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Technologies for Glycan Analysis

UPLC® and Mass Spectrometry Solutions


Glycosylation Functions: Risks and Regulatory Concerns

Mediates biological activity

Glycans impact safety and efficacy

– Correct and consistent structure of the glycans

– Obtaining the desired medical effect

– Avoid adverse immunological reaction

Alteration in glycans may eliminate or alter activity

Immune response triggered by unrecognized glycans

Consistent glycan distribution indicates process stability


Glycoprotein Characterization Requirements

Selectivity for Key Glycans

Sensitivity for Minor Glycoforms

Reproducibility of Results

Quantitative Information

Robust Complete Solution

Site of attachment

n% glycan1

?


Glycan Structures - Key Critical Quality Attributes

Source: NIBRT. http://www.nibrt.ie/


Oligosaccharide Structures N-linked and 0-Linked

O-linked glycosylation to the hydroxy Oxygen of serine or

threonine side chains

N-linked glycosylation to the amide Nitrogen of asparagine side chains


Glycan Complexity Determines Characterization Requirements

TrastuzumAb, 1 N-linked site 150 KDa

Entanercept 3 N-linked sites 13 O-linked sites 51 KDa

Erythropoietin 3 N-linked sites 1 O-linked site 34 KDa

Difficulty

http://en.wikipedia.org/wiki/File:HerceptinFab.jpg


Regulator Readiness – FDA Now Devising Guidelines

BPCI act of 2009 established a framework to develop guidelines

FDA outlined a ‘case-by-case’ principle – i.e. no ‘formula’ for biosimilars:

– ‘it’s unlikely that a “one size fits all” systematic assessment of biosimilarity can be

developed’

– ‘…a “totality of the evidence” approach, … by evaluating more attributes and

combinations of attributes at greater sensitivities with multiple complementary

methods.

– ‘… the scope and extent of [clinical] studies may be reduced further if more extensive

fingerprint-like characterization is used’.

– ‘Immunogenicity remains a critical factor when assessing biosimilarity’


FDA: Fingerprinting Concept Applied to Glycosylation


UPLC and MS Based Analytical Approaches for Comprehensive Glycosylation Analysis


UPLC and MS Based Analytical Workflows for Comprehensive Glycosylation Analysis

Intact Protein

Glycopeptides Released Glycans

MW profiling (ESI MS)

Proteolytic Digestion PNGase F Digestion

Fluorescent and mass profiling of glycans

Relative Quantitation

Structure Elucidation

-- MS based

-- LC retention time only (GU value)

& Glycosidase Array

Relative Quantitation of glycans

Glycan site heterogeneity

Glycan Occupancy


Intact Glycoprotein Analysis


0.5

0.5

0.5

0.5

0.5

0.5

0.2

0.2

0.5

0.5

Flow

(ml/min)

6

6

6

6

6

6

6

6

6

Initial

Curve

54.00

3.50

3.40

2.80

2.70

2.10

2.00

0.51

0.50

0.00

Time

(min)

5

90

5

90

5

90

5

5

5

%B

0.5

0.5

0.5

0.5

0.5

0.5

0.2

0.2

0.5

0.5

Flow

(ml/min)

6

6

6

6

6

6

6

6

6

Initial

Curve

54.00

3.50

3.40

2.80

2.70

2.10

2.00

0.51

0.50

0.00

Time

(min)

5

90

5

90

5

90

5

5

5

%B

Load/Wash

-Divert Flow-

Gradient

Column

Washing

and

Regeneration

A = 0.1% Formic Acid (Water) B = 0.1% Formic Acid (ACN)

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4RT (min)

% M

eCN

+ 0.

1% F

A

0

0.1

0.2

0.3

0.4

0.5

0.6

Flow

(ml/m

in)

Sample loading/Desalting

With this on-line method it takes 2 minutes to load, wash and elute protein

• The desalting column fits into the ACQUITY UPLC® oven – best results for desalting are obtained using temperatures between 75 to 85 degrees – this is a key factor for efficient antibody desalting.

Here we see the fluidic configuration for LC/MS analysis. A post-column salt diversion valve is utilized to divert buffers and nonvolatile salts to waste during the sample loading step.

Intact Glycoprotein Analysis Rapid Protein Desalting


GOF/G0F

G0F/G1F

G1F/G1F G0F/G2F

G1F/G2F

G0/GOF

G2F/G2F

Intact Glycoprotein LC/MS Analysis

0.5 µg IgG1

Post-run Blank

Pre-run Blank

TIC (0.0 – 3.0 min)


LC/MS Analyses of Trastuzumab Assessment of Four Batches

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

Batch 2

Batch 3

(M

an

5)2

(G1F)2 G0F/G2F

Man

5/

Man

6

(G

2F)2

(G

0F)2

G0

F/

G1

F

G1

F/

G2

F

G0

/G

0F

Method is fast, adapted to high-throughput


Released N-Glycan Analysis


Sample Preparation for Released N-Glycan Analysis

Reduction/alkylation using 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 - 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/MS

analysis

1 hr 4 hrs - Overnight

30 min. 1 hr 40 min

1 hr 2 – 3 hrs 30 min

Lyophilize the sample

1 hr


Waters GlycoWorks Kit Sample Preparation Workflow

Step 1 Step 1

Step 2

Step 3

Step 4

Step 5


USP Compendial Methods Preparing for Biosimilars


Labeled Glycan Separation via HILIC Hydrophilic-Interaction Chromatography

Reversed-Phase Mode

– Hydrophilic compounds

– Hydrophobic stationary phase

– Polar Solvents

– Aqueous to organic gradient

Normal Phase Mode

– 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


UPLC and HPLC-based 2-AB Labeled Glycan Analyses

XBridge BEH Glycan 2.5 µm XP

ACQUITY UPLC 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

8 9 10

11

12

14

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

Minutes 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

13

Pc*half-height = 110

Pc*half-height = 78

Pc*half-height = 55

2.1 x 150 mm 0.50 mL/min

2.1 x 150 mm 0.34 mL/min

2.1 x 150 mm 0.24 mL/min

ACQUITY UPLC® H-Class Bio

Alliance HPLC

8700 psi (Column, Max)



50.0

Alliance® HPLC


UPLC Glycan Separation Protocol Waters 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

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)

1x

3x

Acidic Glycans

Glycan Performance Test Standard

+A3 (Trisialylated) Glycans


GU: A Glucose Unit

A GU value is a normalized glycan structure retention time observed in

HILIC, obtained using dextran as a molecular weight ladder

What is a GU Value?

Why are GU Values Generated?

GU Values assist in normalizing glycan retention time information across

different instruments so that data between scientists, labs, and across

different geographies can be compared and shared

…Why don’t we just use retention times?

Retention times are not a reliable and robust approach to interpret glycan data

We need to normalize the retention times


Enabling Standards -2AB labeled Glycans

GU

2

GU

5

GU

10

GU

15

FLR λex = 330 nm λem = 420 nm

2AB-Dextran Ladder, FLR

2AB-labeled Dextran Ladder Standard

Retention time Calibration Std.

Glycan Performance Test Standard

The Glycan Performance Test Standard is mixture of 2-AB labeled glycan that is QC verified to contain the components needed to benchmark and evaluate ACQUITY UPLC BEH Glycan, 1.7 µm Columns.


Calibration of Dextran Allows Retention Time Conversion to GU

Log

Mol

Wt

4.4

4.5

4.6

4.7

4.8

4.9

5.0

5.1

5.2

Retention Time (min) 5 10 15 20

GU = s + aT + bT2 + cT3

+ dT4 + eT5

EU

0

2

4

6

8

10

12

14

16

18

20

Retention Time (min) 5 10 15 20 25

3 4 5

6

7 8

9 10 11 12 13 14 15

16 17

n

BEH Glycan 1.7 um Waters Dextran Ladder

EU

0

2

4

6

8

10

12

14

16

18

20

Retention Time (min) 5 10 15 20 25

4.44 min

GU = s + aT + bT2 + cT3 + dT4 + eT5

5.89GU

Calibration Curve


Assigned GU Units ACQUITY UPLC H-Class Bio & Alliance HPLC


Why Empower and GU Values?

EU

0.0

2.0

4.0

6.0

8.0

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

3 4 5 6 7 8 9 10 11 12 13 14

GU Value

Human IgG1

Glycoprotein X

ACQUITY UPLC® H-Class Bio 1.7 m BEH Glycan (2.1 mm x 250 mm) FLR detection (ex 330 nm, em 420 nm) 53% to 70% Linear gradient (30 min) Solvent A: 50 mM NH4+HCOO-

Solvent B: Acetonitrile

?

?

?

?

? ? ? ?

? ?

?

?

?

?

?

? ?

? ? ? ?

?

? ? ? ? ? ? ? ? ?


GU Values Provide an Orthogonal Approach to Characterization

Structure

Comp Fuc1Hex6HexNAc5NeuAc2 Fuc1Hex6HexNAc5NeuAc2 Fuc1Hex6HexNAc5NeuAc2

m/z 2733.9729 2733.9729 2733.9729

GU ~10.2 ~11.1 ~10.6

Risk? None Immunogenic anaphylaxis?

Prevalent structural isomers makes MS of glycans challenging


Overall Workflow

Glycan Profiling

Glycoprotein Sample

Known Glycans

Unknown Glycans

Structural Characterization

Empower® Automation GU Value Generation

GlycoBase Interrogation

Structure Confirmation (MS and Enzyme Arrays)


Overall Workflow

Glycan Profiling

Glycoprotein Sample

Known Glycans

Unknown Glycans

Structural Characterization

Empower ® Automation GU Value Generation

GlycoBase Interrogation

Structure Confirmation (MS and Enzyme Arrays)

Report Generation


2AB-labeled Glycan Retention Time Base Database - Glycobase 3.1+

Collaboration with NIBRT • Development of a UPLC based 2AB-labled Glycan Retention Time Database


Database Interrogation


Search Database Using GU Value


2AB-labeled IgG1 Glycans HILIC –UPLC/FLR

Waters Application Note: 720003576en


Acquire Both FLR and MS Data From the Same Injection

HILIC-UPLC/FLR/QTof MS

(2AB-labeling) G0

- GN

G0F

- GN

G0

G2FMan

5G1

a

G1Fa

G1Fb

Man

6

G0F

Man

7

G2FS

1M

an8

G2FS

2

FLR

MSG1F

- GN

Time (min.)5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00

%

0

5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00

EUx

10e4

200000.016

400000.031

600000.063

800000.063

1000000.063

1200000.125

1400000.125

(1) ACQUITY FLR ChA Ex330,Em420 nmRange: 1743799

1: TOF MS ES+BPI

2.15e3

G2G1b

FLR

BPI MS

Waters Application Note: 720003576en “Trastuzumab Glycan Batch-to-Batch Profiling Using a UPLC/FLR/Mass Spectrometry Platform”


UPLC/FLR/QTOF MS Analysis of 2AB-labeled Enbrel N-linked Glycans

Prozyme 2AB_Enbrel_glycan STD N_1, 1.2 ug Protein/ul

Time5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00

%

0

100

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00

EU

x 1

0e4

0.000

250000.016

500000.031

750000.063

1000000.063

1250000.125

122111_UEA038_YQY_04 (1) ACQUITY FLR ChA Ex330,Em420 nmRange: 1567404

122111_UEA038_YQY_04 1: TOF MS ES+ BPI

9.88e4

FLR

BPI MS

1 2 3

4

5

6 7

8a

8b

10

11

12

13

14

15

16

17

18

19

9a,b

Glycan released from 6.0 µg of protein was injected

2AB-labeled Enbrel® Glycans


Proposed Structure (Enbrel N-linked Glycans)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20


Exoglycosidase Digestion

AMF

BKF

ABS NAN 1

ABS

GUH

SPGBTG

GUH

SPGBTG

BTG

X1-2F

AMF

BKF

Linkage position8

23

6

4

β-linkageα-linkageUnknown linkage

Exoglycosidase specificities

BKF a1-6/2

ABS a2-3/6/8NAN 1 a2-3/8

SPG b1-4BTG b1-3/4/6

GUH b1-2/4/6bMan

X1-2F a1-2AMF a1-3/4

Sialidase

Galactosidase

Fucosidase

Hexosaminidase


GU Value Relates to Glycan Structure

Linkage position

2 3

4

8 6

2 3

4

8 6

2 3

4

8 6

2 3

4

8 6

Linkage position

0.7-0.9Mana1-2,3,6mannose

~1.15Gal of any structure

a2-6NeuNAc


a2-3NeuNAc

0.8-0.9Any structureab1-3,4galactose

0.10-0.15b- mannoseb1-4Bisecting GlcNAc

~0.8GlcNAca1-3,6Outer arm fucose

~0.5Any structurea1-6Core fucose

GUincrement

tolinkagemonosaccharide

0.7-0.9Mana1-2,3,6mannose


a2-6NeuNAc


a2-3NeuNAc

0.8-0.9Any structureab1-3,4galactose

0.10-0.15b- mannoseb1-4Bisecting GlcNAc

~0.8GlcNAca1-3,6Outer arm fucose

~0.5Any structurea1-6Core fucose

GUincrement

tolinkagemonosaccharide

Glc

GlcNAc

Gal

GalNAc

Fuc

NeuNAc

Man

Symbol for sugar

Xylose

NeuNAc

Man

Xylose


Exoglycosidase Array

G.U.

164 14 18 26242220 286 8 10 12

65 7 8 9 10 1211 1413 15 16 17

Min.

(vi) ABS+BKF+SPG+GUH

(v)ABS+BKF+SPG

(iv) ABS+BKF

(i) Pooled Glycans

(iii) ABS

(ii) NAN1

Man3

A2

A4

A3

A2G2

A3G3

A4G4

FA2G2

FA3G3

FA4G4

FA2G2

FA3G3

FA4G4

FA2G2S2

FA3G3S2

FA4G4S2

FA2G2S1

Possible Sulphated


UNIFI™ Biopharmaceutical Platform Solution v. 1.7

Informatics Tool for 2-AB Labeled Glycan Analysis

Empower® 3 Chromatography Data Software

Supports UPLC®/FLR for Glycan analysis Compliant ready chromatography data software package for advanced data acquisition, management, processing, reporting and distribution.

Supports UPLC/FLR/QTof MS for Glycan analysis Workflow specific informatics tool; integrates robust UPLC/optical/MS characterization technology with comprehensive software, for application s in bioseparations, intact protein mass analysis, peptide mapping and released glycan analysis.


Biopharmaceutical Platform Solution with UNIFI

The UNIFI Biopharmaceutical Platform Solution supports LC and MS data workflows in a single solution that encompasses acquisition, data processing, bioinformatics, visualization, reporting and configurable compliance tools.

Analytical Workflow Includes: -Intact Protein Analysis (LC/UV/MS) -Peptide Mapping (LC/UV/MS) -Glycan Analysis (FLR/MS)


Focus on Complete System Solutions

Xevo® TQ-S

Xevo G2-S Q-Tof SYNAPT® G2-Si

ACQUITY UPLC®

H-CLASS

PATROL

Calibrants & Check Standards

Sample Preparation Kits

ACQUITY UPLC®

M-CLASS

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