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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
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
The ABCs of Glycoproteins: How to Combine Analytics, Biology,
and Chemistry to Improve Your Understanding of Glycosylation
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
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
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
The ABCs of Glycoproteins: How to Combine Analytics, Biology,
and Chemistry to Improve Your Understanding of Glycosylation
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
©2013 Waters Corporation 39
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).
©2013 Waters Corporation 40
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
©2013 Waters Corporation 41
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
©2013 Waters Corporation 42
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
©2013 Waters Corporation 43
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)
©2013 Waters Corporation 44
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
©2013 Waters Corporation 45
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
©2013 Waters Corporation 46
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
©2013 Waters Corporation 47
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
©2013 Waters Corporation 48
Relative Quantitation Data
N= 3
©2013 Waters Corporation 49
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
©2013 Waters Corporation 50
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”
©2013 Waters Corporation 51
Glycosylation Site Characterization
Scott McLuckey
ETD CID
©2013 Waters Corporation 52
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
©2013 Waters Corporation 53
ETD of O-linked Glycopeptide - 7 linkage sites are assigned
ETD spectrum
©2013 Waters Corporation 54
CID of the same O-glycopeptide - serial neutral loss ions
T15: SQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPK
7 Modifications
NL = 365.13 Da
Output from BiopharmaLynx
CID spectrum
©2013 Waters Corporation 55
“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
©2013 Waters Corporation 56
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.
©2013 Waters Corporation 57
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)
©2013 Waters Corporation 58
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
©2013 Waters Corporation 59
A Review Panel in UNIFI After Glycan FLR Data Processing
©2013 Waters Corporation 60
UNIFI Reporting
©2013 Waters Corporation 61
UNIFI is able to Acquire Glycan UPLC/FLR/QTOF MS data
2AB-labeled Etanercept N-Glycan
FLR
MS
©2013 Waters Corporation 62
Product Portfolio
www.waters.com/biopharm www.waters.com/glycan Download or order our New biopharmceutical Application Notebook at www.waters.com/biopharmbook
©2013 Waters Corporation 63
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
©2013 Waters Corporation 64
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
The ABCs of Glycoproteins: How to Combine Analytics,
Biology, and Chemistry to Improve Your Understanding of
Glycosylation
Q&A
The ABCs of Glycoproteins: How to Combine Analytics, Biology,
and Chemistry to Improve Your Understanding of Glycosylation
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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