analysis of nanalysis of n--linked linked glycansglycansof ... · biopharmaceuticals monoclonal...

Bio Day Bio Day –– DENMARK DENMARK –– MARCH MARCH 20132013

Analysis of NAnalysis of N--linked linked GlycansGlycans of of

GlycoProteinsGlycoProteins

[email protected][email protected]

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


MW 6,000

Insulin

MW 34,000

EPO

MW 147,000

Glycan Separation TechnologyGlycan Separation Technology


Glycosylation FunctionsGlycosylation FunctionsRisks and Regulatory ConcernsRisks and Regulatory Concerns

� Mediates biological activity

� Alteration may eliminate or alter activity

� Immune response triggered by unrecognized glycan


� 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


Isomeric StructuresIsomeric Structures

– The basic carbohydrate units are called monosaccharides

Hexose

HexNAc


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


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


� Quantitative Results

� Robust Complete Solution

AgendaAgenda

� Importance of Glycan Analysis

�� Current Glycoprotein AnalysisCurrent Glycoprotein Analysis








� Summary

Glycoprotein CharacterizationGlycoprotein CharacterizationWork FlowsWork Flows

Glycoprotein

MW ProfileESI MS


Glycopeptides& Peptides N- Oligosachharides

MALDILabel

Peptide MappingUPLC-UVUPLC-MS

HILIC / Fluorescence Detection

Galactose

Glycoprotein Characterization Glycoprotein Characterization Glycoprotein Glycoprotein

6

3

6


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


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



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


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



Galactose

Mannose

Fucose

Glycopeptides with HILICGlycopeptides with HILIC


720003363en

AgendaAgenda



�� GlycanGlycan Characterization: OligosaccharidesCharacterization: Oligosaccharides







� Summary

Glycoprotein Characterization Glycoprotein Characterization Glycan AnalysisGlycan Analysis

6

3

6

3

6

3

6

3

Glycan Release



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


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)


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


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




GlycanGlycan Analysis: Sample PreparationAnalysis: Sample Preparation


�� GlycanGlycan Analysis: Sample PreparationAnalysis: Sample Preparation




� Summary

GlycoanalysisGlycoanalysis WorkflowWorkflow

Free Glycans

And Proteins

Denaturation Deglycosylation

Isolation

Glycoprotein


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


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


- 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


- 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


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


� 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))


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


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


720004584EN

AgendaAgenda







�� GlycanGlycan Analysis: ChromatographyAnalysis: Chromatography



� 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


– 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


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


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


� 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


– 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


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


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


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


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


size

EU

0.0

10.0

20.0

30.0


2 4 6 8 10 12 14

�

�

�

�

� �

�

�

�

�

�

��

�

�

�

�

�

�

�

�

�

�

�

�

�

�

�

�

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


MS

ACQUITY UPLC H-class/FLR/Xevo G2-S QTOF MS

AgendaAgenda







�� Empower & GU units Empower & GU units


� 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


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


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


Glycan Profiling Structural Characterization

Empower Automation GU Value Generation

GlycoBase Interrogation

Structure Confirmation (MS and Enzyme Arrays)

Glycoprotein Sample

Known Glycans

Unknown Glycans


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


EU

0.0

0.5

1.0

1.5

2.0

2.5

3.0


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


! 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


Integrate the Integrate the DextranDextran PeaksPeaks


Select Select ProcessProcess��CalibrateCalibrate::Retention Times Shift to GU ValuesRetention Times Shift to GU Values


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


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


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








�� NIBRT NIBRT GlycobaseGlycobase 3+3+

� Summary

www.waters.com/glycanswww.waters.com/glycans


Where to next?Where to next?Database interrogationDatabase interrogation


Search Database Using GU ValueSearch Database Using GU Value


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


� 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)


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

analysis of nanalysis of n--linked linked glycansglycansof ... · biopharmaceuticals monoclonal...

Documents