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High-throughput Analysis Of Antigen-specific, Vaccine-induced
Antibody Glycosylation Using Multi-Capillary CE
Madeleine Jennewein
Ragon Institute of MGH, MIT and Harvard
Antibody Modifications Impact Functionality
PDB accession code: 1IGY
Antibodies Mediate Effector Functions
Antibodies Mediate Effector Functions
Antibody Modifications Impact Functionality
Glycosylation
PDB accession code: 1IGY
Antibody N-linked Glycosylation is Critical
GlcNAc
Mannose
Antibody N-linked Glycosylation is Critical
Galactose (G)
GlcNAc
Mannose
Antibody N-linked Glycosylation is Critical
Sialic Acid (S)
Galactose (G)
GlcNAc
Mannose
Antibody N-linked Glycosylation is Critical
Sialic Acid (S)
Galactose (G)
GlcNAc
Mannose
Fucose (F)
Antibody N-linked Glycosylation is Critical
Sialic Acid (S)
Galactose (G)
GlcNAc
Mannose
Fucose (F)
Decreasing Antibody Fucosylation Increases Antibody Efficacy
Trastuzumab
PMID: 20484044
Rituximab
PMID: 16219319
Increasing Antibody Bisection Increases Antibody Functionality
Ferrara, et al. PNAS, 2011Goede et al., NEJM, 2014
What is the Role of Antibody Glycosylation in Control of Infection and Vaccine-Mediated Protection?
Ackerman, M.E. et al. J Clinical Invest. 2013
Mass Spectrometry • Low throughput • Low sensitivity (50-100ug IgG)• Low quantitative value• Expensive equipment• Requires extensive data de-convolution
HPLC• Low throughput• Moderate sensitivity (20ug IgG)• Quantitative• Requires specialized equipment
Historical Glycan Analysis Techniques have drawbacks
Capillary Electrophoresis for Glycan Analysis
• High throughput (>36 samples per hour)• High sensitivity (1-5ug of IgG)• Quantitative• Easy to interpret data• Globally accessible machinery
WashBeads
30 min
Sample preparation
16
“Alter” Method
PNGase FTreat
EthanolPrecipitation
VacuumDry
APTSLabel
ColumnPurify
PNGase FTreat
Add GlycanAssureBeads
EluteGlycans
TealLabel
1 hour
1 hour
20 min 2 hour 3 hour
30 min
GlycanAssure
Data Analysis
17
“Alter” Method
Export .fsafile
GlycanAssure
Convert to.xml
Load intoMatLab Script
Align toreference
Hand collatepeaks
Export.xls file
Copy intoanalysis template
Report relativepeak frequencies
Import intoGlycanAssure
Analysis
Select AnalysisMethod
Export.xls file
Reproducibility of Glycosylation Analysis
Black=Same sample run 20 times
Grey=20 different samples run concurrently
Mahan et al., J. Immunol Methods, 2014
Reproducibility of Glycosylation Analysis
0 10 20 30 40 500
5
10
15
20
25
Average CV
Avera
ge A
rea
Pe
ak A
rea
Average CV
0
5
10
15
20
25
10 20 30 40 50G0
G0
B
G0
F
G1
G1
'/G
0F
B
G1
'B
G1
'F/G
1F
B
G1
B
G1
F
G1
S1
F
G2
G2
F
G2
FB
G2
S1
G2
S1
F
G2
S2
G2
S2
F
M3
N2
F
0
20
40
60
80
100
Inte
rmed
iate
Pre
cis
ion
(%
CV
)
Inte
rme
dia
te P
reci
sio
n (
%C
V)
0
20
40
60
80
100
Antibody Glycosylation is Antigen Specific
Mahan and Jennewein et al. Plos Path 2016
gp120p24HA
Glycosylation Responses To Different Antigens Are Not Correlated
Mahan and Jennewein et al. Plos Path 2016
Glycosylation Correlated to Functionality
relative
row min row max
AD
CD
AD
CC
CD
10
7a
IFN
-M
IP-1
AD
CP
AD
CV
I
Annotation
G2FBG2FG2BG2/G1FBG1FG1FG1BG0FBG1G0FG0BG2S1FG2S1BG2S1G1S1FG1S1G2S2FBG2S2FG2S2BG2S2
*
**
***
**
Mahan and Jennewein et al. Plos Path 2016
HIV-Specific Antibody Glycosylation Varies With HIV Infection
gp120-specific antibodies
Mahan and Jennewein et al. Plos Path 2016
Bulk Antibody Glycosylation Differs Between Geographical Regions
Mahan and Jennewein et al. Plos Path 2016
United StatesKenya/RwandaSouth Africa
Vaccination Elicits a Glycosylation Pattern Distinct from Bulk Glycosylation
Bulk Vaccine-specific
Mahan and Jennewein et al. Plos Path 2016
Bulk Vaccine
The Vaccine-Specific Response is Independent of Regional Glycosylation
Differences
Mahan and Jennewein et al. Plos Path 2016
Non-vaccine Specific Antibody Glycans Are Stable During Vaccination
Mahan and Jennewein et al. Plos Path 2016
Baseline 2-weeks post vaccine 2-weeks post-boost
Different vaccines elicit distinct glycosylation profiles
Mahan and Jennewein et al. Plos Path 2016
IPCAVDVAX003
Concluding Remarks
• CE allows much greater analysis of antibody glycosylation
• This technology is high throughput and can be implemented easily
• Antibody glycosylation is antigen-specific
• Antibody glycosylation is programmable by vaccination
AcknowledgementsAlter Lab: Galit AlterTodd SuscovichAlison MahanKendall DionneJacquelynne TedescoAmy ChungAvery Normandin
Collaborators: Henrik StreeckMaria PauNahekeSchuitemakerDon FrancisPatricia FastDagna LauferBruce WalkerLindsey BadenDan Barouch
ThermoFisher:Baburaj KunnummalWesley McGinn-StraubDominic Y. CaseñasMichael Brewer