waters glycan analysis by uplc-fl-ms€¦ · waters glycan analysis by uplc-fl-ms ... structure...

©2009 Waters Corporation | COMPANY CONFIDENTIAL ©2012 Waters Corporation

St John Skilton

Waters Glycan Analysis by UPLC-FL-MS

with UNIFI™


How Do you Explain it to the FDA?


How Do you Explain it to the FDA? In its constituent parts…


Workflow for Routine Intact Protein and Peptide Map Analysis by LC/MS

Prepare Standards & Samples

Data Acquisition & Processing

Data Management

Create Analysis Method and Sample Sequence

Reports Compiled, Reviewed & Signed


UNIFI 1.7 (1H 2013)

Biopharmaceutical Platform Solution

Intact Protein

Application Solution

Peptide Mapping


Bioseparations

Size Exclusion (UV)

Released

Glycan


DDA (Peptide & Glycan)

Intact Protein: Optical and MS Peptide Mapping: Optical and MSE

Released Glycan(GU): FLR (+MS, NIBRT Library)

QTof DDA MS/MS Bioseparations: Optical Workstation (GxP) or Workgroup (Compliance)


UPLC and MS Based Analytical Approaches for comprehensive Glycosylation Analysis

Export RT and

Import GU

values

Export MS and

MS/MS Spectra

for Structure


UPLC and MS Based Analytical Approaches 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


Routine LC/MS Analysis Of Intact Protein

•ACQUITY UPLC H-Class Bio with BEH C4 RP column •Xevo G2 QTof MS

• MassLynx 4.0 (data acquisition) • BiopharmaLynx v. 1.3 (data processing, reporting) •or UNIFI (acquisition, processing, reporting)

GOF/G0F

G0F/G1F G1F/G1F G0F/G2F

G1F/G2F

G0/GOF G2F/G2F

MaxEnt 1


Automation and Data Processin

Automated Processing using deconvolution and mass assignment

Relative quantification

Data comparison (Control/Analyte)

Batch variations directly measurable

Control

As above, and additionally:

Automated Acquisition, processing, reporting

Workgroup architecture for audit trail, moving to compliant-ready systems (1.7)

Trend plots for multiple samples and components

Analyte

Difference Plot


Batch 1

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

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


LC/MS Analysis of HC (Reduced IgG)

Heavy chain (HC) and light chain (LC) fragments generated by partial reduction of monoclonal antibody with DTT.

2 2 +

LC (~25 kD) HC (~50 kD)


Total Ion Chromatogram of reduced IgG1 antibody

Column: ACQUITY UPLC BEH C4, 1.7 µm, 2.1 x 50 mm

Gradient: 28-32% B in 12 min A: 0.02% TFA in water B: 0.018% TFA in ACN

LC HC

+34 Charge State

HC Mass Spectrum


MaxEnt1 deconvoluted spectra of IgG HC (~50kDa) generated from three different batches of Trastuzumab

Batch 1

Batch 2

Batch 3

G0

F-G

N

G0

G0

-GN

Man

5

G 1

G1

F

Man

6

G2

G2

F

G2

FS

1

G0

F

G1

F-G

N

Reduction of the antibody into monomeric HC improves isoform resolution allowing additional isoform identification and quantitation of the individual glycoform.

HC

A : Trastuzumab HC

A : Trastuzumab HC

A : Trastuzumab HC

Man

5


Intact Erythropoietin

LC/MS chromatogram

32 Glycoforms were accounted for


Comparison of Glycoform Quantitation by MS with 2AB labelled methodology – IgG1

Glycoform quantitation for reduced species by LC/MS can be compared to quantification of species by 2-AB labelled methods by UPLC with FL detection


Automated comparative analysis – intact protein analysis

Protein modifications identified

Deconvoluted annotated mass spectra

Chromatograms for reference and analyte


Tools for sample comparisons

Innovator

“Biosimilar”


Localization to Japanese and Chinese


Investigating Intact Mass Results

Peak 14.81 min

Peak 15.51 min


UNIFI™ Intact Mass Reporting Tools


UPLC and MS Based Analytical Approaches for Comprehensive Glycosylation Analysis

Intact Protein



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


Reminder: How Are Glucose Unit (GU)

Values Generated?

The elution times of glycans are expressed in glucose

units (GU) by reference to a dextran ladder.

Each individual glycan structure has a GU value that is

related to the number, linkage of its monosaccharides.

GU values can be used to predict structures.

The use of arrays of exoglycosidase enzymes in

combination with UPLC-HILIC profiling and Glycobase

enables the individual monosaccharides and linkages to

be determined.


Empower/ UNIFI GU processing Using GPC function

Minutes

40000

50000

60000

70000

80000

90000

100000

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

EU

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

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

2AB-Dextran ladder

“Analyte”


A) B)

C)

UNIFI

Glycan FLR

workflow

Retention Time Database

A) 2AB-Dextran ladder B) Generate Calibration curve C) Apply the curve to an

“unknown” sample

Glycan FLR Analytical Workflow In UNIFI


UNIFI is able to Acquire Glycan UPLC/FLR/QTOF MS data

2AB-labeled Etanercept N-Glycans

FLR

MS

Determine GU Values for Glycobase™


109 Glycans were Identified in an EPO Protein


Glycan Search inside Glycobase Database - IgG


A Review Panel in UNIFI After Glycan FLR Data Processing


UNIFI Reporting – all of the information in one click


Reviewing SEC Calibration Curve Results


Product Portfolio

www.waters.com/biopharm www.waters.com/glycan Download or order our New biopharmceutical Application Notebook at www.waters.com/biopharmbook

http://www.waters.com/biopharm

http://www.waters.com/glycan


Conclusions

Released glycan FLR analysis workflows are a recent

addition to the UNIFI Biopharmaceutical Platform Solution.

The UNIFI application workflow enables a scientist in

regulated or unregulated laboratory environments to

— acquire,

— process

— and report qualitative and quantitative glycan profiling

information,

— automatically


St John Skilton

Taking Glycan Analysis Further

IMS, ETD and TAP



• Quantitation of Individual Glycans

• Isomer discrimination

• Profiling of glycans

• Structure Elucidation

Intact Protein



Enzymatic Digestion

Peptide Mapping for:

• Location of Glycosylation

• O-linked and N-linked glycans

Discrimination of isobaric glycopeptides

Enzymatic Cleavage

Complementary Information with

Mass Spectrometry


Low Abundance Components

2ab Herceptin B 2033, 2.5 ug p/ul, batch 3

Time6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00

%

0

100

6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00

EU

x 1

0e4

0.000

20000.000

40000.000

60000.000

80000.000

100000.000

120000.000

140000.000

YAA_030_YQY_102009_010 (1) ACQUITY FLR ChA Ex330,Em420 nmRange: 1318325

YAA_030_YQY_102009_010 1: TOF MS ES+ BPI192

FLR

BPI MS

G0

F-G

N

G0

-GN

G1

F-G

N

G2

-GN

Man

5

G2

Man

7

Man

6

G2

F+

NA

c

Man

8

G2

F+

2N

Ac G1

FB

G1

G

1

Some Glycans poorly detectable in ESI (limited ionisation)


XIC of G2FS1 and G2FS2 2ab Herceptin B 2033, 2.5 ug p/ul, batch 3

Time24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00

%

0

100

24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00

%

0

100

YAA_030_YQY_102009_010 1: TOF MS ES+ 1099.9 0.15Da

40

YAA_030_YQY_102009_010 1: TOF MS ES+ 1245.5 0.15Da

24

XIC of Attributable glycan: G2FS2

XIC of Attributable glycan: G2FS1

G2FS1 2+

G2FS2 2+

FL detection can reveal glycans to assign by mass and confirm by Extracted Ion Chromatograms

(XICs)

A MS-only method risks missing important impurity and

modification information – UPLC-FL-MS(MS) is an ideal combination


DDA/MSMS of G2FS1 (M+2H)2+

MS/MS Spectra Deconvoluted by MaxEnt 3


UPLC/HILIC/MS of Glycopeptides


HILIC UPLC/MS for Glycopeptide Mapping

6 x magnified

4 x magnified

0 5 10 15 20 25 30 35 40 45

minutes

GLYCOPEPTIDES

MS BPI

UV 280 nm

A

B

*

Tryptic mAb IgG peptides.

BEH Glycan column -2.1 x 150 mm

Mobile phase A -10 mM Ammonium Formate

Mobile phase B -100% Acetonitrile

Gradient -90 – 50% B in 45 min.

Column temp. 40 °C

Non glycopeptides Glycosylated

MS trace (BPI)

UV trace (280nm)


Glycopeptides from Bovine Fetuin

0 5 10 15 20 25 30 35

minutes

MS BPI

A

B

C

MS BPI

XIC MS 657.24

non-glycopeptides

O-linked

glycopeptides

N-linked

glycopeptides

12

non-glycopeptides

34

56

78

1

2

34

5

6

78 9

9

2

3

4

5

6

78

9

1

1012

11

13

14

15

16

10 1211

13

14

1516

17

17

1819

+ H+

PNGase F treated

Individual Glycopeptide MS ion count was used for quantitation.

Oxonium ion


Glycopeptide Quantitation (IgG)

Rel. quantitation of glycopep. % Rel. quantitation of glycopep. %

UV 280 nm RSD % XIC MS RSD %

G0 6.3 ± 0.3 4.6 G0 6.1 0.1 1.4

G0F 35.7 ± 0.2 0.5 G0F 38.3 0.8 2.1

G1 a 3.1 ± 0.1 4.4 G1 a 2.4 0.2 10.3

G1 b 0.8 ± 0.1 14.4 G1 b 1.1 0.1 9.5

Man5 - - - Man5 1.1 0.0 2.8

G1F a 34.4 ± 0.1 0.2 G1F a 31.2 0.6 1.9

G1F b 11.2 ± 0.0 0.2 G1F b 11.6 0.2 1.6

G2F 8.5 ± 0.2 2.0 G2F 8.2 0.3 4.0

EEQYNSTYR


Bovine Fetuin Glycosylation Site Heterogeneity

5 glycosylation sites, 34 glycopeptides were identified.

Gilar et al, Analytical Biochemistry 417 (2011) 80–88


Asp

24

+ FA

4G

4S1

,FA

4G

4S1

Lac

3

Asp

24

+ FA

4G

4S2

Lac

2,F

A4

G4

S4 L

ac1

Asp

24

+FA

4G

4S1

Lac

3,

Asp

2

4+F

A4

G4

S3 L

ac2

, A

sp2

4+F

A4

G4

S4, A

sp

24

+FA

4G

4S2

Lac

2,

Asp

24

+FA

4G

4S1

Lac

3

A

sp 2

4 F

4G

4S4

Asp

24

+FA

4G

4S2

Lac

2,

Asp

24

+FA

4G

4S1

Lac2

A

sp2

4+F

A4

G4

S4 L

ac1

Asp

24

F4

G4

S4

Asp

24

+FA

4G

4S1

Lac

3

Asp

24

+ FA

4G

4S1

Lac

1

Asp

38

+ FA

4G

4S2

Asp

38

+ FA

4G

4S1

, FA

4G

4S2

Asp

38

+ FA

4G

4S1

iso

mer

s

Asp

38

+ FA

4G

4S1

Asp

24

+ FA

4G

4S2

Lac

2

NIBRT - HILIC-LC-MSE analysis of Glu-c Endoproteinase digest of EPO

46


Informatics Tool for Glycan Site Heterogeneity

oxonium ion annotation

carbohydrate annotation

MSE elevated energy fragment ion spectrum


Sequence Confirmation – HB for Vaccine

Hemagglutinin B (rHA_B)

TATQGEVNVTVIPLTTTPTK


Other QTOF MS Tools for Glyco Site Heterogeneity ETD

Z2

Z4

Z5

C6

Precursor ion (3+)

C7

C8

C9

Charge reduced 2+

Z6

Z7

Z8 Z9

C10

C11

C13

EAISPPDAASAAPLR O-linked Glycopepide from EPO







Intact Protein



Enzymatic Digestion Enzymatic Cleavage

ETD for O-Linked Glycans

Targeted Analysis:


• O-linked [and N-linked] glycans



Glycosylation Site Characterization

Scott McLuckey

ETD CID


ETD spectrum of N-linked Glycopeptide

m/z

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500

TOF MSMS 740.30ES+

3.46e4986.81

510.30

277.13423.26

322.20

404.23

740.37

567.30

1480.21

1225.57

1197.07

1115.06

1297.12

1269.59

1401.661298.13

1401.15

1298.631343.14

Precursor ion

[M+4H] 4+

chargereduced

[M+3H]3+

chargereduced

[M+2H]2+

z3c2z2 c3

z4

c4z5

2+

z62+

z72+

z82+

c62+

c72+

c82+

c52+

E E Q Y N S T Y R

Waters Application Note: 720004281en “Electron Transfer Dissociation of N-linked Glycopeptides from an Recombinant mAb Using Synapt G2-S HDMS


ETD of O-linked Glycopeptide - 7 linkage sites are assigned

ETD spectrum


CID of the same O-glycopeptide - serial neutral loss ions

T15: SQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPK

7 Modifications

Output from BiopharmaLynx

CID spectrum

Highly O-linked glycopeptides generate mostly neutral loss!


CID of the same O-glycopeptide - serial neutral loss ions

T15: SQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPK

7 Modifications

NL = 365.13 Da

Output from BiopharmaLynx

CID spectrum

NL = 203.08 Da

NL = 162.05 Da

Highly O-linked glycopeptides generate mostly neutral loss!

Etc.

Etc.







Intact Protein



Enzymatic Digestion Enzymatic Cleavage

Peptide Mapping Technique: “TAP” fragmentation [SYNAPT™] for simultaneous

peptide sequence and glycan confirmation

Untargeted Peptide Mapping for:





Innovative Techniques for Sequencing Glycopeptides

Glycopeptides can be ‘mined’ from the LC/ MSE data

Unique architecture of SYNAPTTM used to simultaneously supply

multiple sets of information

—Glycan information from low-energy CID

—Peptide backbone sequence confirmed from high-energy CID

—Information is obtained simultaneously

—O- and N-linked glycans can be identified

© 2009 American Society for Mass Spectrometry.

Published by Elsevier Inc.

doi:10.1016/j.jasms.2009.07.017

Rapid Commun. Mass Spectrom. 2008; 22: 29–40;

(www.interscience.wiley.com) DOI: 10.1002/rcm.3330


Glycopeptide Identification using LC-MSE approach

Low Energy channel

High Energy channel

Extracted marker ions m/z: 204.1+366.1 to reveal glycopeptides

© 2009 American Society for

Mass Spectrometry.

Published by Elsevier Inc.

doi:10.1016/j.jasms.2009.07.

017


Enhanced Structural Characterisation: Time Aligned Parallel Fragmentation (TAP)

Drift time

Product ions separated by IMS

m/z

Precursor ion fragmented

Drift time

m/z

Precursor and products share same drift time

Ions from Quad region

Refragmentation in TRANSFER Cell –

higher energy to break peptide bonds

Fragmentation in TRAP Cell – LOW energy to cleave Glycans


Glycan Fingerprint from Trap Cell Fragmentation

Low energy Fragmentation in

the Trap cell cleaves glycans

Glycan assignment according to accurate masses of precursors and fragments


P H+

EEQYNSTYR

Peptide Sequence and Glycosylation Site From Transfer Cell Fragmentation

High energy Fragmentation in the Transfer

cell cleaves peptide bonds

Peptide Sequence Confirmation using characteristic b and y ions


TAP fragmentation on the first stage to obtain O-linked glycan sequence

Pep

Pep

Pep

MH+

(EAISPPDAASAAPLR)

G1 data

Example of workflow applied to EPO: showing selected glycans


MS/MS of Peptide

B

y8

y10

EAISPPDAASAAPLR, MH+

y11 y12 y10 y8

y11

Pep

y12

Example showing workflow applied to EPO: glycopeptide sequence







Intact Protein



Enzymatic Digestion

Peptide Mapping for:




Enzymatic Cleavage

IMS of Glycopeptides in an LC/MSE run


Enhancing data clarity during fast peptide mapping with HDMSE

ASMS 2012 Poster: WP 450


Chromatogram from LC/MS run of Bovine Fetuin

Extracted Chromatogram HexNAc Oxonium Ion

Elevated Energy

TIC Chromatogram Low Energy

(X-Axis in Scans)

Craig Dorschel et al, ASMS 2010 Oral presentation


Drift Scope Selection – 3D plot of m/z vs Drift Time

011510_UCA012_Fetuin_02.raw : 1

Glycopeptides are expected in the outlined region. Species have:

- Long Drift Time - High m/z

- High charge states


Normal (3D) View

m/z

Retention Time


IMS Enhanced View

m/z

Mobility


Investigation of the mobility space clearly illustrates two isobaric forms of the O-linked glycopeptide

Delta RT 0.005 minutes

Delta RT 300 msec Delta m

/z 1

.4 a

mu

Delta m

/z 1

.4

am

u

Delta Drift 12


Mobility, m/z Maps for Other Glycoproteins

050510_UAA114_HERcn_01.raw : 1

050510_UAA114_a1AGP_02.raw : 1050510_UAA114_Fetuin_04.raw : 1

050510_UAA114_TRFE_01.raw : 1

Fetuin 1-Acid Glycoprotein

Transferrin Trastuzumab


Summary

Intact Protein Glycoprotein Profiling

Using BiopharmaLynx Bioinformatics

tool

—Batch data processing

—Data comparison (batch to batch,

Innovator/Biosilimar)

FLR labeling Glycan Analysis

—BEH Glycan Column chemistry gives

higher resolution and faster

chromatographic separation

—Exoglycosidic digestion + GU

assignment enable full glycan structure

characterization

UPLC/FLR/Qtof MS Platform offers

—Glycan profiling using FLR chromatogram

—Accurate mass confirmation

—MSMS

— ETD of glycopeptides (N-, O-linked)

gives linkage site information

UNIFI – platform solution for

biopharmaceuticals


It is child’s play…

http://www.ted.com/speakers/ursus_wehrli.html http://de.wikipedia.org/wiki/Ursus_Wehrli


It is child’s play… with the right tools!

http://www.ted.com/speakers/ursus_wehrli.html http://de.wikipedia.org/wiki/Ursus_Wehrli


AcknowledgementS

Waters Corporation

Ying Qing Yu

Joomi Ahn

Eoin Cosgrave

Asish Chakraborty

Stephane Houel

Jonathan Williams

Scott Berger

Beth Gildea

Vern Tisdale

Jennifer Fournier

Craig Dorschel

Weibin Chen

NIBRT (Ireland)

Pauline Rudd

Mark Hilliard

Giorgio Carta

Jonathan Bones


2013 Waters Biopharmaceutical Application Notebook

www.waters.com/ biopharmbook

http://www.waters.com/

waters glycan analysis by uplc-fl-ms€¦ · waters glycan analysis by uplc-fl-ms ... structure...

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