The ABCs of Glycoproteins: How to Combine Analytics ... · The ABCs of Glycoproteins: How to...

The ABCs of Glycoproteins: How to Combine Analytics, Biology,

and Chemistry to Improve Your Understanding of Glycosylation

Broadcast Date: Tuesday, March 15, 2013

Time: 11AM EST, 8AM PST

Sponsored by

The ABCs of Glycoproteins: How to Combine

Analytics, Biology, and Chemistry to Improve Your

Understanding of Glycosylation



Your Moderator

John Sterling Editor-in-Chief

Genetic Engineering & Biotechnology News



Pauline Rudd, Ph.D.

NIBRT Research Professor of Glycobiology

NIBRT Glycobiology group

Multidimensional glycosylation

analysis

Pauline M Rudd

Conway Institute

Eoin Cosgrave, Weston Struwe, Henning Stockmann, John O’Rourke, Jer Hayes, Oscar

Potter, Mark Hilliard, Patrick Jennings, Giorgio Carta, Jayne Telford, Moham Kumar, Jenny

Huffman (Edinburgh), Niaobh McLoughlin, Rebecca Duke, Simone Albrecht, Barbara

Adamczyk, Radka Fahey, Akram Asadi Sheni, Joanne Withers, Tharmala Tharmalingam,

Barbara Keegan

Irish Minister of Science: Sean Sherlock, Ian Marison

Missing– Louise Unwin, Ciara McManus, Eugene Dempsey, Margaret Doherty, Karina Marino,

Jayesh Kattla, Natalia Artemenko, Michael Schomburg, Marie Galligan, Henning Stockmann,

Simone Albrecht

Centres of Innovation award

2012

//upload.wikimedia.org/wikipedia/en/0/04/Northeastern-logo.svg

12/03/2013 5

The National Institute for Bioprocessing Research and

Training , Ireland

6,500 m2

NIBRT: private Institute based on innovative partnership between 4 academic institutions funded by IDA Ireland (€57 million) with a mission to:

Conduct world-class research in key industrial areas of bioprocessing

Train highly skilled personnel for the bioprocessing industry

Provide flexible, multipurpose bioprocessing research and training facilities

Provide BioanalyticsTraining,

Contract research & services: glycan analysis, product characterisation

DNA

From genes to functional proteins

4 bases

Alanine

Arginine

Asparagine

Aspartic Acid

Cysteine

Glutamic Acid

Glutamine

Glycine

Histidine

Isoleucine

Leucine

Lysine

Methionine

Phenylalanine

Proline

Serine

Threonine

Tryptophan

Tyrosine

Valine

Polypeptides fold

into proteins: N-

glycans attached

co-translationally

amino acids

3KM in each cell

Adult: 70 trips from

the earth to the sun

and back EPO

http://en.wikipedia.org/wiki/File:L-alanine-3D-balls.png

http://en.wikipedia.org/wiki/File:L-Alanin_-_L-Alanine.svg

Blood Proteins

Hormones

Growth Factors

Cytokines

Vaccines

Monoclonal Antibodies

Bone Proteins

Other

127 are Glycoproteins (>66%) Walsh (2010). Nature Biotech; 28(9):917-924

Therapeutic Glycoproteins are secreted recombinant proteins

that are intended to interact with the host

N- and O-glycosylation

Nt

Asn-X-Thr

Ser

Asn-X-Ser

N-Glycan

N-Glycan

C

O Glycan

O O Et

N O

P

O

O O C C

O-Inositol

O

O

O

O

P

O

O

Thr O-GalNAc

Asn

O-GalNAc Golgi

ER

ER

Tissue plasminogen

activator Type I and

Type II

IgG

EPO

Golgi

Complex type (bi-, tri-,

tetra- antennary)

Oligosaccharide

transferase

a-Man I

Glycoproteins consist of collections of glycoforms: due to

macroheterogeneity and micro-heterogeneity at each site

Glc Glc

a-Glc I a-Glc II a-Glc II

Asn Xaa Ser/Thr

NH2

Glc

ER

GlcNAc-

TI

a-Man II

DolP precursor

GlcNAc-T2 FucT8

SialylT GalT

IgG

EPO

EPO glycans can:

•Extend half life

•Stabilise and protect

•Modulate activity

•Be antigenic

Ser 126

Glycoproteins can be secreted

Glycosylation is cell, protein, site (glycopeptides) and

environment specific

A4G4FBS(6)4

Asn 24

Asn 83

Asn 38

Erythropoetin

Sugars are also hard to analyse because they can

be linked in different ways

Glc

GlcNAc

Gal

GalNAc

Fuc (deoxy galactose)

NeuNAc

Man

Open symbols: hexose

Filled in: HexNAc

2 3

4

8 6

unknown linkage

b - linkage

a - linkage

Linkage position

Linkage type

Xylose

Symbolic representation must include

monosaccaride sequence and linkage

Sialyl Lewis a

Sialyl Lewis x

R

R

a1,3

a1,6

etc

Plant/

PNGase A

Mammalian/

PNGase F

Adaptable Comprehensive Glycosylation platform needed to

address different analytical requirements

Production & Scale up

• Media composition,

• temperature, pH, hypoxia,

• cell death affect glycosylation,

• protein QC pathways,

• aggregation

Product analysis

Critical features • Regulation

(new products, Biosims)

• Batch release

Product stability

Glyco excipients

At-line monitoring •Trouble shooting

• Purification/formulation

• Glycan analysis of fractions

Process

Aanalytical

Technology

Response to therapy

Cell line selection • High producing

• Appropriate glycosylation

for optimal activity

• Set process parameters

• Identify key control points

2AB

Safety &Efficacy •Antigenicity:

•Half life

a-gal

Testing function • Potential side effects

• Response to therapy

• Identifying responders

•Fundamental research

Quality By Design

Basic research

Importance

of sugars for

function

IgG EPO

Sample reduced

and alkylated. Gel block washed to

remove

contaminants

Glycoprotein

immobilised in gel block set in a 96 well plate

or on PVDF membrane

1. N-linked

glycan release via PNGase F

digestion

2. O-linked

glycan release via chemical

reaction

3. In gel

protease digestion

for peptide

release

Peptides eluted.

Analysis via LC-MS, LC-MSn

MALDI

Wash

NM

DT

gu

ABS, BTG, BKF

Exoglycosidase array

digestions

HILIC: glycan poolAEC

LCMS/CE etc

Extractor

programme:compares 2AB GU

values with

GlycoBase makes preliminary

assignments

autoGU

then refines assignments based on the digest data.

Structural

assignment and quantitation

Total glycome analysis

Fluorescent labelling via

reductive amination (e.g. 2AB) SPE plate for

excess reagent removal

Labelled glycans eluted,

dried and reconstituted in a set volume.

N-G

lyco

lyl

NA

Released glycans

eluted

SA speciation

MSRP HPLC

Five stages (1) Sample prep (2) Glycan release (3) Labelling (4) Separation (5) data

interpretation

A systematic approach to protein glycosylation analysis: a path

through the maze. Nature Chem Biol 6 713- 723 Marino, Rudd et al 2010

CE

Or Solid support

Henning Stockmann Margaret Doherty Niaobh O’Donoghue Michael Schomberg

Pauline Rudd

• Robotic platform: 96-well plate glycan sample prep, release/ labelling

• Linear sequencing robot for at-line sampling

• Academic (biomarkers, GWAS/glycome) and pharma applications

Optimised automated glycan release and labelling,

UPLC separations coupled with computer assisted data

analysis bringing glycomics into line with other ‘omics

Minutes

50 60 70 80 90 100 110 120 130

8 7

6

5

4 3

9 10

Dextran Ladder

Glucose units

Sample

Chromatogram

IgG

Detailed glycan analysis using predictive HILIC columns Waters HPLC, 150 x 4.6 mm, 3μm TSKgel Amide 80 column; acetonitrile gradient

Dextran Calibration: gu values assigned to sample

A2G2S2

8.4gu HILIC column separates

arm specific isomers

ABS

NAN 1

Linkage position

8

2 3

6

4

β-linkage

α-linkage

Unknown linkage

Exoglycosidase specificities

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

Sialidase

ABS

AMF

BKF

X1-2F

AMF

BKF

BKF a1-6/2

X1-2F a1-2 Fucosidase

AMF a1-3/4

SPG BTG

SPG

BTG

BTG

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

GUH

GUH

GUH b1-2/4/6bMan Hexosaminidase

Sub-Collections

Herceptin 20

Fetuin 24

Invertase 37

Erythropoietin 89

Mouse IgG 35

Glycan standard mix 40

Human serum glycome 135

Dynamically

generated charts

Current Waters (UPLC) Collection

Herceptin® (Trastuzumab) N-linked Glycans 4

.30

+β-Hexosaminidase

(ABS+BTG+BKF+GUH)

5.4

1

5.8

8

6.1

8

6.2

8

6.3

6 6

.71

6.8

4

7.2

0

7.6

3

8.3

3

9.1

2

Undigested

4.8

4 5

.41

4.7

1 + Galactosidase

(ABS+BKF+BTG)

6.1

9

6.2

8

5.4

1

5.8

9

6.3

6 6

.71

6.8

4

7.6

3 + Sialidase

(ABS)

4.7

1

4.8

3

5.4

1

5.8

6 6

.28

6.3

9

7.2

1 + Fucosidase

(ABS+BKF) 0.4 GU = Core fucose

1.7 GU = 2 x Galactose

1.1 GU = 2 x N-Acetylglucosamine

0.7 GU = 1 Sialic acids

1.4 GU = 2 Sialic acids

5.0

6.0

7.0

8.0

9.0

Dextran ladder

Everything digests to Man3

HILIC-UPLC

Mark Hilliard

Suppression of ADCC (anti-inflammatory)

activity (Kaneko et al., Science 313, 670-3, 2006)

sialic acid

Placental transport; FcRn; C1q binding

(Kibe et al., J Clin Biochem Nutr 21,57-63,1996)

galactose

Functionality of terminal residues of N-glycans on IgG

Enhanced ADCC activity core fucose (-)

(Okazaki, et al., J Mol Biol 336, 1239-49, 2004)

Ligand for Mannose Binding Protein (complement

activation) GlcNAc/mannose

(Malhotra, et al., Nat Med 1, 237-243, 1995)

a-gal Antigenic

Enhanced ADCC (cytotoxic) activity bisecting

GlcNAc (Umana, et al., Nat Biotechnol 17, 176-180, 1999)

Affects Fc-FcR

Interactions

Wormald, Mimura, Dwek & Rudd

(based on Sondermann et al. Nature 2000)

Fc-FcR Influences Immune Effector Functions

Potential influences on the composition of glycomes and

glycoforms and their function

Need to link the glycome, proteome, transcriptome, metabolome, genome

• Genes/Single Nucleotide Polymorphisms, Epigenetics

• Expression levels/activities of processing enzymes and approx 600 proteins

involved in protein glycosylation

• Levels of monosaccharide nucleotide donors

• Metabolites, lipids

• Organisation of cellular organelles

• Activators of cell signalling pathways: cytokines/hormones, drugs, oncogenes

• Environment/Disease

1. Ammonia concentration

2. Growth rate

3. Dissolved CO2

4. Media composition

5. Feed times

6. Hypoxia

7. Enzyme deletion, inhibition

8. Length of culture

Factors that change glycosylation during

production

Glycan profiling: commercial EPOs

9

0.

0

0

9

5.

0

0

10

0.

00

10

5.

00

11

0.

00

A

B

D

C CHO

cells

CHO

cells

CHO

cells

Human

cell line

EPO complexed

with EPO receptor

GU values

• 1D separation of a glycan

pool by a single technique

• provides signature for

simple snapshot whole

pool or individual critical

quality attributes (CQAs)

• preliminary (unconfirmed)

information may be

surmised.

• Higher resolution the

better the comparison

NMR, HPLC CE lectin chips

Mass Spectrometry.

Waters Alliance HILIC HPLC

Wormald, Dwek,

Rudd

CQA: Identification of antigenic epitope (N-glycolyl

neuraminic acid) on EPO

m/z value

306 Neu5Gc (B-type) 424 Gal-GlcNAc antenna (1,3A-type) 1431 Parent ion [M+H2PO4-H]2-

Saccharide

(-) Mode Waters Micromass QTof

Ultima Global (nanoESI-MS/MS)

FcRIIIA

m/z200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400

%

0

100

DQ090959 2 (0.091) Sb (99,40.00 ); Sm (Mn, 2x3.00); Cm (1:89) TOF MSMS 1430.30ES- 52.3306.08

218.06

1430.931380.37

1247.31307.09 1205.81425.12 586.20 1127.36 1277.87 1432.40

B 1

424 Neu5Gc (B-type)

Gal-GlcNAc antenna

(1,3A-type)

Parent ion

[M+H2PO4-H]2-

Neu5Gc

Neu5Ac: 290 Neu5Gc: 306

Minutes

20 24 28 32 36 40 44 48

Monosialylated

Disialylated

Trisialylated

Tetrasialylated

Total N-Glycan Pool.

Minutes10 14 18 22

AEC-

Fluorescence

HILIC-

Fluorescence

Detailed structural analysis: monosaccharide sequence, linkage, relative

or absolute quantification by high resolution separation technologies:5-D

2D 1D

GU values

Waters Alliance WAX HPLC

Waters Alliance HILIC HPLC

Negative ion CID spectra of tri- and tetra-sialylated

tetra-antennary N-glycans: David Harvey 4D

MALDI MS: acetylation of sialic acid residues on

commercial ESAs: David Harvey 5D

Glycan Analysis of Etanercept (Enbrel®) Molecules

CHO cells - have restricted simple O-glycosylation

TNFα Receptor

Fc (IgG)

Mucin-like Fusion

Domain

Sialylated Core 1 O-glycans

• Human Dimeric fusion protein - TNFαR

- fusion domain

- IgG Fc

• Competitive inhibitor of TNFα - anti-inflammatory for psoriatic

arthritis

• 3 N-glycan sites per chain

• 13 O-glycan sites per chain

At least 32 glycans per molecule

Needs site analysis as well as glycan analysis

Core 1 - Gal(b1,3)GalNAc

Core 3 - GlcNAc(b1,3)GalNAc

Core 2 - Galb1,3[GlcNAcb1,6]GalNAc

Core 4 - GlcNAcb1,3[GlcNAcb1,6]GalNAc

Core 2

Major O-glycan core structures

Etanercept N-glycans by HILIC-Fluorescence-MS/MS

Etanercept N-glycans

Fragmentation for glycan structural characterization

MS

MS/MS

[M-2H]2-

Waters Xevo G2 with FLD

Waters 1.7 m BEH Glycan

(1 mm x 100 mm)

6D Recognition: Mucin O-glycans from Chicken Intestines

Analysis of chicken

intestinal mucin O-

glycans:

• C. jejuni is a commensal

bacteria in chickens

• Chicken mucin attenuated

bacterial binding & is

attributed to mucin

glycosylation (Alemka et al.)

• Large intestine inhibited C.

jejuni binding and

internalization better than

small Intestine and caecum

• Goal: To Identify

glycosylation differences

in the chicken intestinal

tract as it pertains to C.

jejuni colonization

Alimentary health: Detailed O-glycan analysis

Chicken Mucins are Highly Sulphated and Sialylated OH

OH

OH

NHAcO

OH

O

NHAcOH

O

OH

O

OH

OH

SO4-

OH

241

301

444

315

OH

OH

OH

NHAcO

OH

O

NHAcOH

O

O

OH

OH

OH

OH

SO4-

282

505

444

Weston Struwe (STINT grant) Niclas Karlson Catherine Hayes (Gothenburg)

•Building an O-glycan fragmentation data base

11

6

2

11

2

17

2

6

2

10

6

3

3

3

7

9

9

3

11

13

12

11

13

20

10

12

4

19

21

10

35

38

13

13

14

0 10 20 30 40 50 60

Chicken caecum

Human-2 (caecum)

Human-1 (caecum)

Chicken small intestine

Human-2 (ileum)

Human-1 (ileum)

Chicken large intestine

Human-2 (transverse colon)

Human-1 (transverse colon)

O-Glycan Structures

Mu

cin

Ori

gin

Sulphated

Sulphated-Sialylated

Sialylated

Neutral

Chicken gut mucin O-glycans are more highly sulphated than human

Robbe, C et al. Biochem J 1, 843 (2004)

Instrument Acquisitions

Key Personnel

Jonathan

Bones

Mark

Hilliard

Stefan

Mittermayer Weston

Struwe

Margaret

Docherty

Michael

Schomberg

Glycan analysis is complex

The question determines the technological approach

Marino, Rudd et al Nature Chemical Biology 2010

Centre of Innovation

Eoin Cosgrave



Ying Qing Yu Principal Scientist

Waters

©2013 Waters Corporation 38

Ying Qing Yu, PhD

Principal Scientist

Waters Corporation

BIOTHERAPEUTIC PROTEIN GLYCOSYLATION ANALYSIS USING

UPLC/QTOF MS

GEN Webinar March. 15th 2013


Glycosylation is a Key Critical Quality Attribute

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

figures are from Wikipedia

The International Conference on Harmonization Guideline Q6B requires the analysis of carbohydrate content, structural profiles, and characterization of the glycosylation site(s) within the polypeptide chain(s).

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


UPLC and MS Based Analytical Workflows for Comprehensive Glycosylation Analysis

Fluorescent and mass profiling of glycans

Relative Quantitation

Structure Elucidation

MS based

LC retention time only (GU value)

Intact Protein

Glycopeptides Released Glycans

MW profiling (LC-ESI MS)

Proteolytic Digestion PNGase F Digestion

Glycosylation site and

site heterogeneity


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)

GOF/G0F

G0F/G1F G1F/G1F G0F/G2F

G1F/G2F

G0/GOF G2F/G2F

MaxEnt 1


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


Fluorescent Labeled Glycan Analysis

Sample Preparation

HILIC-UPLC/FLR

GlycoWorksTM Sample Preparation Kit -- Free glycan extraction and clean up -- 2AB-labeling and clean up

FLR

http://www.waters.com/webassets/cms/library/docs/720004584EN_GlycoWorks.pdf


2AB-labeled IgG1 Glycans Separated using HILIC –UPLC/FLR

Time (min.)

10 20 30

G0 -

GN

G0F

- G

NG

0

G0

FM

an

5

G1a

G1

F -

GN

G1

Fa

G1F

b

Man

6

G2

G2F

Ma

n 7

Man

8

G2

FS

1

G2

FS

2

Batch 1

Batch 2

Batch 3

G1b

Waters Application Note: 720003576en


Relative Quantitation Data

N= 3


Why UPLC?

Alliance 2695

FLR 2475

TOSOH TSK 3.0 um

2.0 x 150 mm

72-62% B in 50 min.

0.36 mL/min.

UPLC

UPLC FLR

BEH Glycan 1.7 um

2.1 x 150 mm

72-62% B in 45 min.

0.40 mL/min.5 .0 0 1 5 .0 0 2 5 .0 0 3 5 .0 0 4 5 .0 0 m in .

m in .5 .0 0 1 5 .0 0 2 5 .0 0 3 5 .0 0 4 5 .0 0

1

1

2

2

4

4

5

5

6

6

7

7

9

10

10

11

11

12

12

13

13

2AB labeled Human IgG Glycans

8 3


Acquire Both FLR and MS Chromatograms From the Same Injection

HILIC-UPLC/FLR/QTof MS

(2AB-labeling)

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

EU

x10

e4

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

Time22.00 22.50

(1) ACQUITY FLR ChA Ex330,Em420 nmRange: 336117

1: TOF MS ES+BPI256

FLR

MS

m/z953 954 955 956 957 958 959

954.37

FLR

BPI MS

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


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

NL = 365.13 Da

Output from BiopharmaLynx

CID spectrum


“New Tools” for Glycoprotein Characterization-- Standards

Intact mAb Mass Check Standard

G0F+

G1F

G0F+

G0F

G1F+

G1F

G1F+

G2F

G2F+

G2F

G0F+

G1F

ESI- QTOF MS

MaxEnt 1 Deconvolution


More on 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.


Informatics Tool: UNIFI Biopharmaceutical Platform Solution

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)


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


A Review Panel in UNIFI After Glycan FLR Data Processing


UNIFI Reporting


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

2AB-labeled Etanercept N-Glycan

FLR

MS


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


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 – advanced characterization

Reference Standards – system

performance

UNIFI – platform solution for

biopharmaceuticals


Waters Corporation

Joomi Ahn

Asish Chakraborty

Stephane Houel

Jonathan Williams

Scott Berger

Beth Gildea

Vern Tisdale

Jennifer Fournier

NIBRT (Ireland)

Pauline Rudd

Mark Hilliard

Giorgio Carta

Jonathan Bones

Acknowledgement



The ABCs of Glycoproteins: How to Combine Analytics,

Biology, and Chemistry to Improve Your Understanding of

Glycosylation

Q&A



Thank You For Attending

The ABCs of Glycoproteins: How to Combine Analytics,

Biology, and Chemistry to Improve Your Understanding of

Glycosylation

Broadcast Date: Tuesday, March 15, 2013

Time: 11AM EST, 8AM PST

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