Glycan Separation Technology [1 ] Glycan Separation Technology Glycoprotein analysis can involve identifying complex N and 0-Linked structures composed of frequently similar and repeating sugar moieties. Hydrophilic-interaction liquid chromatography (HILIC) with fluorescence detection is a well-recognized and reliable technique that effectively separates and quantitates isolated glycans 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 Fucose 10. 2AB 2 Manose Sialic Acid 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 mannose 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 (glycoproteins) make up 50-70% of human proteins while approximately 30% of the cell surface is made up of glycolipids 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 antibody 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 oligosaccharides 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 1 - 2 Init 0.5 25 75 6 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 neuraminic acid 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.