Glycosylation Analysis with TSKgel Amide-80 2 µm UHPLC Columns

The Application Notebook

The Application Notebook, The Application Notebook-07-01-2015, Issue 0

Glycosylation is one of the most common forms of post-translational modification of proteins. The polysaccharide side chains (glycans) play critical roles in physiological and pathological reactions. Besides the interest in characterizing glycosylation pattern of proteins for structure/function analysis, the thorough characterization of glycosylation is also a major quality parameter in the production of biotherapeutics. Hydrophilic interaction liquid chromatography (HILIC) is a well-recognized technique that effectively separates and quantifies isolated glycans.

 

Glycosylation is one of the most common forms of post-translational modification of proteins. The polysaccharide side chains (glycans) play critical roles in physiological and pathological reactions. Besides the interest in characterizing glycosylation pattern of proteins for structure/function analysis, the thorough characterization of glycosylation is also a major quality parameter in the production of biotherapeutics. Hydrophilic interaction liquid chromatography (HILIC) is a well-recognized technique that effectively separates and quantifies isolated glycans.

Glycoprotein analysis involves characterizing complex N- and O-linked structures composed of sugar moieties. HILIC using amide‑based stationary phases and fluorescence or MS detection is a well-established, robust technique to obtain high-resolution separation of N-linked glycans released from glycoproteins. Tagging the glycans with a fluorescent label such as 2-aminobenzamide (2AB) or aminopyridin (PA) allows the sugars to be detected at femtomole levels.

TSKgel Amide-80 chemistry is ideally suited for the separation of charged and neutral fractions of glycan pools in one run. The retention of labelled polysaccharides by TSKgel Amide-80 enables the identification of glycan structures by comparison to a labelled dextran ladder that is used to normalize retention times to calculate the number of glucose units (GU values) of the separated glycans. The GU values obtained after separation of sequential exoglycosidase digests can be used to predict the glycan structure by database query.

Packed with 2 μm spherical silica particles that are covalently bonded with non-ionic carbamoyl groups, TSKgel Amide‑80 2 µm provides the same unique selectivity as the well‑established TSKgel Amide-80 3 µm or 5 µm. The 2 µm material improves peak capacity and sensitivity for both (U)HPLC and LC–MS analysis and allows a smooth transfer of established methods form HPLC to UHPLC.

Material and Methods

UHPLC Analysis: 

TSKgel Amide-80 2 μm (2.0 mm × 15 cm)
TSKgel Amide-80 3 μm (2.0 mm × 15 cm)

 

A: 200 mmol/L acetic acid + triethylamine (pH 7.3)

B: acetonitrile

75%B (0–5 min), 75–50%B
 (5–80 min, linear)

 

0.5 mL/min

 

40 °C

 

Detection:Fluorescence (EX @ 315 nm, EM @ 380 nm)

50 μL

 

Figure 1: pyridylaminated oligosaccharides released from mAb-1 (mouse)

 

Figure 2: (a) Pyridylaminated oligosaccharides
released from mAb-1 (mouse); (b) Pyridylaminated oligosaccharides released from mAb-2 (human); (c) PA-glucose ladder (3–22 mer) (TaKaRa Bio).

 

 

LC–MS Analysis: 

TSKgel Amide-80 2 μm (2.0 mm × 15 cm)

 

A: 50 mmol/L HCOONH4, pH 7.5
B: acetonitrile

 

75%B (0–5 min), 75–50%B
(5–30 min, linear)

 

0.3 mL/min

 

40 °C

 

(a) Fluorescence (EX @ 315 nm, EM @ 380 nm)
(b)    LC–MS, ESI positive, SIM (Shimadzu LCMS-8030)

 

50 μL

 

2-AB labelled N-glycans released from human IgG (Ludger, cat.# CLIBN-IGG-01)

 

 

Results and Conclusion
The new TSKgel Amide-80 2 µm phase shows a 1.4-fold higher resolution of PA-glycan peaks (Figure 1).

 

 

Maximum pressure drops of TSKgel Amide-80 2 µm do not exceed 55 MPa during gradient at the conditions used (flow rate 0.5 mL/min). The suitability of the new 2 μm material for glycosylation analysis of labelled glycans by both fluorescence detection (Figure 2) and mass spectrometric detection (Figure 3) are demonstrated for various antibody samples.

 

 

 

 

 

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