The C2 strong anion exchange columns were designed for charge-based analysis and purification of 2-AB and 2-AA labeled glycans as part of lot release QC for therapeutic glycoproteins.
The outline of the procedure is as follows :
• The oligosaccharides are labeled by reductive amination with 2-AB or 2-AA.
• Excess labeling reagents are then removed using LudgerCleanTM S cartridges.
• The labeled oligosaccharides are analysed by anion exchange HPLC on a LudgerSep C2 column with fluorescence detection.
LudgerSep C2 HPLC columns contain particles with a macroporous polymeric anion exchange coating optimized for anion exchange chromatography of complex glycan mixtures. Developed as a replacement for weak anion exchange columns, the C2 columns deliver reliable, reproducible separations. Samples are applied in water and are typically eluted with a sodium acetate or ammonium formate aqueous salt gradient.
Picomole quantities of oligosaccharides may be analysed by fluorescent labeling with 2-aminobenzamide (2-AB) using the LudgerTag 2-AB Glycan Labeling Kit (LT-KAB-A2) or with 2-aminobenzoic (2-AA) using the LudgerTag 2-AA Glycan Labeling Kit (LT-KAA-A2), followed by LudgerSep C2 strong anion exchange HPLC. The maximum amount of glycan sample that can be loaded on the column depends on the number and type of glycan components as well as the nature of any non-glycan material. The typical range for successful analytical runs is 1pmol – 1 nmol per sample peak and up to 200 nmol of total glycans.
We recommend using our LudgerSep C buffer (500 mM ammonium formate buffer pH 9, 20 % acetonitrile v/v) rather than lower pH solvents because this higher pH ensures that all anionic glycans (sialic acids, sulphated and phosphorylated sugars) are in a negatively charged state. A lower pH solvent (e.g. pH 4.4) can be used for separation of sialylated glycans, but this is below the pKa value for phosphorylated glycans.
Separation of 2-AB labeled glycan standards (picomolar concentration) using a LudgerSep C2 4.6×150 mm column
Separation of 2-AB human IgG glycans using a LudgerSep C2 4.6×150 mm
HPLC Column System Suitability Application Note
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C2 Strong Anion Exchange Column Specifications
Base Matrix 8 mm polystyrene divinyl benzene with a stable quaternized polyethylene imine coating
Column dimensions – volume
4.6×150 mm – 2.49 ml
4.6×50 mm – 0.83 ml
Typical flow rate 0.3 – 2.0 ml / min
Pressure limit 300 psi
Column tube Stainless steel, Valco compatible end fittings
pH compatibility pH 1 to pH 13
LudgerSep C2 strong anion exchange columns can be used with an HPLC system capable of delivering accurate gradients at a flow rate of 0.3 to 2.0 ml/min. In general, systems which mix eluants at high pressure (after the pump head) have lower dead volumes and supply more accurate gradients that are appropriate at the flow rate needed for the LudgerSep C2 strong anion exchange column.
The loop size to be used depends on the separation mode and amount of sample. For analytical runs it is desirable to minimise the sample volume and, typically, a 10 μl loop is used with sample injection volumes of 1 to 5 μl (partial fill) or > 10 μl (complete fill). For charge mode separations, generally, anionic glycans which are retained by the column (and are therefore effectively concentrated on the column) are reasonably tolerant of larger injection volumes whereas non-anionic glycans are not retained by the column matrix and will elute in a volume proportional to the injection volume.
A fluorescence detector is required (λex = 330 nm, λem=420 nm) for LudgerTag 2-AB or 2-AA labeled oligosaccharides. This could be either a filter or monochromator type. Fluorescence filters for OPA-peptide analysis work well and, typically, with these glycan peaks eluting from the LudgerSep C2 column containing 2 – 5 pmol of 2-AB or 2-AA labeled glycans can be detected with good signal-to-noise.
