analysis of nanalysis of n--linked linked glycansglycansof ... · biopharmaceuticals monoclonal...
TRANSCRIPT
Bio Day Bio Day –– DENMARK DENMARK –– MARCH MARCH 20132013
Analysis of NAnalysis of N--linked linked GlycansGlycans of of
GlycoProteinsGlycoProteins
AgendaAgenda
�� Importance of Importance of GlycanGlycan AnalysisAnalysis
� Current Glycoprotein Analysis
� Glycan Characterization: Oligosaccharides
Glycan Analysis: Sample Preparation
©2013 Waters Corporation 2
� Glycan Analysis: Sample Preparation
� Glycan Analysis: Chromatography
� Empower: GU units
� NIBRT Glycobase 3+
� Summary
BiopharmaceuticalsBiopharmaceuticals
Monoclonal Antibodye.g. Herceptin®
GlycanMixtures
©2013 Waters Corporation 3
MW 6,000
Insulin
MW 34,000
EPO
MW 147,000
Glycan Separation TechnologyGlycan Separation Technology
©2013 Waters Corporation 4
Glycosylation FunctionsGlycosylation FunctionsRisks and Regulatory ConcernsRisks and Regulatory Concerns
� Mediates biological activity
� Alteration may eliminate or alter activity
� Immune response triggered by unrecognized glycan
©2013 Waters Corporation 5
� Glycan distribution indicates process stability
� Glycans impact safety and efficacy – Correct and consistent structure of the glycans
– Obtaining the desired medical effect
– Avoid adverse immunological reaction
Oligosaccharide StructuresOligosaccharide StructuresNN--linked and linked and 00--LinkedLinked
� O-linked glycosylation to the hydroxy oxygen of serine or threonine side chains
� N-linked glycosylation to the amide nitrogen of asparagine side chains
©2013 Waters Corporation 6
Isomeric StructuresIsomeric Structures
– The basic carbohydrate units are called monosaccharides
Hexose
HexNAc
©2013 Waters Corporation 7
D-(+)-GalactoseD-(+)-GlucoseD-(+)-Glucose D-(+)-Mannose
N-Acetyl-D-glucosamine
D-(+)-Fucose
N-Acetyl-D-galactosamine
Sialic acid
DeoxHex
Sialic Acid
Glycan Analysis Glycan Analysis Biopharmaceutical Development CycleBiopharmaceutical Development Cycle
Discovery
Development
Formulations &Stability
Clone Selection
Characterization
Target ID
Comparability
©2013 Waters Corporation 8
Production
Post-Approval
QC/QA
PAT- Process Analytical Technologies
ID Test?
ComparabilityGlycan Analysis
GlycoproteinGlycoproteinCharacterization RequirementsCharacterization Requirements
� Selectivity for Key Glycans
� Sensitivity for Minor Glycoforms
� Reproducibility of Separation
©2013 Waters Corporation 9
� Quantitative Results
� Robust Complete Solution
AgendaAgenda
� Importance of Glycan Analysis
�� Current Glycoprotein AnalysisCurrent Glycoprotein Analysis
� Glycan Characterization: Oligosaccharides
Glycan Analysis: Sample Preparation
©2013 Waters Corporation 10
� Glycan Analysis: Sample Preparation
� Glycan Analysis: Chromatography
� Empower: GU units
� NIBRT Glycobase 3+
� Summary
Glycoprotein CharacterizationGlycoprotein CharacterizationWork FlowsWork Flows
Glycoprotein
MW ProfileESI MS
©2013 Waters Corporation 11
Glycopeptides& Peptides N- Oligosachharides
MALDILabel
Peptide MappingUPLC-UVUPLC-MS
HILIC / Fluorescence Detection
Galactose
Glycoprotein Characterization Glycoprotein Characterization Glycoprotein Glycoprotein
6
3
6
©2013 Waters Corporation 12
N-Acetyl glucosamine
Mannose
Fucose
6
3
Glycoprotein
Glycoprotein CharacterizationGlycoprotein CharacterizationLC/MS analysis of Intact IgGLC/MS analysis of Intact IgG
0.5 µg GOF/G0F
G0F/G1F
G1F/G1FG0F/G2F
©2013 Waters Corporation 13
0.5 µg IgG1
DesaltingCartridge
GOF/G0F
G1F/G2F
G0/GOF
G2F/G2F
Deconvoluted Mass SpectrumIgG1
Glycoprotein Characterization Glycoprotein Characterization GlycopeptideGlycopeptide AnalysisAnalysis
6
3
6
3
Glycoprotein
©2013 Waters Corporation 14
N-Acetyl glucosamine
Galactose
Mannose
Fucose
6
3
6
3
Protease Digestion and Separation
Glycopeptides
Glycoprotein
Antibody Glycopeptide Antibody Glycopeptide MicroheterogeneityMicroheterogeneity
AU
0.0
2.0e-3
4.0e-3
6.0e-3
8.0e-3
1.0e-2
UV
IgG Glycopeptides
©2013 Waters Corporation 15The UPLC™ chromatograms of tryptic digest of mouse IgG show resolution of the glycoforms.
Time36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00 54.00 56.00
%
-7
93
36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00 54.00 56.00
0.0
TIC
IgG GlycopeptidesIgG GlycopeptidesUPLC MS and UVUPLC MS and UV
G1F (M+H)+3
m/z =922.00%
47.40 47.60 47.80 48.00 48.20 48.40 48.60 48.80 49.00
%
2
G2F (M+H)+3
m/z =976.02
©2013 Waters Corporation 16
Time47.40 47.60 47.80 48.00 48.20 48.40 48.60 48.80 49.00
%
1
47.40 47.60 47.80 48.00 48.20 48.40 48.60 48.80 49.001
47.60 47.80 48.00 48.20 48.40 48.60 48.80 49.00
1522
825918
575
G0F (M+H)+3
m/z =867.99
UV 214
IgGIgG Complex Complex NN--GlycansGlycans -- GlycopeptidesGlycopeptides
©2013 Waters Corporation 17
N-Acetyl glucosamine
Galactose
Mannose
Fucose
Glycopeptides with HILICGlycopeptides with HILIC
©2013 Waters Corporation 18
720003363en
AgendaAgenda
� Importance of Glycan Analysis
� Current Glycoprotein Analysis
�� GlycanGlycan Characterization: OligosaccharidesCharacterization: Oligosaccharides
Glycan Analysis: Sample Preparation
©2013 Waters Corporation 19
� Glycan Analysis: Sample Preparation
� Glycan Analysis: Chromatography
� Empower: GU units
� NIBRT Glycobase 3+
� Summary
Glycoprotein Characterization Glycoprotein Characterization Glycan AnalysisGlycan Analysis
6
3
6
3
6
3
6
3
Glycan Release
©2013 Waters Corporation 20
N-Acetyl glucosamine
Galactose
Mannose
Fucose
Glycoprotein Oligosaccharides
Studying Studying GlycansGlycans: The Workflow: The Workflow
1) Release glycans from the protein
2) Label the free glycans for detection
3) Separate a complex pool of glycans
©2013 Waters Corporation 21
4) Assign glycan structures to peak
What does this look like What does this look like at the bench?at the bench?
Sample Preparation Glycan Separation Data Processing Data Interpretation
Glycoprotein Sample Identified Glycan Structure(s)
©2013 Waters Corporation 22
2AB Labeling
Excess label removal
Glycan Dry Down
Reconstitution
Formic Acid Treat Sample Set Run Sample Integration
Glycan Dry Down Data Export
Enzymatic Deglycosylation System equilibration Adjust Dextran Amounts GlycoBase 3.1
Free Glycan Capture Dextran Run Process Sample Set Tentative Assignments
Glycan Dry Down Standard Run Calibrate Dextran MS or Enzyme Analysis?
Where does Waters contribute?...
What Waters Offers for What Waters Offers for GlycanGlycan Analysis`Analysis`
Sample Preparation Glycan Separation Data Processing Data Interpretation
Solid Phase Extraction Offerings
Standards and Reagents Offerings
Informatics Offerings
Instrument Offerings
©2013 Waters Corporation 23
2AB Labeling
Excess label removal
General Lab Supplies
General Lab Supplies
General Lab Supplies Sample Set Run Sample Integration
General Lab Supplies Data Export
Enzymatic Deglycosylation System equilibration Adjust Dextran Amounts GlycoBase 3.1
Free Glycan Capture Dextran Run Process Sample Set Analyst Defined
General Lab Supplies Standard Run Calibrate Dextran Analyst Defined
AgendaAgenda
� Importance of Glycan Analysis
� Current Glycoprotein Analysis
� Glycan Characterization: Oligosaccharides
GlycanGlycan Analysis: Sample PreparationAnalysis: Sample Preparation
©2013 Waters Corporation 24
�� GlycanGlycan Analysis: Sample PreparationAnalysis: Sample Preparation
� Glycan Analysis: Chromatography
� Empower: GU units
� NIBRT Glycobase 3+
� Summary
GlycoanalysisGlycoanalysis WorkflowWorkflow
Free Glycans
And Proteins
Denaturation Deglycosylation
Isolation
Glycoprotein
©2013 Waters Corporation 25
RapiGestTM SF
IAM
DTT
GlycoWorks™HILIC µElution Plate
Isolation
of Glycans
Derivatize
with 2-ABUPLC - FLR
Isolation of
Labeled Glycans
Enzymatic Deglycosylation Considerations
� Reproducible and complete release
� Conditions maintain glycanase enzyme activity
� Conditions denature the protein to enhance glycan cleavage
� Conditions keep the protein in solution
©2013 Waters Corporation 26
RapiGest™SF is an acid-labile surfactant that meets these requirements
The Journey of Complex Glycans The Journey of Complex Glycans (1.5 day sample prep. Process Recommended)(1.5 day sample prep. Process Recommended)
Reduction/alkylationusing DTT/IAM-Denature and unfold the protein
Enzymatic removal of N-linked glycans- using PNGase F
Extraction of free glycans - using HILIC SPE device
Lyophilization of the free glycans
Adding 1% formic acid to the lyophilized glycans and set for 30 minutes
1 hrOvernight*
*In order to ensure complete deglycosylation, it is recommended to leave your
sample overnight
©2013 Waters Corporation 27
- using HILIC SPE deviceglycans and set for 30 minutes- to convert glycans to free reducing form
Lyophilize the sample again
FLR derivatization e.g., using 2AB (heating at 65 C˚ for 2 -3 hrs)
Extraction of the 2AB-glycans - using HILIC SPE device
HILIC-UPLC/FLR/MSanalysis
30 min.1 hr40 min
1 hr 30 min30 min
Lyophilize the sample
1 hr
The Journey of Simplier Glycans The Journey of Simplier Glycans (1 day sample prep. Process, 10 hrs)(1 day sample prep. Process, 10 hrs)
Reduction/alkylationusing DTT/IAM-Denature and unfold the protein
Enzymatic removal of N-linked glycans- using PNGase F
Extraction of free glycans - using HILIC SPE device
Lyophilization of the free glycans
Adding 1% formic acid to the lyophilized glycans and set for 30 minutes
1 hr2-4 hrs*
* This step needs to be optimized to your protein and can be less or more
©2013 Waters Corporation 28
- using HILIC SPE deviceglycans and set for 30 minutes- to convert glycans to free reducing form
Lyophilize the sample again
FLR derivatization e.g., using 2AB (heating at 65 C˚ for 2 -3 hrs)
Extraction of the 2AB-glycans - using HILIC SPE device
HILIC-UPLC/FLR/MSanalysis
30 min.1 hr40 min
1 hr 30 min30 min
Lyophilize the sample
1 hr
22--AB (2AB (2--aminobenzamideaminobenzamide))GlycanGlycan LabelingLabeling
©2013 Waters Corporation 29
What is in the What is in the GlycoWorksGlycoWorks™ ™ Kits?Kits?
� Sample clean-up devices- High-throughput: GlycoWorksHILIC micro-elution SPE plate for free glycan and labeled glycan extraction
- Single Use: GlycoWorks HILIC1cc Cartridge (10 pack)
Protein denaturation
� Standard- GlycoWorks Control Standard
©2013 Waters Corporation 30
� Protein denaturation- RapiGest SF (denaturation)
- DTT and IAM(reductive/alkylation)
� FLR labeling chemistry - Acetic acid
- DMSO
- Reductive reagent, Sodium cyanoborohydride
- GlycoWorks Control Standard
� Additional Standards - Dextran Calibration Ladder (2AB-labeled)
- Glycan Performance Test Standard (2AB-labeled)
� Care and Use with Protocol
10
00
0
Dextran
Conventional Dextran Ladder Conventional Dextran Ladder (supplied by Sigma or (supplied by Sigma or ProzymeProzyme))
©2013 Waters Corporation 31
n
20
00
0
30
00
0
40
00
0
50
00
0
60
00
0
70
00
0
80
00
0
90
00
0
10
00
00
11
00
00
12
00
00
13
00
00
14
00
00
15
00
00
Waters Waters GlycoWorksGlycoWorks™ Dextran ™ Dextran LadderLadder
6
7
8
9
1011 12 13
14 1516
1718
19
20
n
Dextran
©2013 Waters Corporation 32Minutes
4
5
2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0 42.0 44.0
Waters Waters GlycoWorksGlycoWorks™ ™ GlycanGlycan Performance Test StandardPerformance Test Standard
• Based on pooled human serum IgG• Spiked with M5 and M6
• Certificate of Analysis using the ACQUITY BEH Glycan Column
(1) ACQUITY FLR ChA Ex330,Em420 nmRange: 1846473
4
5
8
9 10
13
18
FLR
©2013 Waters Corporation 33
Time (minutes)
10 15 20 25 30
TOF MS ES+BPI
4.95e3
1 26
7
9 1011
1214 15 1617
18
19
MS
New New GlycoWorksGlycoWorks™ Product Guide™ Product Guide
©2013 Waters Corporation 34
720004584EN
AgendaAgenda
� Importance of Glycan Analysis
� Current Glycoprotein Analysis
� Glycan Characterization: Oligosaccharides
Glycan Analysis: Sample Preparation
©2013 Waters Corporation 35
� Glycan Analysis: Sample Preparation
�� GlycanGlycan Analysis: ChromatographyAnalysis: Chromatography
� Empower: GU units
� NIBRT Glycobase 3+
� Summary
HILIC HILIC HHydrophydrophililicic--IInteraction nteraction CChromatographyhromatography
� Reversed-Phase Mode– Mid range hydrophilic to hydrophobic compounds– Hydrophobic stationary phase– Polar Solvents– Aqueous to organic gradient
� Normal Phase Mode– Hydrophobic compounds
©2013 Waters Corporation 36
– Hydrophobic compounds– Hydrophilic stationary phase– Non-polar solvents– Organic to organic gradient
� HILIC mode
– Hydrophilic compounds– Hydrophilic stationary phase– Polar Solvents– Organic to aqueous gradient
HILIC Retention MechanismsHILIC Retention Mechanisms
©2013 Waters Corporation 37
Combination of partitioning and hydrogen bonding
• Polar analyte partitions between bulk mobile phase and the immobilized water layer
• Hydrogen bonding between the analyte and amide hydrophilic surface
HILIC HILIC -- HPLCHPLC22--AB Labeled Murine IgG AB Labeled Murine IgG NN--GlycansGlycans
G0F
G1F
Vicam IgG
8000.000
9000.001
10000.001
11000.001
12000.001
Gly_tos 012308_12a ACQUITY FLR ChA Ex330,Em420 nmRange: 126862 x 100 mm5 µm
Amide Column
©2013 Waters Corporation 38
G2F
Time48.00 49.00 50.00 51.00 52.00 53.00 54.00 55.00 56.00 57.00 58.00 59.00 60.00 61.00 62.00 63.00 64.00 65.00
EU x 10e4
0.000
1000.000
2000.000
3000.000
4000.000
5000.000
6000.000
7000.000
8000.000
FL EU
65.0
Time (minutes)
ACQUITY UPLC BEH GlycanACQUITY UPLC BEH GlycanColumn ChemistryColumn Chemistry
BEHParticle
©2013 Waters Corporation 39
� Ligand type: Trifunctional Amide
� BEH Particle size: 1.7 µm
� Endcap style: None
� Recommended pH range from 2 to 11
Developed, Optimized, and Tested Developed, Optimized, and Tested For Glycan AnalysisFor Glycan Analysis
� ACQUITY UPLC BEH Glycan Column
– HILIC mode separation of carbohydrates
– Amide bonded phase
– Stable BEH Particles
©2013 Waters Corporation 40
– Stable BEH Particles
– 1.7 µm Diameter Particles
– Optimized for use on ACQUITY UPLC® System with fluorescence detection
– Quality Control tested with 2-AB human IgG glycan standards
ACQUITY UPLC BEH Glycan Column ACQUITY UPLC BEH Glycan Column Certificate of AnalysisCertificate of Analysis
Chromatographic Testwith
Glycan Performance Test Standard
Chemical Tests
Individual Column Tests
©2013 Waters Corporation 41
Waters Waters GlycanGlycan Separation TechnologySeparation Technology
5 µm Tosoh TSKgel Amide-804.6 mm x 250 mm
3 h method
3 µm Tosoh TSKgel Amide-80
©2013 Waters Corporation 42
Retention Time (min)
3 µm Tosoh TSKgel Amide-804.6 mm x 150 mm
1 h method
1.7 µm Waters BEH Glycan2.1 mm x 150 mm30 min method
Retention Time (min) 15
1.7 µm Waters BEH Glycan2.1 mm x 150 mm30 min method
1806030
BatchBatch--toto--Batch Reproducibility of ACQUITY Batch Reproducibility of ACQUITY UPLCUPLC®® BEH BEH GlycanGlycan Material Using Material Using 22--AB Labeled Human AB Labeled Human IgGIgG NN--Linked Linked GlycansGlycans
Batch 1
Batch 2
©2013 Waters Corporation 43
Batch 3
Batch 4
2AB Labeled Glycan Standard - Ref 186006349
ACQUITY UPLCACQUITY UPLC®® Conditions
Column: ACQUITY UPLC BEH Glycan1.7µm, 2.1 x 150 mm
Eluent A: 100 mM Ammonium Formate, pH 4.5Eluent B: Acetonitrile
Temperature: 60 °C
Fluorescence: λex = 330 nm, λem = 420 nm
©2013 Waters Corporation 44
Sample amount: 15 pmol
UPLC Gradient:
Time Flow rate(min) (mL/min) % A % BInit 0.5 25 75 46.5 0.5 40 60 48 0.25* 100 0 49 0.25* 100 050 0.5 25 7563 0.5 25 75
*Flow rate lowered during aqueous regeneration
Neutrals and Charged Structures Neutrals and Charged Structures are Separated in a Single Injectionare Separated in a Single Injection
40.0
50.0
�
�
�
�
�
� Fucosylated� Sialylated� High Mannose Structures� Terminal Galactose
� Afucosylated
©2013 Waters Corporation 45
size
EU
0.0
10.0
20.0
30.0
Retention Time (min)
2 4 6 8 10 12 14
�
�
�
�
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�
�
�
�
�
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�
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Why choose Why choose GlycoWorksGlycoWorks and and UPLCUPLC ??Looked at the FLR/MS chromatogram.Looked at the FLR/MS chromatogram.
2AB-labeled Etanercept N-Glycan profile (released from 4 µg of protein)
FLR
©2013 Waters Corporation 46
MS
ACQUITY UPLC H-class/FLR/Xevo G2-S QTOF MS
AgendaAgenda
� Importance of Glycan Analysis
� Current Glycoprotein Analysis
� Glycan Characterization: Oligosaccharides
©2013 Waters Corporation 47
� Glycan Analysis: Sample Preparation
� Glycan Analysis: Chromatography
�� Empower & GU units Empower & GU units
� NIBRT Glycobase 3+
� Summary
Why Empower and GU Values?Why Empower and GU Values?EU
4.0
6.0
8.0
3 4 5 6 7 8 9 10 11 12 13 14
GU Value
Human IgG1
Waters H-Class
1.7 µm BEH Glycan (2.1 mm x 250 mm)FLR detection (λex 330 nm, λem 420 nm)53% to 70% Linear gradient (30 min)Solvent A: 50 mM NH4+HCOO-
Solvent B: Acetonitrile
©2013 Waters Corporation 48
0.0
2.0
EU
0.0
1.0
2.0
3.0
4.0
5.0
RetentionTime (min)
2 4 6 8 10 12 14 16 18 20
Glycoprotein X
?
?
??
??????
?
?
?
?
?
??
????
?
???? ???? ?
GU Values Provide and Orthogonal GU Values Provide and Orthogonal Approach to CharacterizationApproach to Characterization
Prevalent structural isomers makes MS of glycans challenging
©2013 Waters Corporation 49
Structure
Comp Fuc1Hex6HexNAc5NeuAc2 Fuc1Hex6HexNAc5NeuAc2 Fuc1Hex6HexNAc5NeuAc2
m/z 2733.9729 2733.9729 2733.9729
GU ~10.2 ~11.1 ~10.6
Risk? None Immunogenic anaphylaxis?
Overall WorkflowOverall Workflow
Glycoprotein Sample
Known Glycans
Unknown Glycans
©2013 Waters Corporation 50
Glycan Profiling Structural Characterization
Empower Automation GU Value Generation
GlycoBase Interrogation
Structure Confirmation (MS and Enzyme Arrays)
Glycoprotein Sample
Known Glycans
Unknown Glycans
©2013 Waters Corporation 51
Glycan Profiling Structural Characterization
Empower Automation GU Value Generation
GlycoBase Interrogation
Structure Confirmation (MS and Enzyme Arrays)
Report Generation
GU Values Aid Dramatically in GU Values Aid Dramatically in Identifying Identifying GlycanGlycan StructuresStructures
4 5 6 7 8 9 10 11
3.0
3.5
4.0
GU Value
11
©2013 Waters Corporation 52
EU
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Retention Time (min)
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1
2
3
5
4 6
710
1315
161819
22
23
2425
26
28
29
3132
33
34
35363739
40414344 45
8
9
1214
17
21
2730 38 42
20
GU Values Aid Dramatically in GU Values Aid Dramatically in Identifying Identifying GlycanGlycan StructuresStructures
Sialylated Neutral Phosphorylated
©2013 Waters Corporation 53
! No Mass Spec Used for Glycan Characterization !
Brief Summary Empower and GU Brief Summary Empower and GU valuesvalues
1) Ensure GPC option is installed in Empower
2) “GU” custom calculation needs be added to your project
custom fields
3) Dextran ladder standard is required to calibrate GU calculation
©2013 Waters Corporation 54
Integrate the Integrate the DextranDextran PeaksPeaks
©2013 Waters Corporation 55
Select Select ProcessProcess��������CalibrateCalibrate::Retention Times Shift to GU ValuesRetention Times Shift to GU Values
©2013 Waters Corporation 56
For Samples, Integrate For Samples, Integrate Peaks of InterestPeaks of Interest
5.584 8.832 12.215 15.384
18.235
20.776
23.027
EU
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
©2013 Waters Corporation 57
9.904
10.370
11.733
12.498
12.933
13.220
14.132
14.484
15.032
15.271
15.557
16.237
16.665
17.253
18.075
18.958
19.892
0.0
5.0
EU
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Minutes
6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
Select Select ProcessProcess��������QuantitateQuantitate::Retention Times Shift to GU ValuesRetention Times Shift to GU Values
40000
50000 60000 70000 80000
90000
100000
EU
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
©2013 Waters Corporation 58
Minutes
53538
54904
57183
58917
61199
62514
63387
65565
66208
67317
68005
69069
69845
70781
73053
74512
76567
78203
78715
79529
82842
86500
87367
89266
98516
0.0
5.0
EU
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
AgendaAgenda
� Importance of Glycan Analysis
� Current Glycoprotein Analysis
� Glycan Characterization: Oligosaccharides
©2013 Waters Corporation 59
� Glycan Analysis: Sample Preparation
� Glycan Analysis: Chromatography
� Empower: GU units
�� NIBRT NIBRT GlycobaseGlycobase 3+3+
� Summary
www.waters.com/glycanswww.waters.com/glycans
©2013 Waters Corporation 60
Where to next?Where to next?Database interrogationDatabase interrogation
©2013 Waters Corporation 61
Search Database Using GU ValueSearch Database Using GU Value
©2013 Waters Corporation 62
Interesting Application Notes/postersInteresting Application Notes/posterson Glycan Analysison Glycan Analysis
� A new column for improved resolution for glycan analysis App Note 720003112en
� Batch to Batch Profiling using UPLC/FLR/MS App Note 720003576en
� Method Development of 2AB labeled Glycans 720003238en
� Analysis of Procaïnamide labeled N-Glycans 720004212en
©2013 Waters Corporation 63
� Analysis of Procaïnamide labeled N-Glycans 720004212en
� Analysis of N-linked Glycans from Coag Factor IX 720004019en
� Usage of UPLC of 2AB labeled Fetuin Glycans Removed by Exoglycosidase 720003865EN
� A Holistic Workflow for Acquiring, Processing, and Reporting Fluorescent-Labeled Glycans w Unify 720004619en
� Type green reference in search box on www.waters.com
SummarySummary
� Waters provides you with a total solution from sample handling, UPLC separation, data workflow and interpretation
� GlycoWorks™ provides you a easy to use sample handling workflow in a complete kit from one single vendor (offering flexibility by allowing the customer to chose enzyme and fluorescent label)
©2013 Waters Corporation 64
fluorescent label)
� Glycan Separation Technology Columns provide you high resolution separation, with consistent batch to batch performance
� Empower provides the necessary tools for calculations and calibrations
� Waters cooperation with NIBRT : Glycobase database for glycan interpretation and trainings
©2013 Waters Corporation 65