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N-link glycan analysis by HPLC

N-Link Glycan Analysis by HPLC

Analysis of the N-linked glycan structures of glycoproteins determines their glycan type and abundance.

·         Glycan antennary: 1-antennary, 2-Antennary, 3-antennary and 4-antennary

·         Fucose residues, antennary fucose or core fucose.

·         Sialic acids distribution

·         Glycan termini residues: N-acetyl-galactoseamine (GalNAc) and bisecting N-acetyl-glucosamine (GlcNAc), and the antigenic structure alpha 1-3 galactose

·         High mannose and hybrid structures

N-linked glycan analysis is applicable to all stages of biopharmaceutical development and manufacturing processes including cell line and clone selection, upstream and downstream process development, manufacturing in-process control, batch consistency and release.

N-linked glycan analysis is required by ICH Q6B guidelines for biopharmaceutical characterization.
N-linked glycan profiling is obtained using a spectrum of biochemical methods, including HPLC and MALDI-MS:
N-linked glycan profiles are obtained by enzymatic release of the glycans from the glycoprotein using PNGase F, glycan labeling with the fluorescence dye 2-Aminobenzamide (2AB), and glycan separation by normal phase chromatography (NP-HPLC).

In order to obtain full structural analysis, enzyme array of exoglycosidases is applied to the labeled glycans, followed by re-separation on the NP column. The peaks are verified according to their retention time using in-house-characterized standard database.

In addition, for sialic acid quantitation, N-linked glycan charge distribution is determined by HPLC separation of the 2AB labeled glycans using Weak Anion Exchange (WAX) column.
MALDI-MS is applied for further verification of each de-sialylated NP-HPLC peak (off line).

Our reports provide comprehensive information accompanied by professional guidance, upon our customer’s request.

 

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