Glycan Analysis using HILIC with Fluorescence Detection

INTRODUCTION

Monoclonal antibodies(mAbs) are one of the fastest growing areas of therapeutic interest in the biopharmaceutical industry today.They are produced from cell lines and are made up of two heavy and two light polypeptide chains which are joined together by disulphide bonds.The Fab termini of the heavy and light chains contain a variable region which determines the specificity of the mAb for a target antigen(known as the complementary determining region) .

mAbs are heavily glycosylated proteins， which means they have oligosaccharide species(glycans) attached at specific locations on the molecule.All mAbs contain at least two glycosylation sites(usually on the heavy chain) ， as depicted in figure 1， with the presence of glycans affecting the safety and efficacy of the drug.For this reason， the glycosylation pattern must be thoroughly characterised during mAb production to ensure the target drug has been correctly synthesised.In fact，

glycosylation is a post-translational modification which is considered a Critical Quality Attribute and therefore should be monitored according to the ICHQ6B guidelines.

Figure1：Schematicshowingglycosylationregionsof amonoclonal antibody

Fc=Crystal lis able fragment， Fab=Antigen-binding fragment，

Hc=He au y chain， Lc=Light chain

2-AB Labelling

Enzymatic release of the glycan species with PNG as eF allows separation of closely related carbohydrates using HPLC.Since glycans do not contain a chromophore， they must be labelled with a fluorescent compound before being detected.In the application shown in figure 3， 2-amino benzamide(2-AB) was used as the fluorescent label.Figure 2 shows the formation of a 2-AB labelled glucose monomer.Due to the highly polar nature of released glycans， hydrophilic interaction liquid chromatography(HILIC) is employed for their separation，the details of which are shown in tables 1 and 2.

 
HILIC Separation

Figure 3 shows the analysis of a glycan ladder standard containing 15 glucose homopolymers of increasing length.Method details are shown in tables 1 and 2.This method demonstrates the ablity of the ACE Glycan phase to separate structurally similar glycan species and can be used as a calibration reference when identifying more complex glycans based on relative retention times.ACE Excel Glycan色譜柱甘油分析

Figure 3：Separation of glucose polymers.(ACE Application Note#7160)

ACE Excel Glycan色譜柱甘油分析ORDERING TABLE

Product:ACE Excel 3 Glycan,UHPLC column

Size:150x2.1mm

Part number:EXL-1116-1502