UPLC and HPLC Separation Strategies for Successful ... · Terminal Terminal Glycan Glycan...

UPLC and HPLC Separation Strategies for UPLC and HPLC Separation Strategies for

Successful Characterization of Glycans Successful Characterization of Glycans

Derived from Therapeutic ProteinsDerived from Therapeutic Proteins

©2013 Waters Corporation 1

Derived from Therapeutic ProteinsDerived from Therapeutic Proteins

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Today’s SpeakerToday’s Speaker


Bill Warren has been with Waters Corporation for more than 20 years,

having worked in both technical and marketing capacities. He is

currently responsible for strategic and tactical implementation of

programs that support new bioseparations products and technologies

which help accelerate customer productivity in the biopharmaceutical

market segment.

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


N-Linked Glycosylation &


N-Linked Glycosylation &

Biopharma Significance

GlycosylationGlycosylation plays a plays a critical role in biologycritical role in biology

Nearly 50% of all proteins are glycosylated.What do the glycans do?


N-linked glycans play a role in…

Protein foldingCell-cell communicationBiological activityProtein half-lifeCell attachmentetc…

IgG-FcγRIIIa Interaction

PDB file 3SGJ

NN--linked linked glycanglycan biosynthesis is a biosynthesis is a highly complex processhighly complex process


Struwe WB, Cosgrave EFJ, and Rudd PM. (2011). Glycoproteomics in Health and Disease. Functional and Structural Proteomics of Glycoproteins.

GlycansGlycans are Highly Heterogeneousare Highly Heterogeneous

High Mannose Complex Hybrid


Number of Approved “Biotherapeutics”Number of Approved “Biotherapeutics”in Europe and USin Europe and US


190 EMA and/or FDA Approved

Walsh (2010). Nature Biotech; 28(9):917-924

127 of 190 approved are 127 of 190 approved are GlycoproteinsGlycoproteins


127 are Glycoproteins (>66%)

Walsh (2010). Nature Biotech; 28(9):917-924

NN--Linked Glycosylation Is Critical to Linked Glycosylation Is Critical to MabMab Biological ActivityBiological Activity

CH2a

CH2b

Asn-297(a)Asn-297(b)


IgG Fc region

IgG Glycosylation

Asn-297

CH3aCH3b

Asn-297

Structure based on PDB file 1H3Y from Krapp S et al. J Mol Biol, 2003, 325(5): 979-989

Asn

Desialylation of IVIg abrogates anti-inflammatory properties in K/N miceKaneko et al (2006). Science; 313(5787): 670-673

Terminal Terminal GlycanGlycan Function:Function:SialicSialic AcidAcid


Terminal Terminal GlycanGlycan Function:Function:GalactoseGalactose

Involved with placental transport of IgGIgG galactosylation increased in pregnant womenKibe et al (1996). J Clin Biochem Nutr; 21(1): 57-63

Asn


Terminal Terminal GlycanGlycan Function:Function:Core Core FucoseFucose

Loss of core α(1,6) fucose on IgG results in enhanced ADCC activityOkazaki et al (2004). J Mol Biol; 336(5): 1239-1249

Asn


BiopharmaBiopharma Attempts to Replicate Attempts to Replicate Human Human GlycosylationGlycosylation


Struwe WB, Cosgrave EFJ, and Rudd PM. (2011). Glycoproteomics in Health and Disease. Functional and Structural Proteomics of Glycoproteins.

Negative Attributes to Mammalian Negative Attributes to Mammalian Cell Culture Cell Culture GlycosylationGlycosylation

Asn

Asn

50% of non-allergic blood donors contain antibodies againstβ(1,2)-xylose and α(1,3)-core fucoseBardor et al (1995). Glycobiology; 13(6): 427-434


Asn

AsnPresence of gal-α(1,3)-gal can induce anaphylaxisChung et al (2006). N Engl J Med; 358(11): 1109-1117

N-glycolylneuraminic acid is an oncofetal antigen in humansMuchmore et al (1989). J Biol Chem; 264(34): 20216-20223

Regulatory Agencies Are Dutifully Regulatory Agencies Are Dutifully Aware of Aware of GlycosylationGlycosylation


Effects that Augment Effects that Augment GlycosylationGlycosylationCan Have ConsequencesCan Have Consequences


2013 Waters Glycan Analysis Survey2013 Waters Glycan Analysis Survey(N = 176)(N = 176)

Question: For what purpose(s) is your laboratory performing glycan / monosaccharide analyses ? (Multiple Responses Allowed)


Agenda:Agenda:

� Overview of Glycan Characterization Strategies

� Intact Glycoprotein LC/MS Analysis

� Released, N-Glycan Profiling by UPLC and HPLC and Effective

Use of GU-Based Data Reduction


Use of GU-Based Data Reduction

� Structural / Linkage Data Using Exoglycosidase Digestions

� Summary

Fractionation Methods to Fractionation Methods to Characterize GlycoproteinsCharacterize Glycoproteins

6

3

6

3

Glycan Release

Glycoprotein

6

3

6

3


6

3

6

3

Protease Digestion

Glycopeptides

GlycoproteinOligosaccharides

(Glycans)

Monosaccharides

2013 Waters Glycan Analysis Survey2013 Waters Glycan Analysis Survey(N = 176)(N = 176)

Which of the techniques listed below does your laboratory perform? (Multiple Responses Allowed)


Waters Glycan Separation Waters Glycan Separation TechnologyTechnology

Sample Preparation Informatics

UPLC-based

HPLC-based


SeparationsChemistry

Mass Spectrometry

Intact Glycoprotein LC/MS AnalysisIntact Glycoprotein LC/MS Analysis


G0F/G1F

Intact Glycoprotein LC/MS AnalysisIntact Glycoprotein LC/MS Analysis

Pre-runBlank

TIC (0.0 – 3.0 min)


GOF/G0F

G0F/G1F

G1F/G1FG0F/G2F

G1F/G2F

G0/GOF

G2F/G2F

0.5 µg IgG1

Post-runBlank

LC/MS Analyses of four batches of mAb LC/MS Analyses of four batches of mAb marketed Trastuzumab) showing variations marketed Trastuzumab) showing variations in the proportions of major glycoformsin the proportions of major glycoforms


Intact protein glycosylation profile

BiopharmaLynx™ mirror plot for comparison to Reference compound

Intact protein glycosylation profile of a monoclonal Antibody in mirror mode

• Automated Processing using deconvolution and mass assignment

• Relative quantification


• Reference and sample easily compared

• Batch variations directly measurable

Tabular assignment of Glycoforms in BiopharmaLynx™

Batch 1

MaxEnt1 Deconvoluted Spectra of Intact IgG1 MaxEnt1 Deconvoluted Spectra of Intact IgG1 from three Batches Processed by BiopharmaLynxfrom three Batches Processed by BiopharmaLynx

(M

an5)2

(G1F)2G0F/G2F

(G

2F)2

(G

0F)2

G0F/G

1F

G1F/G

2F

G0/G

0F


Batch 2

Batch 3

Man5/M

an6

Method is fast, adapted to high-throughput

Released, NReleased, N--Glycan ProfilingGlycan Profilingby UPLC and HPLCby UPLC and HPLC


Challenges:Challenges:“Glycan” Analyses“Glycan” Analyses

A Difficult and Complex Analytical Problem

– Sample Preparation

o Complex frequently involving enzymatic glycan release from isolated glycoproteins followed by labeling


– Separation

o May involve intact protein, peptide, and / or isolated glycan analysis

o “Complete” characterization MOST difficult and time consuming

– Detection

o No chromophore on isolated glycans

o LC/MS for compound identification

Waters GlycoWorksWaters GlycoWorksTM TM AndAndSample Preparation WorkflowSample Preparation Workflow

Step 1 Step 1


Step 2

Step 3

Step 4

Step 5

15

20

% Abundance

ControlBefore SPE

AC

GlycoWorks HILIC SPE with the GlycoWorks HILIC SPE with the optimized elution conditionsoptimized elution conditions


0

5

10

Peak 3 G0F

Peak 16 A3

% Abundance

GlycoWorks HILIC SPE ProcessedElution with 100 mM NH4OAc, 5% ACN

B3

16

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

ACQUITY UPLC BEH GlycanACQUITY UPLC BEH GlycanColumn ChemistryColumn Chemistry

BEHParticle


� Ligand type: Trifunctional Amide

� BEH Particle size: 1.7 µm, 2.5um, and 3.5um

� Endcap style: None

� Recommended pH range: 2 to 11

Effective Separation of Neutral and Effective Separation of Neutral and Charged 2Charged 2--AB Labeled Glycans on AB Labeled Glycans on Waters BEH Glycan, 1.7um ColumnWaters BEH Glycan, 1.7um Column

Peak 2-AB Labeled Glycan

1 G0-GN

2 G0

3 G0F

4 Man5

5 G0FN

6 G1F

7 G1F

8 G1FN

9 Man6

10 G2

11 G2F

12 G2FN

13 G1FS1

14 G2FS1

1x

Glycan Performance Test Standard


14 G2FS1

15 A3

16 A3

1

2

3

4

5

6,7

8

9

10

11

12

13

14

15,16

Neutral Glycans

Nature 446, 1023-1029(26 April 2007)

3x

Acidic Glycans

+A3 (Trisialylated) Glycans

UPLC and HPLCUPLC and HPLC--based, BEH Glycan Column based, BEH Glycan Column Certificate of AnalysisCertificate of Analysis

Chromatographic Testwith

Glycan Performance Test Standard

Chemical Tests

Individual Column Tests


Late 1970’s10µ Irregular micro-porous

1000-2500 psi25,000 plates/meter

3.9 x 300mm

Particle Particle Size Size Evolution:Evolution:Increased Rs with Smaller ParticlesIncreased Rs with Smaller Particles

Early 1970’s40µ pellicular non-porous coated

100-500 psi1000 plates/meter

1m columns

10 min


10 min

1980’s thru 20035 – 2.5µ spherical micro-porous

1500-4000 psi50,000 - 80,000 plates/meter

3.9 x 150mm

10 min

10 min

Broad BandBroad PeakLess SensitivityLess Resolving Power

HPLC

Advantages of UPLC vs. HPLCAdvantages of UPLC vs. HPLC--Based Particle and Instrument Based Particle and Instrument Technologies for BioseparationsTechnologies for Bioseparations

Narrow PeakIncreased SensitivityIncreased Resolving Power

Waters UPLC®

Technology


Power

UPLC and HPLCUPLC and HPLC--based,based,22--AB Labeled Glycan AnalysesAB Labeled Glycan Analyses

XBridge BEH Glycan 2.5 µm XP

ACQUITY BEH Glycan 1.7 µm

EU

0.00

2.00

4.00

6.00

10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

1 2

4

3

5

6

7

8910

11

12

14

20.00

Alliance HPLC

UPLC

13

Pc*half-height = 110

Pc*half-height = 78

2.1 x 150 mm0.50 mL/min

2.1 x 150 mm

UPLC-based

HPLC-based

8700 psi (Column, Max)



EU

0.00

10.00

20.00

15.00 20.00 25.00 30.00 35.00 40.00

XBridge BEH Glycan 3.5 µm

EU

0.00

5.00

10.00

Minutes25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

Alliance HPLCPc

*half-height = 55

2.1 x 150 mm0.34 mL/min

2.1 x 150 mm0.24 mL/min

HPLC-based



50.0

20

25

% Abundance

1.7 um, 0.50 mL/min)

2.5 um XP, 0.34 mL/min

3.5 um, 0.24 mL/min

Relative Abundance DeterminationsRelative Abundance Determinations

1

2

3

4

5

6,7

8

9

10

11

12

13

14

15,16


n=30

5

10

15

Peak 1

G0-GN

Peak 2

G0

Peak 3

G0F

Peak 4

Man5

Peak 5

G0FN

Peak 6

G1F

Peak 7

G1F

Peak 8

G1FN

Peak 9

Man6

Peak 10

G2

Peak 11

G2F

Peak 12

G2FN

Peak 13

G1FS1

Peak 14

G2FS1

Peak 15

A3

Peak 16

A3

% Abundance

*Peaks 8 and 9 – 3.5 µm resolution insufficient, accuracy of the integration is poor

Glucose Unit Concept(Way to Normalize RT Variations)


Dextran Calibration Ladder Standard and the Assignment of GU Values (UNIFI)


Using GU Values:BEH Glycan Column Scaling


Using UPLC and MS


Using UPLC and MS

For Glycan Analysis

GlycanGlycan Analysis by LC or MS Alone Analysis by LC or MS Alone Is Insufficient for CharacterizationIs Insufficient for 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?

GlycanGlycan Analysis by LC or MS Alone Analysis by LC or MS Alone Is Insufficient for CharacterizationIs Insufficient for Characterization

Co-elution of structures in LC makes identification challenging


� Fucosylated� Sialylated

� High Mannose Structures

� Terminal Galactose

Waters Glycan Performance StandardWaters Acquity H-Class Bio

1.7 µm Waters BEH Glycan

2.1 mm x 150 mm30 min gradient

Retention Time (min)

2 4 6 8 10 12 14

Combining the techniques can Combining the techniques can answer many important questionsanswer many important questions


UPLC-FLR-ESI-MS/MSUnifi 1.6

Structural / Linkage Data Using Structural / Linkage Data Using Exoglycosidase DigestionsExoglycosidase Digestions


Structural / Linkage Data Using Structural / Linkage Data Using Exoglycosidase DigestionsExoglycosidase Digestions

� Released glycan pool is highly complex

– Requires use of sequential enzymatic workflows to release glycans at

known cleavage positions

– Presence of branching & linkage isomers

� Chromatographic resolution is a limiting factor


– HPLC separations w/ HILIC chemistries often yield coelutions

– Results interpretation are compromised

� Glycan identification

– Must first identify enzymatically-released glycan

– Then piece together the complete carbohydrate structure based on

enzymatic workflow evidence

Glycan ProfilingGlycan ProfilingApplying Exoglycosidase Enzymes Applying Exoglycosidase Enzymes

Intact feting 2-AB-labeled glycans

2-AB-labeled bovine feting glycans

No exoglycosidase digestion

α2-3,6,8 Sialidase

Mannose

N-Acetylglucosamine

Galactose

1

2

Man3

A2

A3


Workflow of the enzymatic array digestion and the structures of the released bovine fetuin N-glycans

A3G(4,4,4)3

3

4

5

Sialidase + β1-4 Galactosidase

Sialidase + β1-3,4 Galactosidase

Sialidase + Galactosidase +

Hexosaminidase

A2G(4)2

A3G(3)1

A3G(3,4,4)3

4

32

β-linkage

α-linkage

Glycan ProfilingGlycan ProfilingMeeting the Resolution ChallengeMeeting the Resolution Challenge

Intact 2 AB labeled fetuin N glycans

1

2Removed:

Sialic acid

linkage isomers

UPLC HPLC


3

4

5

Removed:

Sialic acid,

Galactose β1-4

Removed:

Sialic acid,

Galactose β1-3, β1-4

Removed:

Sialic acid,

acetylglucosamine

Galactose β1-3, β1-4

Loss of Terminal Loss of Terminal MonosaccharidesMonosaccharidesCan Be Tracked Using GU ValuesCan Be Tracked Using GU Values

Monosaccharide Linkage To GU Increment

Core fucose α(1,6) Any structure 0.5

Outer arm fucose α(1,3)α(1,6)

GlcNAc 0.8

Outer arm fucose α(1,2) Gal 0.5

Mannose α(1,2)α(1,3)

Man 0.7-0.9

Approach: Exoglycosidase Arrays with UPLC-FLR Analysis


α(1,3)α(1,6)

GlcNAc β(1,2)β(1,4)β(1,3)β(1,6)

Any structure 0.5-0.6

Bisecting GlcNAc β(1,4) α-mannose 0.1-0.15

Galactose α/β(1,3)α/β1,4)

Any structure 0.8-0.9

NeuAc α(2,3) Gal of any structure ~0.7

NeuAc α(2,6) Gal of any structure ~1.15

SUMMARY SLIDE SUMMARY SLIDE

� Glycosylation of many biotherapeutic proteins (e.g., mAbs) is critically important since it effects the efficacy and potential undesired immunogenicity of the prescribed drug.

� The effective characterization of protein associated glycans is complex and frequently requires use of several different separation technologies.


� US and various international regulatory agencies require submission of glycan analysis data in order to approve / license a biotherapeutic or biosimilar drug.

� Waters offers a range of HPLC and UPLC-based sample preparation, column chemistries, and glycan / sialic acid / monosaccharide application solutions to help in the discovery, development, and manufacturing quality testing required to deliver safe and effective biotherapeutic proteins.

Thank You!Thank You!

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