Separation of Glycans by Shodex™ NH2P Amino HILIC Column

In this application, we present the feasibility of NH2P-50 4E, polymer-based amino HILIC column, for the structure analysis of N-glycans in biological samples. Characterization of glycan is important since different biological properties of glycoprotein play unique roles on pharmacokinetics, stability, and antigenicity.

A mixture of eight different N-glycans modified with 2-aminopyridine (PA) was used as a test sample. Pyridylamino derivatization is one of the common labelings used for the analysis of very low-concentration glycan. The method provides a detection limit of 30 fmol using a fluorescence detector (1) or even lower with a mass spectrometer (MS).

Figure 1: Separation of eight PA-sugar chain by NH2P-50 4E. Eluent; (A) CH3CN (B) 200 mM Acetic acid (pH 7.3) Gradient A/B = 65/35 to A/B = 50/50 over 60 min. Flow rate; 0.5 mL/min, Temp.; 40 °C, Detector; Florescence detector (Ex: 310 nm, Em: 380 nm). Sample PA-sugar chain stds: 0.125 pmol/µL each, 5 µL. 1: Std.012, 2: Std.013, 3: Std.014, 4: Std.001, 5: Std.015, 6: Std.002, 7: Std.010, 8: Std.004.

Various LC separation modes have been developed for glycan analysis. HILIC mode is one of the separation modes generally used in combination with others: Because of the complexity and the diversity of glycans possibly present in a biological sample, multiple separation modes are often required.

Experimental Conditions

Separation was carried by Asahipak NH2P-50 4E (4.6 × 250 mm, 5 µm); column heated at 40 °C and flow rate at 0.5 mL/min. A mixture of (A) acetonitrile and (B) 200 mM acetic acid (pH = 7.3, pH adjusted by triethylamine) was used as a mobile phase. The linear gradient method was programmed from A/B = 65/35 to A/B = 50/50 over 60 min. Sample contained 0.125 pmol/µL each of eight different PA-sugar chain standards (Takara Bio Inc., Otsu, Shiga, Japan), diluted with mobile phase (A/B = 65/35). Injection volume of 5 µL was used for the experiment. A fluorescence detector was used for the measurement using excitation wavelength at 310 nm and emission wavelength at 380 nm.

Figure 2: Structures of PA-sugar chain analyzed in this study.

Results

Good separation was obtained for most PA-sugar chains that were analyzed. The PA-sugar chains were separated based on their structure differences. A baseline separation was not achieved for PA-sugar chain 001 and 014, however the structure analysis can be completed if a MS was used as a detector in place of fluorescence detector.

NH2P series column is available in different sizes. Our newest member of the series, NH2P-40 3E (3.0 × 250 mm, 4 µm) can be used with a conventional HPLC system with improved resolution, while NH2P-50 2D (2.0 × 150 mm, 5 µm) is recommended for MS analysis.

In addition to the PA labeled sugar chains, the NH2P series column is also appropriate for the separation of sugar chains modified with other labels: N-glycans in Human IgG, labeled with 2-aminobenzoic acid (2AA) were separated using NH2P40-2D (2.0 × 150 mm, 4 µm) (Data available in Shodex™ catalog).

Conclusions

Shodex™ polymer-based amino HILIC column, NH2P-50 4E, can be used to separate selected N-glycans modified with fluorescent label. The method provides femtomole level analysis of N-glycans with a fluorescence detector.

References

(1) Takara Bio Inc. html:/catalog.takara-bio.co.jp.

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