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Separation of 2AB Labeled N-Glycans from Bovine Fetuin on a Novel Mixed-Mode Stationary Phase Udayanath Aich, Xiaodong Liu, Srinivasa Rao, and Chris Pohl Thermo Fisher Scientific, Sunnyvale, CA, USA

IntroductionGlycans are involved in a wide range of biological and physiological processes, including recognition and regulatory functions, cellular communication, gene expression, cellular immunity, growth, and development. The functions of glycans are often dependent on the structure and types of oligosaccharides attached to proteins. Glycans are commonly investigated as important species in therapeutic protein drug development because there is strong evidence that bioactivity and efficacy are affected by glycosylation. Understanding, measuring, and controlling glycosylation in glycoprotein-based drugs, the oligosaccharide content of glycoprotein products, as well as thorough characterization of biosimilars has become increasingly important. The structures of glycans are highly diverse, complex, and heterogeneous due to post-translational modifications. Thus, it is challenging to comprehensively characterize glycan profiles and determine their structures [1].

Various HPLC separation modes have been used for the analysis of glycans, including normal phase (NP) or hydrophilic interaction (HILIC) chromatography, ion-exchange (IEX) chromatography, and reversed-phase (RP) chromatography. Because glycans are highly hydrophilic polar substances, neutral glycans may be commonly separated using amide HILIC columns, such as the Thermo Scientific™ Accucore™ 150-Amide-HILIC column [2], which separates glycans by hydrogen bonding, resulting in a size and composition-based separation. This type of column is particularly useful for

Key WordsGlycanPac AXH-1, mixed-mode column technology, N-glycans, proteins, HILIC, anion-exchange, charge based separation, glycan quantification

AbstractThis application note demonstrates the analysis of fluorescently labeled N-glycans released from proteins (bovine fetuin) and separated by hydrophilic interaction liquid chromatography (HILIC) / weak anion exchange mixed-mode chromatography. The separation is carried out with a 1.9 µm Thermo Scientific™ GlycanPac™ AXH-1 column using fluorescence detection. The method exhibits excellent separation based on charge, size, and polarity. This phase also provides a unique tool for quantifying glycans based on charge.

the separation of glycans released from monoclonal antibodies, the majority of which are neutral glycans [3]. Based on novel mixed-mode surface chemistry, the GlycanPac AXH-1 column combines both weak anion-exchange (WAX) and HILIC retention mechanisms for optimal selectivity and high resolving power [4]. The WAX functionality provides retention and selectivity for negatively charged glycans, while the HILIC mode facilitates the separation of glycans of the same charge according to their polarity and size. As a result, the GlycanPac AXH-1 column provides unparalleled capabilities for glycan separations. In addition, this column has the flexibility to be used in a purely ion exchange, charge-based separation mode for the separation of various glycans without discrimination of size and polarity. This makes it a suitable tool for accurate quantification of glycans based on charge, which cannot be achieved with any other HPLC/UHPLC columns on the

market. The GlycanPac AXH-1 column is designed and tested for use with LC-fluorescence detection and LC-MS applications using volatile aqueous buffers (e.g., ammonium acetate or ammonium formate) and acetonitrile. The substrate of the GlycanPac AXH-1 column is based on high purity spherical silica. The column is available in both 1.9 μm and 3.0 μm sizes for UHPLC and standard HPLC applications, respectively.

2

Experimental Details

Consumables Part Number

GlycanPac AXH-1, 1.9 μm, Analytical column (2.1 × 150 mm) 082472

Deionized (D.I.) water, 18.2 MΩ-cm

Fisher Scientific™ HPLC grade acetonitrile (CH3CN) AC610010040

Fisher Scientific LC/MS grade formic acid A117-50

Ammonium formate

Equipment

Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLC Analytical LC system consisting of a DGP-3600RS pump, TCC-3000RS thermal compartment, WPS-3000TRS autosampler, FLD3400RS fluorescence detector with Dual-PMT, and a 2 µL micro flow cell

Buffer Preparation

Ammonium formate (100 mM, pH = 4.4): Dissolve 6.35 ± 0.1 g of ammonium formate and 0.70 g of formic acid in 999.43 g of D.I. water. Sonicate the resulting solution.

Sample Preparation

1. Unlabeled nonreduced glycans are released from glycoproteins with PNGase F enzyme. The released glycans are conjugated with 2-amino benzamide (2AB) label group with reported procedure of Bigge et. al. [5]

2. Dissolve 2AB labeled N-glycan from fetuin or individual labeled standards (approximately 5000 pmol each) in 25 µL D.I. water in a 250 µL autosampler vial.

3. Add 75 µL acetonitrile to the same vial and mix till uniformity. Note: Store the standard at -20 °C.

Separation Conditions

Column: GlycanPac AXH-1 (1.9 µm, 2.1 × 150 mm) column

Mobile phase: A: Acetonitrile

B: D.I. water

C: Ammonium formate (100 mM, pH =4.4)

Gradient: Time %A %B %C Flow Rate Curve (min) (mL/min)

-10 78 20 2 0.4 5

0 78 20 2 0.4 5

30 70 20 10 0.4 5

35 60 20 20 0.4 5

40 50 20 30 0.4 5

Column backpressure: 350 bar

Column temperature: 30 °C

Injection volume: 1 µL

Fluorescence detector: λEx

= 320 nm & λEm

= 420 nm

Data Processing

Chromatographic software: Thermo Scientific™ Chromeleon™ 6.8 Chromatography Data System

3ResultsGlycan Separation by Charge, Size, and Polarity Figure 1 shows the separation of neutral and acidic 2AB labeled N-glycan from bovine fetuin using a GlycanPac AXH-1 (1.9 μm, 2.1 × 150 mm) column. The glycan elution profile consists of a series of peaks grouped into several clusters in which the neutral glycans elute first, followed by monosialylated, disialylated, trisialylated, tetrasialylated, and finally pentasialylated species. Analytes in each cluster represent the glycans of the same charge. Within each cluster, the glycans having the same charge are further separated according to their sizes and polarity by HILIC interaction. The structural assignment for each identified peak is based on MS data and listed in Table 1. Figure 2 shows a high throughput separation of 2AB labeled N-glycans from bovine fetuin, with analysis time shortened from 40 min to 20 min.

Peak Compound Stucture (2AB labeling are not shown)

1

2

4

5

6

7

8

9

10

11a

11b

11c

12a

12b

13

14

15

Peak Compound Stucture (2AB labeling are not shown)

16

17

18

19

20

21

22

23

24

25

26

Table 1: Structural characterization of 2AB labeled N-glycans from bovine fetuin using GlycanPac AXH-1 column

N-acetyl glucosamine (GlcNAc)

Mannose (Man)

Galactose (Gal)

N-acetyl neuraminic acid (Neu5Ac)

N-glycolyl neuraminic acid (Neu5Gc)

L-fucose (L-Fuc)

4 Charge-Based Quantitative Determination of Glycans Accurate and easy quantitative analysis of glycans of each charge state is essential for assessing glycan variation in different protein batches and to compare diseased cell glycosylation profiles to normal ones. In addition, quantitative analysis of glycans based on charge state provides a unique tool for determining the relative amounts of glycans based on the number of sialic acid linkages after enzymatic digestion with sialidase S and sialidase A. Figure 3 shows an example of charge-based separation of 2AB labeled N-glycans using a GlycanPac AXH-1 column (1.9 μm, 2.1 × 150 mm) with fluorescence detection.

Column: GlycanPac AXH-1 (1.9 µm)Dimension: 2.1 × 150 mmMobile Phase A: Acetonitrile (100%) Mobile Phase B: WaterMobile Phase C: Ammonium formate (100 mM, pH = 4.4)Flow Rate: 0.4 mL/minInjection Volume: 50 PmolesTemperature: 30 °CDetection: Fluorescence at 320/420 nm Sample: 2AB Labeled N-glycan from bovine fetuin

Figure 1: Separation of 2AB labeled N-glycans from bovine fetuin by charge, size and polarity

Fluo

resc

ence

Cou

nts

400

0

7E5

10 20 30

14

5

78

1112

13

14

15

17

16

18

20

2122

23

242 3 6 25 269 10

neutral

monosialylated

disialylated

19

trisialylated

tetrasialylated

pentasialylated

Minutes

Time (min) % A % B % C Flow

(mL/min) Curve

-10 78 20 2 0.4 5

0 78 20 2 0.4 5

30 70 20 10 0.4 5

35 60 20 20 0.4 5

40 50 20 30 0.4 5

Column: GlycanPac AXH-1 (1.9 µm)Dimension: 2.1 × 150 mmMobile Phase A: AcetonitrileMobile Phase B: Ammonium formate (50 mM, pH = 4.4)Mobile Phase C: Water

Flow Rate: 0.4 mL/minInjection Volume: 40 pmoleTemperature: 30 °CDetection: Fluorescence at 320/420 nmSample: 2AB Labeled N-glycans from bovine fetuin

Figure 3: Charge-based quantitative separation of 2AB labeled N-glycans from bovine fetuin

3

2

7.00 8.00

1

Minutes 9.00 10.00 11.00

16E6

Fluo

resc

ence

Cou

nts

12.00

6

5

4Peak Glycan Type Relative %

1 Neutral 0.4

2 MonoSialic 8.6

3 DiSialic 38.4

4 TriSialic 45.4

5 TetraSialic 7.0

6 PentaSialic 0.2

Time (min) % A % B % C Flow

(mL/min)-5 90 10 0 0.4

0 90 10 0 0.4

6 50 20 30 0.4

12 50 20 30 0.4

Figure 2: High-throughput separation of 2AB labeled N-glycans from bovine fetuin

Column: GlycanPac AXH-1 (1.9 µm)Dimension: 2.1 × 150 mmMobile Phase A: Acetonitrile (100%) Mobile Phase B: WaterMobile Phase C: Ammonium formate (100 mM, pH = 4.4)Flow Rate: 0.4 mL/minInjection Volume: 50 PmolesTemperature: 30 °CDetection: Fluorescence at 320/420 nm Sample: 2AB Labeled N-glycan from bovine fetuin

10.0 20.0

0 1 2 3 4

5 6 7 8

9 10

11 12

13

14

15

16 17

18

19

20

21

22 23

24 25 26

3.0

neutralmonosialylated

disialylated

trisialylated

tetrasialylated

pentasialylated

Fluore

scence

Count

s

35E6

Minutes

Time (min) % A % B % C Flow

(mL/min) Curve

-10 75 20 5 0.4 5

0 75 20 2 0.4 5

30 75 20 10 0.4 5

35 60 20 20 0.4 5

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thermoscientific.com/glycanpac © 2013 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.

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ConclusionThe Thermo Scientific GlycanPac AXH-1 column is a high- performance, silica-based HPLC/UHPLC column for simultaneous separation of glycans by charge, size, and polarity. It is designed for high-resolution and high-throughput analysis with unique selectivity for biologically important glycans. We have demonstrated that this column provides unique selectivity and excellent resolution for glycans released from fetuin. In addition, it is suitable for quantification of glycans based on charge [4,6].

Reference[1] Bertozzi, C. R.; Freeze, H.H.; Varki, A. and Esko, J.D. Glycans in Biotechnology and the Pharmaceutical Industry, Essentials of Glycobiology. 2nd edition. Chapter 51, Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009.

[2] Thermo Scientific Accucore HPLC Columns Technical Guide: http://thermo.dirxion.com/ accucore

[3] Thermo Scientific Application Note 20703: “Analysis of Human IgG Glycans on a Solid Core Amide HILIC Stationary Phase”: https://static.thermoscientific.com/images/D22217~.pdf

[4] Thermo Scientific GlycanPac AXH-1 Product Specification and Data Sheet: http://www.dionex.com/en-us/webdocs/114170-PS-GlycanPac-AXH1-Column-PS20695_E.pdf

[5] Bigge, J. C. et al., Non-selective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. Analytical Biochemistry, 1995, 230, 229-238.

[6] Thermo Scientific GlycanPac AXH-1 Product Manual: http://www.dionex.com/en-us/products/ columns/bio/Glycan/lp-114099.html