Glycan Analysis Services - Level 2

This module is an extension of the Level 1 HILIC profiling work to gain more detailed information on the glycan structures and their relative proportions. This can be achieved by Exoglycosidase digestion followed by HILIC-UPLC and/or by HILIC-UPLC-FLR-ESI-MS/MS analyses.

Level 2 Exoglycosidase digestion followed by HILIC-UPLC

Exoglycosidases are the enzymes that cleave the glycosidic linkage of a terminal monosaccharide from glycan. By using positionally specific exoglycosidases in a customised enzyme array, the removed glycan residues can be identified by monosaccharide type, linkage, and sequence. Because each type of monosaccharide is removed separately a series or a mixture of exoglycosidases is required to identify the glycan structure.

Ludger Intact Released Glycan Analysis

This module provides HILIC-UPLC N- and/or O-glycan profiles before and after specific exoglycosidase digestions together with glycan structures and their relative quantitation. The analysis will be performed on single or triplicate N- and/or O-glycan samples, released and fluorescently labelled under the Level 1 HILIC profiling, alongside Ludger positive and negative controls and system suitability standards.

This module is suitable for:

  • glycan assignment (monosaccharide type, order, linkage)
  • quality control - profile comparisons to monitor known structures
  • monitoring batch to batch consistency
  • comparability studies

In order to gain more detailed information and increase confidence in the glycan structures assignment and their relative proportions additional Level 2 HILIC-UPLC-FLR-ESI-MS/MS is recommended.

Sample types:

Released and fluorescently labelled N- and/or O-glycans from:

  • Biopharmaceuticals: mAbs, glycoprotein hormones (e.g. follicle stimulating hormone (FSH) and erythropoietin (EPO), Fc fusion proteins, vaccines)
  • Cells: mammalian cell lines, bacterial cell components
  • Biological fluids, tissues and others
  • Glycoproteins set in SDS-gel (N-glycans only)
  • COVID-19 patient samples (e.g. plasma, tissues)
  • SARS-CoV-2 infected cell lines

Workflow for Level 2 Exoglycosidase digestion followed by HILIC-UPLC

Ludger - Glycan Analysis - Workflow for Level 2 Exoglycosidase digestion followed by HILIC-UPLC

click to enlarge 

Released and fluorescently labelled glycans (Level 1 HILIC profiling) are digested with a series or a mixture of specific exoglycosidases purified and analysed by HILIC-HPLC or HILIC-UHPLC.

Report

Final report contains:

  • HILIC-UPLC profiles for system suitability standards and Ludger positive controls before and after exoglycosidase digestions
  • HILIC-UPLC profiles for client samples and buffer negative controls before and after exoglycosidase digestions
  • Glucose unit (GU) values
  • Glycan structures (including monosaccharide type, order and linkage) and their relative proportions
  • Summary of findings (e.g. G0:G1:G2; % high mannose; % fucosylation; % Galα1-3Gal)

Module Level 2 Exoglycosidase digestion followed by HILIC-UPLC has:

2 options for N-glycans characterisation to choose between:

  1. G-L2N-Ex - Option 1: Exoglycosidase sequencing with HILIC-UPLC profiling on a single released aliquot of each sample
  2. G-L2N-Ex-t - Option 2: Exoglycosidase sequencing with HILIC-UPLC profiling on aliquots of triplicate releases of each sample

2 options for O-glycans characterisation to choose between:

  1. G-L2O-Ex - Option 1: Exoglycosidase sequencing with HILIC-UPLC profiling on a single released aliquot of each sample
  2. G-L2O-Ex-t - Option 2: Exoglycosidase sequencing with HILIC-UPLC profiling on aliquots of triplicate releases of each sample

Relevant Application Note

Application Note - Alpha-Gal-containing biologics

Alpha-Gal-containing biologics


Relevant Posters

Ludger poster - Analysis of Glycosylation Critical Quality Attributes (GCQAs) of monoclonal antibody (mAb) therapeutics

Analysis of Glycosylation Critical Quality Attributes (GCQAs) of monoclonal antibody (mAb) therapeutics

Kozak RP, Kotsias M, Hendel JL, Urbanowicz PA
Presented at: WCB 2019
Reston, USA. Nov 11-13th 2019

Ludger poster - Targeted Analysis of Glycosylation Critical Quality Attributes from Glycoprotein Therapeutics

Targeted Analysis of Glycosylation Critical Quality Attributes from Glycoprotein Therapeutics

Urbanowicz PA, Hendel JL, Kotsias M, Kozak RP
Presented at: BioProduction 2019
Frankfurt, Germany. Nov 5-7th 2019

Ludger poster

Procainamide labelling as part of a flexible glycoprofiling system for monitoring of Gal-a1-3Gal related Glycosylation Critical Quality Attributes (GCQAs) of monoclonal antibody (mAb) therapeutics throughout the product life cycle

Kozak RP, Royle L, Liew LP, Spencer DI, Fernandes DL
Presented at WCBP 2016: 20th Symposium on the Interface of Regulatory and Analytical Sciences for Biotechnology Health Products
Washington DC, United States. January 2016

Keywords: Gal alpha 1-3 gal, IgG, monoclonal antibody (mAb), glycosylation critical quality attribute (GCQA), QbD, procainamide, UHPLC, ESI-MS/MS