab is a chimeric mouse-human IgG1 monoclonal antibody against the epidermal growth factor receptor. It is used to treat head and neck as well as colorectal cancers. The antibody is N-glycosylated both in the Fc and Fab regions, which have been shown to impact safety and quality of the drug. Thus, understanding glycosylation patterns is exceptionally important. As shown in this application report, the BIOshell Glycan column is able to elucidate the complex glycosylation of this biotherapeutic, allowing a better understanding of the drug’s efficacy.

Additional Product Information
BIOshell U/HPLC Columns
Request a BIOshell Glycan Evaluation Column

Table 1. Reagents and Consumables for Glycoprotein Analysis
  Supplier Prod. No.
Step 1: Glycan Release
IgG from human serum Sigma I4506
Trizma HCl Sigma T5941
Urea Sigma U0631
Ammonium Bicarbonate Sigma-Aldrich
09830
PNGase F Sigma P7367
30k centrifugal filter unit Millipore MRCF0R030
Step 2: Labeling
Sodium Cyanoborohydride Aldrich 156159
Procainamide Hydrochloride Sigma P9391
Dimethyl Sulfoxide Sigma D8418
Acetic Acid Sigma-Aldrich 320099
Water Fluka 39253
Dextran Hydrolysate Sigma-Aldrich
31417
Step 3: Cleanup
Acetonitrile Sigma-Aldrich 34851
Discovery Glycan SPE 50 mg Cartridges Supelco 55465-U
Step 4: UPLC-FLR-MS Analysis
BIOshell™ Glycan, 15 cm x 2.1 mm, 2.7 µm Supelco 50994-U
Ammonium Formate Fluka 17843
Formic Acid Fluka 94318

Fused-Core is a registered trademark of Advanced Materials Technology, Inc.
FLUKA is a registered trademark of Honeywell Specialty Chemicals Seelze GmbH

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