Barry Boyes 1,2, Shujuan Tao 2, and Ron Orlando 2

Transcription

1 Barry Boyes 1,2, Shujuan Tao 2, and Ron Orlando 2 1 Advanced Materials Technology, Inc. Wilmington, DE USA 2 Complex Carbohydrate Research Center University of Georgia, Athens, GA USA bboyes@advanced-materials-tech.com

2 Agenda Protein N-glycan analysis by release, labeling and HILIC General workflow Fused-Core Particles for highly efficient separations New polar bonded phase for HILIC Analysis of N-Glycans by Penta-HILIC Standards and separation optimization strategy Complexity in action: isobaric glycan separations Implications of Complexity on Analysis of Released Glycans

3 Protein N-linked Glycan Analysis Release of glycoprotein Asn-linked glycans by PNGase F is a well established approach, allow analysis of complex mixtures of glycans by a variety of methods. Various additional exoglycosidases and endoglycosidases can be helpful in structure elucidation. Considerable work has been accomplished for N-linked analysis of native glycans, as well as permethylated glycans using MS (ToF and ESI), and MS coupled to high resolution separation methods, particulary CE and LC. Glycoprofiling of N-linked glycans, using HILIC (NP) combined with fluorescence detection and/or MS is becoming a standard for biopharmaceutical glycoproteins, including antibodies.

4 Analysis of PNGase Released and Labeled N-Glycans Release of protein N-linked glycans using PNGase F releases oligosaccharides with a free reducing terminus (alditol) that is readily labeled by amines via formation of a Schiff s base, which can be reduced readily. Many amines have been applied to labeling glycans, (Harvey, 211, J. Chrom. 879). In the current work Procainamide is favored due to reported improvements in ESI-MS detection (Klapoetke, et al., 21, J. Pharm. Biomed. Anal. 53) Typical Labeling Conditions Glycan in water (up to 1% volume) 9+% volume of:.4 M procainamide 1M sodium cyanoborohydride in 3% acetic acid/7% DMSO hr reaction at 37 C H 2 N SEC cleanup on Sephadex G-1 minicolumn Absorbance Detection 3 nm or Fluorescence Ex 33/Em 38 nm O N H Mass: glycan N

5 Superficially Porous Particles (Fused-Core ) Porous Shell SEM Particle Cross-section Solid Core 1.7 µm 2.7 µm.5 µm Low back pressure due to the particle design (solid core with a porous shell) Specialized high pressure HPLC equipment is optional Not necessary to filter samples and mobile phase since 2 µm frits are not as small as needed for sub-2-µm High resolution is maintained at high flow rates (flat C-term in van Deemter plot)

6 Silane Surface Modifications with Polar Functional Groups Support effective HILIC retention Exhibit desired kinetic advantages of SPP morphology Reduce unfavorable ionic/coloumbic interactions Hydroxylic Functional Groups FG Mono-OL O O H O H L k Di-OL O H H O R 1 Si R 2 Tri-OL R L O H H O R L = silanol reactive leaving R 1 = R 2 or R 1 = R 2, R 1 = R L Eg. -Me, -OMe, -Cl, -DiiPr H O Pentan-OL AKA Halo Penta-HILIC H O O H O H O H

7 Effect of Linear Velocity on Penta-HILIC Column Efficiency 4.6 mm ID x 5 mm; 9% AcN/1 mm NH 4 Form 3., 25 C; 1 ml, 5 ng Adenosine 2D Graph 2 6 Data fitted to Knox Equation 5 Reduced Plate Height 4 3 Silica HILIC 2 1 Penta-HILIC (minima 1.7;.9 ml/min) Mobile Phase Velocity, mm/sec

8 Column Efficiency Comparisons Pam-G5 2.1 (2. mm) ID x 15 mm, 6 C, k 6, 5 mm Ammonium Formate Aqueous, ph ul Injection (5 pmol), Abs. 3 nm Plate Height (µm) 4 Penta-HILIC, 72% AcN P = 147 bar 1.7 µm Amide, 69% AcN 3 3. µm Amide, 67% AcN P = 331 bar 2 P = 718 bar Flow Rate

9 Penta-HILIC Separations of Labeled Oligosaccharides and Glycans 2.1 mm ID x 15 mm; 5 mm Ammonium Formate, ph 4.4, % AcN (B) in 52.5 min., 6 C; 6 ml/min. Detection: 3 nm Abs; ESI-MS (MS-22, (+) 4.2 kv, 4-2 with SIM) 6. mv Detector A 3nm Pam-G min 7 mv Detector A 3nm Man 5 Man Linear Dextran Standard Rs 11,1 = 4.3 Peak Capacity G22-G2 = 214 RNAase B N-linked Glycans Man Man Man Pam-G min

10 Penta-HILIC Retention of Labeled Oligosaccharides and Glycans 2.1 mm ID x 15 mm; 5 mm Ammonium Formate, ph 4.4, % AcN (B) in 52.5 min., 6 C; 6 ml/min. Detection: 3 nm Abs; ESI-MS (MS-22, (+) 4.2 kv, 4-2 with SIM) HILIC Retention of Glycans 6 Dextran RT (min) ManX Retention Time (min) 4 2 B T Fet Antenn Hydroxyls

11 Penta-HILIC Separations of Abundant bov. a1-agp N-glycans 2.1 mm ID x 15 mm; 5 mm Ammonium Formate, ph 4.4, % AcN (B) in 52.5 min., 6 C; 6 ml/min. MALDI of Permethylated Glycans % Intensity (M-CH 2 +Na) mv Detector A 3nm Mass (m/z) HILIC of End-labeled Glycans min

12 HILIC Separations of a1-agp N-glycans 2.1 mm ID x 15 mm; 5 mm Ammonium Formate, ph 4.4, % AcN (B) in 52.5 min., 6 C; 6 ml/min. Detection: 3 nm Abs; ESI-MS ((+) MS-22, 4.2 kv, 4-2 with SIM) 15 mv Detector A 3nm min :176.5(+) : HexNAc 4 Hex 5 NeuAc 1 2:184.5(+) : HexNAc 4 Hex 5 NeuGc 1 2:1222.7(+) : HexNAc 4 Hex 5 NeuAc 2 2:123.(+) : HexNAc 4 Hex 5 NeuAc 1 NeuGc 1 2:1238.(+) : HexNAc 4 Hex 5 NeuGc 2 BNeuAc 2 BNeuAc 1 NeuGc 1 BNeuGc 2 4 BNeuAc BNeuGc min

13 High Resolution HILIC Separations of asialo-fetuin N-glycans 2.1 mm ID x 3 mm; 5 mm Ammonium Formate, ph 4.4, 7-55% AcN (B) in 9min., 6 C; 6 ml/min. Detection: 3 nm Abs; ESI-MS ((+) MS-22, 4.2 kv, 4-2 with SIM) :931.4(+): HexNAc 4 Hex 5 2:1114.1(+): HexNAc 5 Hex 6 2:1187.2(+): HexNAc 4 Hex 5 dhex 1 T B ? min 9 mv Detector A 3nm min mv Detector A 3nm min

14 High Resolution HILIC Separations of Fetuin N-glycans 2.1 mm ID x 3 mm; 5 mm Ammonium Formate, ph 4.4, 7-55% AcN (B) in 9min., 6 C; 6 ml/min. Detection: 3 nm Abs; ESI-MS ((+) MS-22, 4.2 kv, 4-2 with SIM) 1. mv Detector A 3nm min :1551.(+): HexNAc 5 Hex 6 NeuAc 3 2:1696.6(+): HexNAc 5 Hex 6 NeuAc 4 2:1842.2(+)(1.): HexNAc 5 Hex 6 NeuAc TNeuAc TNeuAc TNeuAc min

15 High Resolution HILIC Separations of Fetuin N-glycans 2.1 mm ID x 3 mm; 5 mm Ammonium Formate, ph 4.4, 7-55% AcN (B) in 9min., 6 C; 6 ml/min. Detection: 3 nm Abs; ESI-MS ((+) MS-22, 4.2 kv, 4-2 with SIM) mv Detector A 3nm min :931.4(+): HexNAc 4 Hex 5 2:17.(+): HexNAc 4 Hex 5 NeuAc 1 2:1222.7(+): HexNAc 4 Hex 5 NeuAc BNeuAc (ISD) min

16 HILIC Separations of bov. a1-agp N-glycans (tri-sialated) 2.1 mm ID x 15 mm; 5 mm Ammonium Formate, ph 4.4, % AcN (B) in 52.5 min., 6 C; 6 ml/min. Detection: 3 nm Abs; ESI-MS ((+) MS-22, 4.2 kv, 4-2 with SIM) 5 mv Detector A 3nm min :1367.6(+): HexNAc 4 Hex 5 NeuAc 3 2:1375.6(+): HexNAc 4 Hex 5 NeuAc 2 NeuGc 1 2:1383.6(+): HexNAc 4 Hex 5 NeuAc 1 NeuGc 2 2:1391.6(+): HexNAc 4 Hex 5 NeuGc 3 BNeuAc 2 NeuGc BNeuAc 3 BNeuAc 1 NeuGc BNeuGc min

17 Conclusions A novel hydroxylated Fused-core silica HILIC packing material has high utility for end-labeled Glycan separations. The HILIC material and method described can be operated in high resolution mode, permitting analysis of complex mixtures of released N- linked protein glycan samples to be resolved, or in very high speed mode, to enable high throughput glycan analysis. High resolution HILIC resolves isobaric glycans, which are abundant (and complex). Variations in isobaric glycan profiles between proteins is very possible. Quantitation by end-labeling using UV or Fluorescence detection only could be problematic, detection by MS only will not easily reveal isomers.

18 Acknowledgements Penta-HILIC AMT Thank you for your Attention! Dr. J. DeStefano, Dr. J.J. Kirkland, Dr. S. Schuster NIH for Financial Support NIH NIGMS GM99355, GM93747 Shimadzu Corp early access to high sensitivity flow cell