Separation Technology

[1 ] Glycan Separation Technology

Glycoprotein analysis can involve identifying complex N and 0-Linked structures composed of frequently similar and repeating moieties. Hydrophilic-interaction liquid (HILIC) with fluorescence detection is a well-recognized and reliable technique that effectively separates and quantitates isolated after their derivatization with fluorescent labels. Waters ACQUITY UPLC® BEH Glycan column was specifically developed and QC tested to provide superior UPLC® component resolution in less time for a range of glycan structures. When coupled to Waters ACQUITY UPLC system, our column chemistry is ideally suited to help users get the correct answers faster.

■ Improved component resolution in less time compared to existing HPLC-based methods

■ Optimized for use with ACQUITY UPLC system with fluorescence detection

■ Based on Waters BEH particle and bonding technology for stable and reproducible labeled glycan separations

■ Quality-control tested with relevant labeled glycan standards for consistent batch-to-batch reproducibility ACQUITY UPLC BEH Glycan Column Separation 1. 2AB 6,7. 2AB 12. 2AB of 2-AB Labeled Human IgG Glycans

2. 2AB 13. 2AB 8. 2AB

3. 2AB 9. 2AB

6 4. 2AB N-Acetylglucosamine 10. 2AB 2 Manose

Galactose 5. 2AB 11. 2AB 11

7

13 3 4 8 5 9

1 10 12

10 15 20 25 30 35 min

UPLC Conditions Sample: 2AB-labeled human-IgG N-glycans Gradient: Time Flow rate (ProZyme, San Leandro, CA, P/N GKSB-005) (min) (mL/min) % A % B Curve Sample Concentration: 10 pmol/µL Init 0.5 25 75 6 Sample Volume: 1.5 µL 46.5 0.5 40 60 6 Injection Mode: Partial Loop 48 0.25 100 0 6 Column: ACQUITY UPLC BEH Glycan, 2.1 x 150 mm 49 0.25 100 0 6 Eluent A: 100 mM Ammonium Formate, pH 4.5 50 0.5 25 75 6 Eluent B: Acetonitrile 63 0.5 25 75 6 Temperature: 60 °C Instrument Configuration: Mixer (P/N 205000403). Detection: Fluorescence: λex = 330 nm, λem = 420 nm Needle (P/N 205000507). No in-line filter. No VanGuard™ column.

The N-linked glycans from pooled human IgG contain high as well as neutral, and sialylated complex structures. The chromatogram shows 35 minutes of a one hour analysis. This challenging sample includes high mannose, bisecting GlcNAc and sialylated species. GLYCAN separation technology

Glycans Critical in Many Biological Processes

Glycosylation is one of most common forms of post-translational modification (PTM) of human and other eukaryotic proteins. Glycosylated proteins () make up 50-70% of human proteins while approximately 30% of the cell surface is made up of and glycoproteins. Consequently glycans play critical roles in a myriad of physiological and pathological reactions ranging from immunity, to blood clotting, to cell development, and death.

Facilitates binding to viruses and bacteria

Modulates function Assists in cell-to-cell Affects antibody communication stability

Part of receptor that interacts with cell growth factors

Modifies Protein Function

[4 ] GLYCAN separation technology

High Quality Separations of Varied Glycan Structures

Several complementary analytical techniques are routinely used to characterize, identify, and quantitate isolated from glycoproteins. Hydrophilic-interaction liquid chromatography (HILIC) is a well-recognized and reliable technique that can effectively separate fluorescently-labeled glycan. Waters ACQUITY UPLC BEH Glycan column was specifically designed for use on Waters ACQUITY UPLC System to give improved component resolution compared to traditional LC techniques for more certain identification and reliable quantitation.

ACQUITY UPLC BEH Glycan Column Separation of 2AB-Labeled Dextran Ladder

UPLC Conditions Sample: 2AB-labeled Dextran Ladder in Glucose Units Sample Concentration: 10 pmol/µL Numbers on peaks are GU units, Sample Volume: 1.5 µL not compounds on page 3 key. Injection Mode: Partial Loop 11 12 9 10 Column: ACQUITY UPLC BEH Glycan, 2.1 x 150 mm 13 8 14 Eluent A: 100 mM Ammonium Formate, pH 4.5 7 15 Eluent B: Acetonitrile 6 1 16 Temperature: 60 °C 2 5 Detection: Fluorescence: λex = 330 nm, λem = 420 nm 3 4 17 Gradient: Time Flow rate 18 (min) (mL/min) % A % B Curve 19 Init 0.5 25 75 6 20 21 46.5 0.5 50 50 6 22 48 0.25 100 0 6 49 0.25 100 0 6 50 0.5 25 75 6 63 0.5 25 75 6

51015202530354045 min

The fast, high-resolution separation of a homopolymeric series of glucose oligomers illustrates the wide range of glycan sizes, extending beyond 22 glucose units that can be separated with the ACQUITY UPLC BEH Glycan column. These labeled glycans can provide calibration reference points that are helpful in identifying more complex glycans based on relative retention times.

ACQUITY UPLC BEH Glycan Column Separation of 2AB-Labeled High Mannose Glycans from Ribonuclease b

UPLC Conditions 2AB 4. Mannose 8 2AB Sample: 2AB-labeled ribonuclease b N-glycans 1. Mannose 5 Sample Concentration: 100 pmol/µL Sample Volume: 1.5 µL Injection Mode: Partial loop 2. Mannose 6 2AB 5. Mannose 9 2AB Column: ACQUITY UPLC BEH Glycan, 2.1 x 150 mm Eluent A: 100 mM ammonium formate, pH 4.5 1 Mannose 7 Eluent B: Acetonitrile 3a, 3b. 2AB isomers Temperature: 60 °C Detection: Fluorescence: λex = 330 nm, λem = 420 nm Gradient: Time Flow rate (min) (mL/min) % A % B Curve 2* Init 0.5 25 75 6 46.5 0.5 40 60 6 48 0.25 100 0 6 49 0.25 100 0 6 63 0.5 25 75 6 4* 3* 5

10 15 20 25 30 35 min

A series of high mannose oligosaccharides was released from bovine ribonuclease b and labeled with 2-AB. The HILIC separation performed on ACQUITY UPLC Glycan column shows both different numbers of mannose residues and isomeric structures.

[5 ] GLYCAN separation technology

ACQUITY UPLC BEH Glycan Column Separation of 2AB Labeled Glycans from Fetuin

2AB UPLC Conditions 1 - 2 Sample: 2AB-labeled Fetuin N-glycans (ProZyme, San Leandro, CA, P/N GKSB-002) Sample Concentration: 10 pmol/µL 2AB 1 - 4 Sample Volume: 1.5 µL Injection Mode: Partial Loop Column: ACQUITY UPLC BEH Glycan, 2.1 x 150 mm Eluent A: 100 mM Ammonium Formate, pH 4.5 Triantennary Eluent B: Acetonitrile Temperature: 60 °C Detection: Fluorescence: λex = 330 nm, λem = 420 nm Gradient: Time Flow Rate (min) (mL/min) % A % B Curve 2AB Init 0.5 25 75 6 1 - 2 46.5 0.5 40 60 6 48 0.25 100 0 6 2AB Biantennary 49 0.25 100 0 6 1 - 4 63 0.5 25 75 6

30 32 34 36 38 40 42 44 46 48 min

Bovine fetuin provides a challenge to glycan analysis because the population of glycans includes charged species with different numbers of charges on the substituents. Waters BEH Glycan column is able to separate and resolve the different number of branches based on degree of N-acetyl substitutions. Note that the isomeric triantennary structures are particularly well-resolved.

Absence of Detectable Carryover between ACQUITY UPLC BEH Glycan Column Injections

UPLC Conditions Sample: 2AB-labeled IgG N-glycans (ProZyme, San Leandro, CA, P/N GKSB-005) Sample Concentration: 10 pmol/µL Sample Volume: 1.5 µL Injection Mode: Partial Loop Column: ACQUITY UPLC BEH Glycan, 2.1 x 150 mm Eluent A: 100 mM Ammonium Formate, pH 4.5 Eluent B: Acetonitrile Temperature: 60 °C Detection: Fluorescence: λex = 330 nm, λem = 420 nm Gradient: Time Buffer A Buffer B (min) (100 mM ammonium formate) (acetonitrile) Initial 25% 75% 46.5 40% 60% 47 25% 75% 60 25% 75% 106.5 40% 60%

20 40 60 80 100 min

Gradient without sample injection

The 2AB-labeled human IgG mixture was chosen to test column sample carryover because it includes a wide range of chemically disparate structures. Following the analysis of the standard injection shown here, the column was re-equilibrated. The gradient was repeated immediately without an injection. Peaks that elute near the same elution solvent strength in this second gradient analysis represent carryover. No carryover can be detected for the structures in this complex mixture.

[6 ] GLYCAN separation technology

Improved Labeled Glycan Resolution and Solvent Saving Benefits of Waters UPLC Technology

Combining Waters ACQUITY UPLC systems and column chemistries reduce analysis time and cost-per-sample, while improving resolution. A key component to achieving these results is our patented sub-2 μm hybrid particle technology that outperforms traditional HPLC using standard 3 µm or larger particle sizes. Tests comparing UPLC to HPLC for the analysis of 2AB-labeled glycans from human IgG show that UPLC is three times faster. ACQUITY UPLC labeled glycan analyses with a 150 mm length column offer 8X solvent savings over traditional HPLC methods. For separations requiring less resolution, the ACQUITY UPLC BEH Glycan 1.7 µm, 2.1 x 50 mm column can be an effective solution yielding an additional 3X solvent savings compared to 150 mm columns.

2AB-labed Glycan chromatography on ACQUITY UPLC BEH Glycan, 1.7um vs. Traditional, 3 µm HPLC Column

UPLC Conditions Sample: 2AB-labeled human-IgG N-glycans Eluent A: 100 mM Ammonium Formate, pH 4.5 (ProZyme, San Leandro, CA, P/N GKSB-005) Eluent B: Acetonitrile Sample Concentration: 10 pmol/µL Temperature: 60 °C Sample Volume: 1.5 µL Detection: Fluorescence: λex = 330 nm, λem = 420 nm Injection Mode: Partial Loop Column: ACQUITY UPLC BEH Glycan, 2.1 x 150 mm

UPLC Gradient:

Time Flow rate (min) (mL/min) % A % B Curve Init 0.5 25 75 6 46.5 0.5 40 60 6 UPLC 48 0.25 100 0 6 49 0.25 100 0 6 1.7 µm 50 0.5 25 75 6 63 0.5 25 75 6

10 15 20 25 30 35 min

HPLC Gradient:

Time Flow rate (min) (mL/min) % A % B Curve HPLC Init 0.2 25 75 6 3 µm 115 0.2 40 60 6 117 0.2 100 0 6 119 0.2 100 0 6 120 0.2 25 75 6 145 0.2 25 75 6

30 40 50 60 70 80 90 min

The 2AB labeled human IgG N-glycans were separated on ACQUITY UPLC BEH Glycan column (1.7 µm) and a typical 3 µm HPLC amide column. The conditions were scaled to give a constant gradient slope while remaining within the pressure limits of the columns. Both columns were tested on the same UPLC system to ensure the best resolution. The UPLC column gives better resolution for more robust analytical methods, greater confidence in peak identity, and enhanced accuracy and precision. The UPLC run time is also significantly shorter, providing higher throughput analyses.

[7 ] GLYCAN separation technology

Column Lengths that Satisfy Resolution and Sample Throughput Requirements

UPLC Conditions Sample: 2AB-labeled human-IgG glycans Eluent A: 100 mM Ammonium Formate, pH 4.5 (purchased from ProZyme) Eluent B: Acetonitrile Sample Concentration: 1 pmol/µL Temperature: 60 °C Sample Volume: 5 µL Detection: Fluorescence: λex = 330 nm, λem = 420 nm Injection Mode: Full Loop Column: ACQUITY UPLC BEH Glycan, 2.1 x 150 mm

2.1 x 150 mm column Gradient: Time Flow rate 3 (min) (mL/min) % A % B Curve Init 0.4 28 72 6 2 4 6 45 0.4 38 62 6 2.1 x 150 mm 45.1 0.25 80 20 6 11 50.0 0.25 80 20 6 50.1 0.4 28 72 6 3 7 60 0.4 28 72 6 4 13 8 9 1 5 10 12

2.1 x 100 mm column 3 Gradient: Time Flow rate 4 (min) (mL/min) % A % B Curve Init 0.4 28 72 6 30 0.4 38 62 6 2.1 x 100 mm 30.1 0.4 80 20 6 35.0 0.4 80 20 6 35.1 0.4 28 72 6 42 0.4 28 72 6

2.1 x 50 mm column 3 Gradient: Time Flow rate (min) (mL/min) % A % B Curve 4 Init 0.4 28 72 6 15 0.4 38 62 6 2.1 x 50 mm 15.1 0.4 80 20 6 18.0 0.4 80 20 6 18.1 0.4 28 72 6 25.0 0.4 28 72 6

0 5 10 15 20 25 30 35 40 min

ACQUITY UPLC BEH Glycan columns are available in three distinct column lengths for various application needs that balance resolution, speed of analysis, and solvent consumption. Shown here are three separations of the same 2-AB labeled IgG glycan sample noting comparative Rs for respective peaks 3 and 4 from each analysis.

[ 8 ] GLYCAN separation technology

Manufacturing and Application Specific Testing for Increased CUSTOMER Assurance

Waters second generation hybrid particles are the basis for our new labeled glycan analysis UPLC column. Using a proprietary bonding process, we’ve developed a robust, amide-based, ligand chemistry for this demanding application. In addition, extensive quality control testing with relevant labeled glycan standards, help us ensure consistent column-to-column performance.

■ Well characterized, state-of-the-art bonding procedures for short amine-containing ligand

■ Particle structure and bonding chemistry stable at low pH and at elevated temperature

■ Quality-control tested with a relevant labeled glycan standard mixture

■ Consistent labeled glycan separations from batch-to-batch

ACQUITY UPLC BEH Glycan Columns Certificate of “Application Specific” Analysis

Each batch of BEH Glycan column material is rigorously tested to ensure consistent performance. The Certificate of Analysis included with each column reports physical, chemical, and chromatographic tests obtained from the actual separation of a qualified 2-AB Labeled Human IgG standard.

Anal. Chem. 2003, 75, 6781-6788, U.S. Patent No. 6,686,035 B2 [ 9 ] GLYCAN separation technology

Batch-to-Batch Manufacturing Consistency of ACQUITY UPLC BEH Glycan Columns

UPLC Conditions Sample: 2AB-labeled human-IgG glycans 6 Sample Concentration: 1.0 pmol/µL 2 11 Sample Volume: 2.0 µL 7 Injection Mode: Full Loop 3 13 Batch A 4 5 8 9 Column: ACQUITY UPLC BEH Glycan, 1 10 12 2.1 x 150 mm Eluent A: 100 mM Ammonium Formate, pH 4.5 Eluent B: Acetonitrile Weak Needle Wash: Acetonitrile/HPLC grade water Batch B Temperature: 60 °C Detection: Fluorescence: λex = 330 nm, λem = 420 nm Gradient: Time Flow rate (min) (mL/min) % A % B Curve Init 0.5 22.0 78.0 6 Batch C 38.5 0.5 44.1 55.9 6 39.5 0.25 100.0 0.0 6 44.5 0.25 100.0 0.0 6 46.5 0.5 22.0 78.0 6 Total Run Time: 50 minutes Batch D

Batch E

Batch F

10 12 14 16 18 20 22 24 26 28 30 min

Each batch of ACQUITY UPLC BEH Glycan column packing material is physically, chemically, and chromatography tested using a well characterized 2AB-labeled glycan standard. Results are rigorously evaluated to ensure that each batch of material will create columns that provide consistent and predictable performance for this demanding application.

Glycan and 2AB-Labeled Glycan Sample Preparation

Efficient protein deglycosylation and glycan isolation are two key requirements for the successful LC separation and quantitation of these compounds. The MassPREP Glycoanalysis Kit offers a robust sample preparation strategy for the LC with fluorescent detector analysis of 2-AB labeled glycans.

For more information, visit www.waters.com/biosep

Waters RapiGest™ SF, MassPREP™ Glycoanalysis Kit, and HILIC µElution plate technology are available to assist in preparing samples prior to analysis.

[10 ] Ordering Information

Ordering information for Waters labeled-Glycan offerings for advanced UPLC glycan separations are shown below. More information can be found at www.waters.com/GST

Column Description Part Number ACQUITY UPLC BEH Glycan, 1.7 μm, 2.1 x 50 mm 186004740 ACQUITY UPLC BEH Glycan, 1.7 μm, 2.1 x 100 mm 186004741 ACQUITY UPLC BEH Glycan, 1.7 μm, 2.1 x 150 mm 186004742 ACQUITY UPLC BEH Glycan, 1.7 μm, VanGuard Pre-Column (3 pack) 186004739

Note: ACQUITY UPLC BEH Glycan, 1.7 μm columns are designed for use with the ACQUITY UPLC system. The benefits of the small particle packing in ACQUITY UPLC BEH Glycan, 1.7 μm columns are only realized with the low system volume and low detector dispersion of an ACQUITY UPLC system.

Sample Preparation Description Part Number RapiGest SF* 1 mg vial 186001860 RapiGest SF 1 mg vial (5 pack) 186001861 RapiGest SF 10 mg vial 186002123 RapiGest SF 50 mg vial 186002122 RapiGest SF custom 186002118 MassPREP Glycoanalysis Kit including RapiGest SF 186002817 MassPREP HILIC µElution Plate 186002780

* RapiGest SF Surfactant is a reagent used to enhance the quality and coverage of enzymatic digestion of proteins.

ACQUITY UPLC System with Fluorescence Detector

[11 ] Sales Offices

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©2009 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY UPLC, UPLC, The quality management system of Waters’ manufacturing facilities VanGuard, BEH Technology, RapiGest, and MassPREP are trademarks of Waters Corporation. in Taunton, Massachusetts and Wexford, Ireland complies with the Interna- tional Standard ISO 9001:2000 Quality Management and Quality Assurance ProZyme is a registered trademark of ProZyme, Inc. Standards. Waters’ quality management system is periodically audited by the registering body to ensure compliance. 720002981EN March 2009 IH-AC

[12 ]