Native and IM-MS

characterization of ADCs

Alain VAN DORSSELAER

BioOrganic Mass Spectrometry Laboratory, IPHC

University of Strasbourg, CNRS

Strasbourg - France

1 ASMS 2015 Waters User’s meeting

mAbs as therapeutics

mAbs : a very important class of human therapeutics - > 50 mAbs & derivatives currently approved (EMA/FDA)

- indications: inflammation, autoimmunity, cancer, transplantation,…

- > 400 mAbs & derivatives in pre-clinical development and clinical trial

anti-cancer mAbs in clinical studies - 84 unmodified IgG (51%)

- 10 bispecific (6%)

- >35 ADC (15%)

- 17 engineered (10%)

- 16 fragments (10%)

mAbs are complex therapeutic proteins

(150 kDa glycoproteins)

Next generation empowered mAbs :

Bispecific mAbs (bsAbs) and Antibody-Drug-Conjugates (ADCs)

mAb + cytotoxic Double-barreled shotgun:

two antigens targetted

A single shot gun

A highly

cytotoxic drug IgG4

(natalizumab)

IgG4

(6F4-2)

+ GSH

Bispecific mAb (bsAb)

(natalizumab/6F4-2)

+

2 different half mAbs

Intact mAb

analysis

(150 kDa)

Fragment analysis

(25-100 kDa) Peptide mapping

(700-7000 Da)

Higher Order Structure

Aggregates

mAb/Ag complexes

(>150 kDa)

Size

mAb characterization is a nightmare

Native IM-MS Top-down MS

(FT-ICR + Orbital trap)

LC-MS

(nano)LC-MS/MS

enzymatic digestion Native MS

HDX-MS Crosslinking MS

The MS

toolbox

4

1991

Native

MS

2006

Ion Mobility MS

2012

High

resolution

native MS

Native MS: a brief history

2000

Automated

nanoESI

2002

Haemocyanins

2.3 MDa

Katta et al. JACS 1991

1991

Holo-myoglobine

17.5 kDa

McKay et al. JACS 2006

2006

Ribosomes

2.3 MDa

2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 24000m/z0

100

%

x20x1010474.19

10236.41

10008.84

4422.33

4173.91

3949.99

9791.424696.49

14088.06

13870.68

10723.23 13659.90

13455.13

10982.64 13255.21

14313.58

16702.72

16496.93

16298.87

16101.28

15914.78

16912.72

17126.64

17349.11

19404.00

18990.11

18796.87

19615.76

19830.56

20053.1921493.64

2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 24000m/z0

100

%

x20x1010474.19

10236.41

10008.84

4422.33

4173.91

3949.99

9791.424696.49

14088.06

13870.68

10723.23 13659.90

13455.13

10982.64 13255.21

14313.58

16702.72

16496.93

16298.87

16101.28

15914.78

16912.72

17126.64

17349.11

19404.00

18990.11

18796.87

19615.76

19830.56

20053.1921493.64

Sanglier et al. JASMS 2002

2013

Bacteriophage HK97

capsid

18 MDa

Increase in size,

nb of partners

and heterogeneity

Binding

Conformation

1996

nanoESI

Snijder et al. Angew. Chem 2013

Synapt

G1

Synapt

G2

Native MS analysis

Native MS gives information on assemblies maintained

by noncovalent interactions

ESI-MS

instrument

3000 3500 m/z

Gas phase Solution

Sample preparation step

= desalting step Instrumental setting

optimizations

- Buffer exhange

- Use of volatile aqueous

buffer at neutral pH

- Ammonium buffer

(0-1M) with controled pH

- Control of the energy

communicated to the

ions in the 1st pumping

stage region of the

instrument

(Vc, Pi adjustment)

Data interpretation

- Binding stoichiometries

- Binding specificity

- Solution affinities

- Dynamics of

assembly/disassembly

Be carefull !

Native Mass Spectrometry: a recursive question

6

We dream to get from Native MS and IM-MS experiments :

• Accurate mass measurements, stoichiometries,…

• Information on interactions forces

• Subunit spatial arrangement, structural information, shape, conformation changes,…

… but all this information concerns molecules in water solution

Toutankhamon

1- Electrospray mecamism

2- Evaporation in interface

Dried, in vacuo

Specific

non-covalent interactions

salts

In water solution

?

Native MS workflow for mAb analysis

For mAbs minimal and easy sample preparation are well descibed

Purified

mAb, bsAb or ADC

Deglycosylation Step

(PNGase F, IgZero, etc.)

Data Interpretation

144190

Mass

143700 144000 144300 144600

Mass

146700 147200 147700 148200

147239

147077 147401

147563

PNGase-FG0F/G0F

G1F/G0F

G1F/G1F

G2F/G0F

G2F/G1F

144190

Mass

143700 144000 144300 144600

Mass

146700 147200 147700 148200

147239

147077 147401

147563

PNGase-FG0F/G0F

G1F/G0F

G1F/G1F

G2F/G0F

G2F/G1F

If necessary

Buffer Exchange (spin columns, microconcentrators,

SEC etc.)

Native MS and IM-MS (with ESI, needles or Chip-based introduction)

Synapt G2 HDMS (Waters)

+ nanoMate Triversa (Advion)

How do we jumped into the mAb field?

8

- mAb native MS

- mAb/Ag binding stoichiometries

- Native IM-MS

2008-2012: Reference Monoclonal Antibodies

(mAb)

- Native MS for monitoring of bsAb formation

- IM-MS for bsAb

- Real-time IM-MS to monitor bsAb formation

2013: towards bispecific mAbs

(bsAbs)

- Native MS mandatory for ADCs’ analysis

- Native MS for drug load profiles, average

DAR

- IM-MS for ADC heterogeneity assessment

- IM-MS for drug load profiles, average DAR

2014: immunoconjugate analysis

(ADC)

1. Native MS and IM-MS

for reference mAb analysis

9

2. Native MS and IM-MS

for bsAb formation

3. Native MS and IM-MS

for ADC formation

Intact mAb analysis : benefits of native MS for intact mAb analysis

trastu 5uM ACNH4 150mM pH7.5 Viva6 120v 6mbar

m/z1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 10000

%

0

100

6000 5000 4000 3000 2000 m/z 9000 7000 8000

25+

dimer

3%

Trastu IgGZero denat 2uM

m/z600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800

%

0

100

3000 2500 2000 1500 1000 m/z 4500 3500 4000

45+

Denaturing Conditions mAb 2µM in H2O:ACN:FA (50:50:1)

Native Conditions mAb 5µM in AcNH4 150mM pH7.5

Full MS spectrum

Deconvoluted

mass spectrum

Deconvoluted

mass spectrum

Full MS spectrum

Beck et al. J Mass Spectrom. 2015 Feb;50(2):285-97.

In Native conditions

sensitivity and mass accuracy

are as good as

in Denaturing conditions.

But spectra are more simple,

and you see interactions !!!

Non covalent dimer

not detected

Native MS analysis of immune mAb/Antigen complexes

Direct determination of mAb/Ag binding stoichiometries

m/z5000 5200 5400 5600 5800 6000 6200 6400 6600 6800 7000 7200 7400 7600 7800 8000 8200 8400 8600 8800 9000 9200 9400 9600 9800

%

0

100

mass130000 140000 150000 160000 170000 180000 190000 200000 210000 220000 230000 240000 250000 260000

%

0

100

147099 5 Da

23+

6000 7000 8000 9000 m/z 0

%

100

kDa 150 200 250

0

%

100

mAb (5 µM)

m/z5000 5200 5400 5600 5800 6000 6200 6400 6600 6800 7000 7200 7400 7600 7800 8000 8200 8400 8600 8800 9000 9200 9400 9600 9800

%

0

100

mass130000 140000 150000 160000 170000 180000 190000 200000 210000 220000 230000 240000 250000 260000

%

0

100

220878 15 Da

245485 15 Da

+3 x 24.6 kDa

+4 x 24.6 kDa 30+

6000 7000 8000 9000 m/z 0

%

100

kDa 150 200 250

0

%

100

mAb (5 µM)

+

JAM-A (40 µM)

m/z5000 5200 5400 5600 5800 6000 6200 6400 6600 6800 7000 7200 7400 7600 7800 8000 8200 8400 8600 8800 9000 9200 9400 9600 9800

%

0

100

196368 8 Da

171783 15 Da

mass130000 140000 150000 160000 170000 180000 190000 200000 210000 220000 230000 240000 250000 260000

%

0

100

+2 x 24.7 kDa 27+

6000 7000 8000 9000 m/z 0

%

100

kDa 150 200 250

0

%

100

+1 x 24.7 kDa

mAb (5 µM)

+

JAM-A (10 µM)

Atmanene C. et al. Anal Chem. 2009, 81(15):6364-73.

Unexpected 1:4

mAb:Ag stoichiometry

Native Ion Mobility MS (IM-MS) for conformational studies

By IM-MS : simultaneous measurement of drift times and m/z ratios

ESI

source

IM

cell

MS

analyzer Detector

Instrumental setting

optimizations

- Wave height and velocity

- Pressure in the IM cell

Data interpretation

- Conformation of

biomolecules

- Conformational changes

Trap

T-Wave

Transfer

T-waveQuadrupoleIon

Guide

Pusher Detector

Primary

pumpingTurbomolecular pump

INTERFACE

TOF ANALYZER

Reflectron

Source

NanoESI

Ar He ArN2

IMS

Cell

Vanne

ArAr

Drift time

m/z

12

Native Ion Mobility MS (IM-MS) for conformational studies

- Ions separation takes place according to ion mobility

Ion Mobility cell

2+ 2+ 1+

Drift time +

1+ Ion separation according to

ion mobility

Size, shape Charge

Compactness

Drift time

z

Drift time

2+ 1+ 2+ 1+

Drift time

N2

z

x

y

z

x

y

z

x

y

z

x

y

x

y

zx

y

z

L

EtDz

MMTkP

T

N

e

iongasb

112760

15.27316

3

Drift times can be related to collisional cross sections (CCS)

Information on ion gas phase conformation (2D representation of 3D structure) 13

Ion Mobility Mass Spectrometry (IM-MS)

For mAb conformational characterization

SM classique IM-MS

SMSupra IM-MS

Denaturing MS

Native MS Native IM-MS

Denaturing IM-MS

Beck A, Sanglier-Cianférani S, Van Dorsselaer A. Anal Chem. 2012, 84(11):4637-46

IM-MS to probe mAb conformational heterogeneity

2010: the first

separation of

2 mAb isoforms

by IM-MS

reported

Lc214 S-S Hc127

Hc232 S-S Hc232

Hc233 S-S Hc233

Lc214 S-S Hc232

Hc127 S-S Hc233

1. Native MS and IM-MS

for reference mAb analysis

16

2. Native MS and IM-MS

for bsAb formation

3. Native MS and IM-MS

for ADC formation

17

Native MS analysis for analysis for bsAb formed by Fab Arm Exchange

IgG4

(natalizumab)

IgG4

(6F4-2)

+ GSH

Bispecific mAb (bsAb)

(natalizumab/6F4-2)

+

2 mAb features are important for the exchange

reaction of IgG4:

- CH3 domain

- Ser at position 228

In vivo, IgG4s can exchange half molecules by a

dynamic process called Fab-arm exchange (FAE).

In vitro, the FAE process can be mimicked by a

reaction with glutathione (GSH)

Serine 228 in IgG4 introduces flexibility in the core hinge region. Besides the usual disulfide

bonds connecting two heavy chains (covalent between HC), intrachain disulfide bonds may

form instead (only non-covalent bonds between HC).

covalent bonds

between HC

The 2 arms are

only maintained

by non-covalent bonds

between HCs Variable

rate

Native MS and IM-MS for bispecific mAb characterization

Debeane et al. Anal. Chem. 2013, Anal Chem. 2013, 85, 9785-92

IgG4 Fab Arm Exchange monitoring

Sample : Natalizumab and Hz6F4-2 IgG4

bsAb

6F4-2

natalizumab

Native MS

+ GSH

Time resolved

IM-MS

O14611FDE.raw : 1

bsAb

bsAb

O14605FDE.raw : 1

23+

24+

+ GSH

+

Fab arm Exchange

+ GSH

Native MS and IM-MS to monitor:

- Dynamics of Fab Arm Exchange

- Bispecific antibody formation

Debaene F. et al. Anal Chem. 2013 85(20):9785-92.

1. Native MS and IM-MS

for reference mAb analysis

19

2. Native MS and IM-MS

for bsAb formation

3. Native MS and IM-MS

for ADC formation

ADCs are next generation empowered therapeutic mAbs

ADCs are tripartite molecules, made by the chemical conjugation of cytotoxins to mAbs

Cytotoxic drug Linker

*Beck A. and Reichert JM., mAbs 2014

cell-killing

capabilities

+

targeting delivery

of the conjugated

drug to tumor cells

Highly

cytotoxic drug

2 already approved ADCs’ on the market:

- brentuximab vedotin (Adcetris®, Seattle Genetics)

- ado-trastuzumab emtansine (Kadcyla®, Genentech)

- > 30 in clinical trials*

3 main types of conjugation:

- at cysteine -SH groups after reduction of the interchain disulfide bonds (brentuximab vedotin)

- at lysine side chains amine (ado-trastuzumab emtansine)

- at engineered cysteine residues at specific sites without reduction (thiomabs)

20

Drug load profile

Unconjugated D0

Average

DAR

Higher order

structures Ion mobility

MS?

Native

MS ?

- Cristallography

- HDX-MS

- SEC-MS

- Bottom up

(nano)LC-MS/MS

- Hydrophobic Interaction (HIC)

- Ion Exchange (IEC)

- Size exdclusion chromatography

- Reversed phase (rpHPLC)

- CE-SDS

Minutes3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

D0

D2 D4

D6

D8

- Middle- and Top-down MS

(MS/MS on intact ADCs )

The analytical toolbox for ADC analysis

- Middle-up

LC-MS

8

0

8

0

n

n

DAR

DAR

A

nA

DAR = 4.0

Site of

conjugation

21

Determining the

Drug Antibody Ratio (DAR)

with MS ?

ADCs’ analytical characterization is more challenging

than unconjugated mAbs

Cysteine-conjugation generates heterogeneous population of drug-loads

22

S-S

Reduction

step

Drug

Conjugation

step

Covalent

species

Non-covalent

species

Need for translational complementary analytical techniques

Compared to “unconjugated” mAbs, ADCs have increased level of complexity:

- a mixtures of species with different nb of drug molecules (0, 2, 4, 6, 8) at different positions

- a mixture of covalent and non-covalently associated populations

At least

one inter-chain

S-S bond missing

Cysteines

Our reference Cys-linked ADC: brentuximab vedotin

mAb

Brentuximab

anti-CD30

(chIgG1)

-Cys-SH (Hinge)

Linker

Peptide protease-clivable linker

-Cit-Val-

Drug

antimicrotubule drug

monomethylauristatin E

indicated for treatment of hematological malignancies (Hodgkin lymphoma, systemic

anaplastic large cell lymphoma)

Cysteines

HIC : the gold standard for Cys-linked ADC

Minutes 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

D4 D2

D6

D0

8

0

8

0

n

n

DAR

DAR

A

nA

DAR = 4.0

3 main ADC quality attributes (QA) can be determined by Hydrophobic Interactions (HIC)

- average DAR

- drug load distribution

- Relative amount of unconjugated mAb (D0)

D8

Cysteines

05-Dec-2013Brentuximab IgZero Viva5 replicat 4 5uM AcNH4 150mM pH7.4 80v 6mbar

m/z500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500

%

0

100

m/z500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500

%

0

100

6000 7000 m/z1000 3000 500040002000

37+

15+

37+

Full scan mass spectrum

Native MS: mandatory for cysteine-linked ADC analysis

- Very minor ion signals correspond to intact ADC

- Non-covalent interactions are disrupted

in denatured conditions

25

100 80 40 60 20 120 140 Mass (kDa)

145,910 Da

123,500 Da

101,072 Da

75,586 Da

25,042 Da

53,177 Da

MaxEnt deconvoluted mass spectrum

Denatured conditions

IgGZero treated ADC @ 2 µM in H2O:ACN:FA (50:50:1)

Cysteines

Full scan mass spectrum

26

Native conditions

IgGZero treated ADC @ 5 µM in 150 mM AcONH4 pH 7.5

05-Dec-2013Brentuximab IgZero Viva5 replicat 4 5uM AcNH4 150mM pH7.4 80v 6mbar

m/z500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500

%

0

100

m/z500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500

%

0

100

6000 7000 m/z 1000 3000 5000 4000 2000

Contamination (PEG)

free LC + 1

payload

Clarification/simplification of native mass spectra due

to fewer charges

(increased sensitivity)

Native MS: mandatory for cysteine-linked ADC analysis Cysteines

Intact ADC signals:

D0, D2, D4, D6, D8 ?

MaxEnt deconvoluted mass spectrum

27

Native conditions

IgGZero treated ADC @ 5 µM in 150 mM AcONH4 pH 7.5

mass144000 145000 146000 147000 148000 149000 150000 151000 152000 153000 154000 155000 156000 157000 158000 159000

%

0

100

O19456FDE 24 (1.627) M1 [Ev-62181,It48] (Gs,10.000,5175:6905,10.00,L10,R10); Sm (Mn, 2x2.00); Sb (25,5.00 ); Cm (2:29) TOF MS ES+ 5.05e5

146 Mass (kDa)

150 148 154 152 158 156

D4

D2

D6

D8 D0

MWexp

(Da) mass

(Da)

Error

(ppm)

D0 145903 7.3 50.2

D2 148539 8.1 54.2

D4 151172 5.8 38.3

D6 153814 12.5 81.4

D8 156446 9.2 59.1

- ADC is almost detected as an intact molecule

- Mass measurement affords drug binding stoichiometries to be assessed

- Native MS maintains non-covalent interactions in the gas phase

Native MS: mandatory for cysteine-linked ADC analysis Cysteines

Native MS

profile

HIC

profile

Native MS analysis to assess average DAR

- Native MS data are in good correlation with HIC data for all QA

- Native MS affords in one single run

1) drug load profile determination and 2) average DAR calculation 28

= 4.0 DAR

Comparison between HIC and Native MS for brentuximab vedotin

= 3.9 DAR

Cysteines

Off-line HIC native MS coupling

Native MS allows to confirm mass homogeneity of each HIC fraction :

no mixtures of different drug binding stoichiometries are observed within one HIC fraction

Min

ute

s

3.0

0

4.0

0

5.0

0

6.0

0

7.0

0

8.0

0

9.0

0

10

.00

1

1.0

0

D0

1

3

D4

D2

2

D6

4

D8

5

2014-09-19 Bretux IgZero HIC Fraction1 3uM AcNH4 150mM pH7.4 6mbar 180v

mass140000 141000 142000 143000 144000 145000 146000 147000 148000 149000 150000 151000 152000 153000 154000 155000 156000 157000 158000 159000

%

0

100

O23505FDE 16 (1.090) M1 [Ev0,It27] (Gs,10.000,5261:7072,7.00,L30,R30); Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (1:29) TOF MS ES+ 4.22e5

A: 145939.97±0.85

145904 ± 2 Da

D0

2014-09-23 Bretux IgZero HIC Fraction5 3uM AcNH4 150mM pH7.4 6mbar 80v

mass140000 141000 142000 143000 144000 145000 146000 147000 148000 149000 150000 151000 152000 153000 154000 155000 156000 157000 158000 159000

%

0

100

O23515FDE 11 (0.755) M1 [Ev-68567,It25] (Gs,10.000,5168:7016,7.00,L30,R30); Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (1:29) TOF MS ES+ 8.24e4

151173 ± 1 Da

D4

2014-09-21 Bretux IgZero HIC Fraction3 0.5uM AcNH4 150mM pH7.4 6mbar 180v

mass140000 141000 142000 143000 144000 145000 146000 147000 148000 149000 150000 151000 152000 153000 154000 155000 156000 157000 158000 159000

%

0

100

O23509FDE 24 (1.627) M1 [Ev-81894,It25] (Gs,10.000,5383:7158,7.00,L30,R30); Sm (Mn, 2x5.00); Cm (1:29) TOF MS ES+ 3.27e5

A: 148558.11±0.99

148539 ± 1 Da

D2

2014-09-25 Bretux IgZero HIC Fraction7 3uM AcNH4 150mM pH7.4 6mbar 180v

mass140000 141000 142000 143000 144000 145000 146000 147000 148000 149000 150000 151000 152000 153000 154000 155000 156000 157000 158000 159000

%

0

100

O23521FDE 6 (0.419) M1 [Ev0,It76] (Gs,10.000,5168:7016,7.00,L30,R30); Sm (Mn, 2x5.00); Cm (1:29) TOF MS ES+ 2.50e5

A: 156515.52±3.37

156457 ± 3 Da D8

150 155 145 Mass (kDa)

1

3

2

5

2014-09-24 Bretux IgZero HIC Fraction6 3uM AcNH4 150mM pH7.4 6mbar 180v

mass140000 141000 142000 143000 144000 145000 146000 147000 148000 149000 150000 151000 152000 153000 154000 155000 156000 157000 158000 159000

%

0

100

O23518FDE 17 (1.157) M1 [Ev-97372,It21] (Gs,10.000,5168:7016,7.00,L30,R30); Sb (2,40.00 ); Cm (1:29) TOF MS ES+ 2.61e5

153815 ± 2 Da

D6 4

29

Cysteines

Advantages of native MS compared to HIC

2 3 4 5 6 7

8 9 10 11 min.

D0

D2?

D4? D1?

Hz1613F12 SG3249DAR2 IgZero 5uM AcNH4 150mM pH7.5 NAP2 6mbar 120v

m/z145500 146000 146500 147000 147500 148000 148500 149000 149500 150000 150500 151000 151500 152000 152500 153000 153500

%

0

100146 152 mass (kDa) 145 150 148 147 151 149 153

D0

D1

D3

D2

D4

= 2.0 DAR

Native MS profile

HIC profile

- Broad HIC peaks

- Difficulty to unambiguously assess

drug binding

- No odd drug load expected

= 1.8 DAR - Native MS mass accuracues allows

unambiguous drug binding

stoichiometry assessment

- odd drug load are clearly detected

- No peak broadening as on HIC

Native MS allowed to precise unclear HIC results 30

D1’?

Cysteines

Native IM-MS of intact cysteine-linked ADC

Deglycosylated Parent mAb O25596FDE.raw : 1

24+

12 16 20 22 tD (ms)

7500

7000

6000

5500

m/z

181410

6500

5000

23+

21+

IgGZero ADC

O23564FDE.raw : 1

12 16 20 22 tD (ms)

7500

7000

6000

5500

m/z

181410

6500

5000

Deglycosylated ADC

SynaptG2, 3µM, AcNH4 150mM pH7,4 Vc=80v, Pi=6mbar, Bias25, WH=40V, WV=923m/s, He=130mL/min, N2= 45mL/min

31

Cysteines

Native IM-MS of intact cysteine-linked ADC

Deglycosylated Parent mAb O25596FDE.raw : 1

24+

12 16 20 22 tD (ms)

7500

7000

6000

5500

m/z

181410

6500

5000

23+

21+

IgGZero ADC

O23564FDE.raw : 1

12 16 20 22 tD (ms)

7500

7000

6000

5500

m/z

181410

6500

5000

Deglycosylated ADC

32

SynaptG2, 3µM, AcNH4 150mM pH7,4 Vc=80v, Pi=6mbar, Bias25, WH=40V, WV=923m/s, He=130mL/min, N2= 45mL/min

IM-MS enlighten and characterize the heterogeneity of ADCs

Cysteines

Native IM-MS of intact cysteine-linked ADC

ADC heterogeneity in drug binding is observed on native IM-MS plots

Each individual even-drug load can be separated in IM-MS

No positional isomers were detected (to low IM cell resolution on intact ADC)

Deglycosylated Parent mAb Deglycosylated ADC O25596FDE.raw : 1

16 17 18 19 tD (ms)

6700

6600

6500

6400

m/z

23+

22+

O23564FDE.raw : 1

16 17 18 19 tD (ms)

6700

6600

6500

6400

m/z

DO 23+

D0 22+

D2 23+

D6 24+

D4 23+

D8 24+

33

SynaptG2, 3µM, AcNH4 150mM pH7,4 Vc=80v, Pi=6mbar, Bias25, WH=40V, WV=923m/s, He=130mL/min, N2= 45mL/min

Cysteines

Native IM-MS of intact cysteine-linked ADC

resolving power of IM cell

tD/tD at FWHM : 16.4 ± 0.8

14.2

D014.2

D214.9

D415.6

D616.3

D817.0

13 14 15 16 17 18 19 tD (ms)

34

Each individual even-drug load can

be separated in IM-MS

Parent

mAb (24+

charge state)

ADC (24+ charge state)

(brentuximab vedotin)

- D0 D2 D4 D6 D8

tD (ms) 14.2 ± 0.0 14.2 ± 0.1 14.9 ± 0.1 15.6 ± 0.1 16.3 ± 0.1 17.0 ± 0.1

(tD)

(ms) - - + 0.7 + 0.7 + 0.7 +0.7

CCS from IM-MS (nm²) 68.0 ± 0.0 68.1 ± 0.1 68.8 ± 0.1 69.5 ± 0.1 70.3 ± 0.1 71.1 ± 0.1

CCS

(nm²) - - + 0.7 + 0.7 + 0.8 +0.8

Predicted*

CCS (nm2) 68.2 68.2 69.0 69.8 70.6 71.4

Predicted CCS

(nm²) - - + 0.8 + 0.8 + 0.8 +0.8

Drug binding induces constant and reproducible tD and CCS differences

Cysteines

Semi-quantitative analysis of native IM-MS

For each charge state, intensities of the extracted drift peaks were plotted across a series

for each drug binding stoichiometry (D0, D2, D4, D6 and D8) and a Gaussian curve fitted.

To give a relative intensity of each conformer, the area underneath each curve was

calculated as a percentage of the total observed signal, here (D0+D2+D4+D6+D8).

= 3.7 ± 0.1 DAR

(triplicates)

35

Norm

aliz

ed

Inte

nsity

(%)

Charge States22 24 26 28

0

20

40

60

80

100 D0: 14%D2: 27%D4: 30%

D8: 10%D6: 19%

Cysteines

Minutes 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

D0

D2 D4

D6

D8

Average DAR determination

from semi quantitative native MS and IM-MS

MaxEnt1 deconvolution

Quantitation based on ion

intensities

n=8

D0

D2 D4

D6

D8

DAR

= 3.9 ± 0.1

DAR

= 3.7 ± 0.1 DAR

= 4.0

Number of

drug load

Calculation based on area

measured under fited gaussian

of charge states extracted ions

n =3

Native MS

HIC

Average DAR determination and drug-load profiles obtained

from native MS or native IM-MS are in good agreement with HIC data

mass144000 145000 146000 147000 148000 149000 150000 151000 152000 153000 154000 155000 156000 157000 158000 159000

%

0

100

O19456FDE 24 (1.627) M1 [Ev-62181,It48] (Gs,10.000,5175:6905,10.00,L10,R10); Sm (Mn, 2x2.00); Sb (25,5.00 ); Cm (2:29) TOF MS ES+ 5.05e5

146 Mass (kDa) 150 148 154 152 158 156

D4

D2

D6

D8 D0

Native IM-MS

36 Debaene F. et al. Anal Chem. 2014, 86(21):10674-83.

Cysteines

Benefits of native MS of Lysyne-conjugates

mAbs have more than 60 surface exposed Lys residues (compared to 32 Cys) available for reactions but

only 32 cysteines, among which 8 only are involved in the interchain disulfide bridges of chimeric, humanized

and human IgG1.

Lysine conjugation generates even more heterogeneous samples even if all species are covalent

Lysines

Benefits of native MS for lysine conjugate analysis

trastuzumab emtansine + IG0 native dil5X 5uM

m/z1000 2000 3000 4000 5000 6000 7000

%

0

100

m/z1000 2000 3000 4000 5000 6000 7000

%

0

100

6000 7000 m/z 1000 3000 5000 4000 2000

trastuzumab emtansine + IG0 native dil5X 5uM

m/z1000 2000 3000 4000 5000 6000 7000

%

0

100

m/z1000 2000 3000 4000 5000 6000 7000

%

0

100

6000 7000 m/z 1000 3000 5000 4000 2000

Native Conditions IgGZero treated ADC @ 5 µM in 150 mM AcONH4 pH7.5

Denatured conditions IgGZero treated ADC @ 2 µM in H2O:ACN:FA (50:50:1)

25+

16+

45+

Full MS spectrum Deconvoluted

mass spectrum

Full MS spectrum Deconvoluted

mass spectrum

Very complex mass spectrum :

overlapping charge state distribution

hamper appropriate Dn quantification

Deconvolution fails in detecting D8 species

Decomplexifictaion of the mass

spectrum

Deconvolution fails in detecting D8

species

* + 220 Da linker adducts

Lysines

Benefits of charge state reduction for lysine ADC analysis

trastuzumab emtansine + IG0 native dil10X 2.8uM + 10 mM Imid

m/z5000 6000 7000 8000 9000 10000 11000

%

0

100

m/z5000 6000 7000 8000 9000 10000 11000

%

0

100

trastuzumab emtansine + IG0 native dil10X 2.8uM + 10 mM Imid

m/z5000 6000 7000 8000 9000 10000 11000

%

0

100

m/z5000 6000 7000 8000 9000 10000 11000

%

0

100

25+

23+

16+

T-DM1 (2µM)

+ 10 mM imidazole

T-DM1 (2µM)

Detection of

D8 species

MaxEnt1 deconvolution

n=8

Deconvoluted

mass spectrum

= 3.0 DAR

= 3.5 DAR

* + 220 Da linker adducts

Lysines

Native MS and IM-MS for Lysine-linked ADCs

Marcoux et al. Protein Science. 2015, Protein Sci. 2015 Feb 18. doi: 10.1002/pro.2666

Sample : trastuzumab emtansine (Kadcyla)

Average DAR and drug load profile

from IM-MS

Average DAR and drug load profile

from native MS

DAR = 3.5 ± 0.1

Number of drug loads

Lysines

Special feature:

Head comparison of trastuzumab and

cetuximab with corresponding biosimilar and

biobetter candidates

Beck A, Debaene F, Diemer H, Wagner-Rousset E, Colas O,

Van Dorsselaer A and Cianférani A, JMS; 2015 in press

What’s coming next ?

41

originator

Trastuzumab

Trastuzumab-B

originator biosimilar

Take home message

42 From May C. et al., Biochemical Pharmacology 2012.

Native MS

HDX-MS Ion Mobility -MS

Native MS is a rapid, robust and reliable method for first-

line semi-quantitative mAb characterization

Native IM-MS for semi-quantitative

global conformational

characterization of mAbs

HDX for structural mAb characterizartion

(epitope mapping, comparability studies, etc.)

Many Thanks for your attention !

The Antibody Physico-Chemistry group

Elsa WAGNER-ROUSSET

Olivier COLAS

Nathalie CORVAÏA

Amandine BŒUF (past)

Daniel AYOUB (past)

Alain BECK

The native MS group of LSMBO

Julien MARCOUX

Guillaume TERRAL

Johann STOJKO

François DEBAENE (past)

Cédric ATMANENE (past)

Alain VAN DORSSELAER

Sarah CIANFERANI

43