native and im-ms characterization of adcs

Native and IM-MS

characterization of ADCs

Sarah CIANFERANI

BioOrganic Mass Spectrometry Laboratory (LSMBO),

Pluridisciplinary Hubert Curien Institute (IPHC)

Analytical Sciences Department

University of Strasbourg, CNRS UMR7178, 67087 Strasbourg - France

[email protected] 1

http://iphc.in2p3.fr/index.php

http://www.unistra.fr/

http://www.cnrs.fr/accueil.html

mAbs as therapeutics

mAbs : the most important class of human therapeutics - more than 40 mAbs & derivatives currently approved (EMA/FDA)

- used for multiple clinical indications (inflammation, autoimmunity, cancer, transplantation, etc.)

- more than 400 mAbs & derivatives in pre-clinical development and clinical trial

mAbs as anti-cancer drugs in clinical studies (Jan 2012)*

- 84 unmodified IgG (51%)

- 25 ADC (Antibody Drug Conjugates) (15%) + 7 (dec 2012)

- 10 bispecific (6%)

- 17 engineered (10%)

- 16 fragments (10%)

*Reichert JM. and Dhimolea A., Drug Discovery Today 2012; Pauwels P. et al., mAbs 2012

mAbs are complex therapeutic proteins

(150 kDa glycoproteins)

Next generation empowered mAbs : bispecific mAbs (bsAbs), antibody-drug-conjugates

(ADCs)

IgG4

(natalizumab)

IgG4

(6F4-2)

+ GSH

Bispecific mAb (bsAb)

(natalizumab/6F4-2)

+bsAb

(2 different half mAbs)

ADC

(mAb + cytotoxic)

Native IM-MS

Intact mAb analysis

(150 kDa)

Middle up mAb

analysis

(25-100 kDa)

Bottom up mAb

analysis

(peptide mapping)

(700-7000 Da)

Top-down MS

(FT-ICR + Orbital trap)

LC-MS

(nano)LC-MS/MS

enzymatic digestion

Higher Order Structure

Aggregates

mAb/Ag complexes

(>150 kDa)

Native MS

Complexity Size

MS toolbox for mAb characterization

HDX-MS Crosslinking MS

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,

Number of partners

And Heterogeneity

Binding

Conformation

1996

nanoESI

Snijder et al. Angew. Chem 2013

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

5

Native MS workflow for mAb analysis

Minimal and easy sample preparation

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?

7

- 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

8


for bsAb formation


for ADC formation

Native MS analysis : benefits for intact mAb analysis

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

Trastuzumab Nap2 denat 2uM meth denatAb 120v ADC TfCE5

3325 3330 3335 3340 3345 3350 3355 3360 3365 3370 3375 3380 3385 3390 3395 3400 3405 3410 3415 3420 3425 3430 3435 3440 3445 3450 3455 3460 3465 3470 3475 3480 3485 3490 3495 3500m/z0

100

%

A: 147917.13±1.11B: 148061.73±0.82C: 148222.42±0.86D: 148383.83±0.80E: 148544.33±0.95

44+

1

2 3

4

5

44+ 1: 147917.1 ± 1.1 Da: G0/G0F

2: 148061.7 ± 0.8 Da: (G0F)2 – error 30 ppm

3: 148222.4 ± 0.9 Da: G0F/G1F

4: 148383.8 ± 0.8 Da: (G2F)2

5: 148544.3 ± 1.0 Da: G1F/G2F

Trastuzumab Nap2 denat 2uM meth denatAb 120v ADC TfCE5

1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200 5400 5600 5800m/z0

100

%

A: 147917.13±1.11B: 148061.73±0.82C: 148222.42±0.86D: 148383.83±0.80E: 148544.33±0.95

2000 3000 5000 4000 6000 7000 m/z

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

%

0

100

O11359FDE 6 (0.419) Sm (Mn, 2x10.00); Cm (1:53) 1: TOF MS ES+ 179

6000 7000 m/z 2000 3000 5000 4000

26+

Trastruzumab 5uMACNH4 150mM pH7.2 NAP2 viva2 FDEHighMass TDC 150v 6mbar

5860 5870 5880 5890 5900 5910 5920 5930 5940 5950 5960 5970 5980 5990 6000 6010 6020 6030 6040 6050 6060 6070 6080 6090 6100 6110 6120 6130 6140 6150 6160 6170 6180 6190 6200 6210 6220 6230 6240 6250 6260m/z0

100

%

A: 148070.30±1.89B: 148230.48±3.31C: 148389.72±2.18D: 148549.48±2.35

2 3

4

5

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

26+

Data analysis is straightforward :

- accurate mass measurement

- Purity assessment

- Homogeneity determination

- Clarification/simplification of native

mass spectra du to fewer charges

(increased sensitivity)

2: 148071.7 ± 0.5 Da: (G0F)2 – error 100 ppm

Native MS analysis for aggregation studies

Detection of oligomeric forms 21-Jan-2014

m/z3500 3750 4000 4250 4500 4750 5000 5250 5500 5750 6000 6250 6500 6750 7000 7250 7500 7750 8000 8250 8500 8750

%

0

100

m/z3500 3750 4000 4250 4500 4750 5000 5250 5500 5750 6000 6250 6500 6750 7000 7250 7500 7750 8000 8250 8500 8750

%

0

100

O23060FDE 8 (0.553) Sm (Mn, 2x20.00); Cm (2:29) TOF MS ES+ 2.16e3


21-Jan-2014

m/z3500 3750 4000 4250 4500 4750 5000 5250 5500 5750 6000 6250 6500 6750 7000 7250 7500 7750 8000 8250 8500 8750

%

0

100

m/z3500 3750 4000 4250 4500 4750 5000 5250 5500 5750 6000 6250 6500 6750 7000 7250 7500 7750 8000 8250 8500 8750

%

0

100



+25

+25

6000 5000 8000 7000 4000 m/z

6000 5000 8000 7000 4000 m/z

+39

+39

mAb dimer: 7%

mAb dimer : 3%

Trastuzumab

Batch # 1

Trastuzumab

Batch # 2

Quantification based on peaks intensities; 22+ to 28+ for monomer; 36+ to 42+ for dimer ; MWdimer : 296526 ± 18Da

Native MS

for batch to batch comparisons

Native MS allows assessment of higher

order structure/oligomeric state

Native MS analysis of immune mAb/Ag 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)

Unexpected 1:4

mAb:Ag stoichiometry

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

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

16 Pritchard C et al. Anal Chem. 2013, 85(15):7205-12

IM-MS for batch-to-batch conformational comparison



17


for bsAb formation


for ADC formation

18

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

non-covalent bonds

between HC

Native MS analysis of bsAB formed by FAE

19

bispecif nat/mut3 5uM AcNH4 150mM pH7.5 6mbar180v

1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500m/z0

100

%

O14364FDE 26 (1.761) TOF MS ES+ 417

A: 148142.72±0.97B: 148300.17±2.77C: 148465.80±0.87D: 148618.45±1.60

bispecif nataluzumab/6F4-2 mutant3 2uM 50v denat

500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000 3250 3500 3750 4000 4250 4500 4750 5000 5250 5500 5750 6000 6250 6500 6750 7000 7250 7500 7750m/z0

100

%

O14353FDE 26 (1.761) TOF MS ES+ 3.21e5

A: 73728.33±0.19B: 73890.42±0.24C: 74407.87±0.35D: 74569.96±0.26E: 147947.25±4.86F: 148139.83±0.37G: 148301.66±0.36H: 148462.72±0.34I: 148621.50±0.58J: 148773.14±2.01K: 148928.86±2.08

3000 2000 5000 4000 1000 m/z

+45 bispecif nataluzumab/6F4-2 mutant3 2uM 50v denat

3260 3265 3270 3275 3280 3285 3290 3295 3300 3305 3310 3315 3320 3325 3330 3335 3340 3345m/z0

100

%

O14353FDE 26 (1.761) TOF MS ES+ 3.17e5

A: 73728.33±0.19B: 73890.42±0.24C: 74068.96±1.71D: 74407.87±0.35E: 74569.96±0.26F: 74731.84±0.32G: 147947.25±4.86H: 148139.83±0.37I: 148301.66±0.36J: 148462.72±0.34K: 148621.50±0.58L: 148773.14±2.01M: 148928.86±2.08

+45

G0F/G0F

G0F/G1F

G1F/G1F

G2F/G0F

G2F/G1F

7000 5000 8000 7000 4000 m/z 3000

bispecif nat/mut3 5uM AcNH4 150mM pH7.5 6mbar180v

6070 6080 6090 6100 6110 6120 6130 6140 6150 6160 6170 6180 6190 6200 6210 6220 6230 6240 6250 6260 6270 6280 6290 6300 6310 6320 6330 6340 6350m/z0

100

%

O14364FDE 26 (1.761) TOF MS ES+ 417

A: 148142.72±0.97B: 148300.17±2.77C: 148465.80±0.87D: 148618.45±1.60

G0F/G0F

G0F/G1F

G1F/G1F

G2F/G0F

+24

+24

Hz6F4-2v3: 73729 ± 1 Da

natalizumab: 74408 ± 1 Da

Hz6F4-2

148302 ± 1 Da

Intact Hz6F4-2v3

148300 ± 3 Da

∼17 % half-antibody

a high proportion of S-S bond between

half-mAbs recombine

an almost exclusive homogeneous ion

population is native MS

Native Conditions

Denatured conditions

Native MS to monitor FAE dynamics and bsAb formation

20

t = 0

t = 4 h

t = 24h

Bispe 24h PBS 180v 6mbar

5400 5500 5600 5700 5800 5900 6000 6100 6200 6300 6400 6500 6600 6700 6800 6900m/z0

100

%

0

100

%

0

100

%

0

100

%

Bispe 24h PBS 180v 6mbar

5400 5500 5600 5700 5800 5900 6000 6100 6200 6300 6400 6500 6600 6700 6800 6900m/z0

100

%

0

100

%

0

100

%

0

100

%

6200 6400 660060005800 6800 m/z

+23+24+25

Native MS can serve for online monitoring

of FAE and bsAb formation

2D Graph 4f = a*exp(-b*x)f = a*exp(-b*x)f = a*exp(-b*x)

f=a*(1-exp(-b*x))f = a*exp(-b*x)

Time (min.)

0 200 400 600 800 1000 1200 1400

Norm

aliz

ed Inte

nsi

ty (

%)

0

10

20

30

40

50

Col 1 vs Col 2 x column 2 vs y column 2 Col 4 vs Col 5 Col 4 vs Col 5: 1080.0000 x column 5 vs y column 5 Col 7 vs Col 8 Col 7 vs Col 8: 120.0000 x column 6 vs y column 6

natalizumab

bsAb

6F4-2

Apparent rate constant:

4.66 x 10-5 s-1*

* compared to 7-10 x 10-5 s-1 as reported in

Rispens, T. et al. J Am Chem Soc 2011, 133, 10302-11

Rose, R. J. et al. Structure 2011, 19, 1274-82.

Detection of a 3rd population after 4h

corresponding to bsAb

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

tD (ms)22 242018

21.3 0.2

20.8 0.2

20.4 0.2

+24

t = 8h

+ GSH

t = 8h

- GSH

control

t = 0

+ GSH

• All 3 mAbs can be detected simultaneously

in the IM cell

• Native IM-MS can serve for online monitoring

of FAE and bsAb formation

natalizumab

Bispecific mAb

6F4-2

O14561FDE.raw : 1

B.

O14611FDE.raw : 1

+23

+24

O14605FDE.raw : 1

Native IM-MS to monitor FAE dynamics and bsAb formation

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



22


for bsAb formation


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 sulfhydril 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)

23

ADCs’ analytical characterization is more challenging

than unconjugated mAbs

Cysteine-conjugation generates heterogeneous population of drug-loads

24

S-S

Reduction

step

Drug

Conjugation

step

Covalent

species

Non-covalent

species

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

- formation of heterogeneous mixtures of species with DARs

- heterogeneity coming from the presence of a mixture of covalent and non-covalently

associated populations due to the presence of drugs at the interchain cysteine residues

Need for translational complementary analytical techniques

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

25

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)

HIC chromatography : 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 HIC

- average DAR

- drug load distribution

- Relative amount of unconjugated mAb (D0)

D8

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 is mandatory for cysteine-linked ADC analysis

- Very minor ion signals correspond to intact ADC

- Non-covalent interactions are disrupted

in denatured conditions

28

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)

Full scan mass spectrum


29

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

Polymer

contamination

free LC + 1

payload

Intact ADC signals

Clarification/simplification of native mass spectra due

to fewer charges

(increased sensitivity)

MaxEnt deconvoluted mass spectrum


30

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 profile

HIC profile

Semi-quantitative 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 31

= 4.0 DAR

Comparison between HIC and Native MS for brentuximab vedotin

= 3.9 DAR

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


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

%

0

100


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


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


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

32

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

100 146 152 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 33

D1’?

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

34


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

35



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+

36



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)

37

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

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)

38

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%

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


146 Mass (kDa) 150 148 154 152 158 156

D4

D2

D6

D8 D0

Native IM-MS

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

Take home message

40 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.)

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

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

42

http://www.oseo.fr/