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