Presentation by Dr. Sarah Cianférani-Sanglier, University of Strasbourg, Strasbourg, France. Talk given at Waters Antibody Drug Conjugates (ADC) 2014 Meeting, Nov. 20-21, Wilmslow UK.

1. 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
sarah.cianferani@unistra.fr 1

2. 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)
4
umab)
IgG4
(6F4-2)
+ GSH
Bispecific mAb (bsAb)
(natalizumab/6F4-2)
+ bsAb
(2 different half mAbs)
ADC
(mAb + cytotoxic)

3. 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. 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 24000
m/z0
100
%
x20x1010474.19
10236.41
10008.84
4422.33
4173.91
3949.99
9791.42
4696.49
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
20053.19
21493.64
2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 24000
m/z0
100
%
x20x1010474.19
10236.41
10008.84
4422.33
4173.91
3949.99
9791.42
4696.49
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
20053.19
21493.64
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

5. 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

6. 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
148200
PNGase-F
144190
143700 144000 144300 144600
Mass
146700 147200 147700 148200
147239
147077 147401
147563
PNGase-F
G0F/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)

7. 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)

8. 1. Native MS and IM-MS
for reference mAb analysis
8
2. Native MS and IM-MS
for bsAb formation
3. Native MS and IM-MS
for ADC formation

9. 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 3500
m/z0
100
%
A: 147917.13±1.11
B: 148061.73±0.82
C: 148222.42±0.86
D: 148383.83±0.80
E: 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
M meth denatAb 120v ADC TfCE5
1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200 5400 5600 5800
m/z
A: 147917.13±1.11
B: 148061.73±0.82
C: 148222.42±0.86
D: 148383.83±0.80
E: 148544.33±0.95
2000 3000 50004000 6000 7000 m/z
m/z
2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500
9) Sm (Mn, 2x10.00); Cm (1:53) 1: TOF MS ES+
179
6000 7000 m/z2000 3000 50004000
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
0
100
%
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

10. Native MS analysis for aggregation studies
Detection of oligomeric forms21-Jan-2014
m/z
3500 3750 4000 4250 4500 4750 5000 5250 5500 5750 6000 6250 6500 6750 7000 7250 7500 7750 8000 8250 8500 8750
%
0
100
m/z
3500 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
O9766FDE 1 (0.084) Sm (Mn, 2x20.00); Cm (1:74) TOF MS ES+
2.65e3
21-Jan-2014
m/z
3500 3750 4000 4250 4500 4750 5000 5250 5500 5750 6000 6250 6500 6750 7000 7250 7500 7750 8000 8250 8500 8750
%
0
100
m/z
3500 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
O9766FDE 1 (0.084) Sm (Mn, 2x20.00); Cm (1:74) TOF MS ES+
2.65e3
+25
+25
60005000 800070004000 m/z
60005000 800070004000 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

11. Native MS analysis of immune mAb/Ag complexes
Direct determination of mAb/Ag binding stoichiometries
m/z
5000 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
mass
130000 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/z
5000 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
mass
130000 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/z
5000 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
mass
130000 140000 150000 160000 170000 180000 190000 200000 210000 220000 230000 240000 250000 260000
%
0
100
+2 x 24.7 kDa27+
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.

12. 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-waveQuadrupole
Ion
Guide
Pusher Detector
Primary
pumping
Turbomolecular pump
INTERFACE
TOF ANALYZER
Reflectron
Source
NanoESI
Ar He ArN2
IMS
Cell
Vanne
ArAr
Drift time
m/z
12

13. 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

14. 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

15. IM-MS to probe mAb conformational heterogeneity

16. 16Pritchard C et al. Anal Chem. 2013, 85(15):7205-12
IM-MS for batch-to-batch conformational comparison

17. 1. Native MS and IM-MS
for reference mAb analysis
17
2. Native MS and IM-MS
for bsAb formation
3. Native MS and IM-MS
for ADC formation

18. 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

19. Native MS analysis of bsAB formed by FAE
19
AcNH4 150mM pH7.5 6mbar180v
2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500
m/z
TOF MS ES+
417
A: 148142.72±0.9
B: 148300.17±2.7
C: 148465.80±0.8
D: 148618.45±1.6
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
0
100
%
O14353FDE 26 (1.761)
30002000 500040001000 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 3345
m/z0
100
%
O14353FDE 26 (1.761) TOF MS ES+
3.17e5
A: 73728.33±0.19
B: 73890.42±0.24
C: 74068.96±1.71
D: 74407.87±0.35
E: 74569.96±0.26
F: 74731.84±0.32
G: 147947.25±4.86
H: 148139.83±0.37
I: 148301.66±0.36
J: 148462.72±0.34
K: 148621.50±0.58
L: 148773.14±2.01
M: 148928.86±2.08
+45
G0F/G0F
G0F/G1F
G1F/G1F
G2F/G0F
G2F/G1F
70005000 800070004000 m/z3000
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 6
0
100
%
O14364FDE 26 (1.761) TO
A: 148
B: 148
C: 148
D: 148
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

20. Native MS to monitor FAE dynamics and bsAb formation
20
t = 0
t = 4 h
t = 24h
v 6mbar
0 5600 5700 5800 5900 6000 6100 6200 6300 6400 6500 6600 6700 6800 6900
m/z
v 6mbar
0 5600 5700 5800 5900 6000 6100 6200 6300 6400 6500 6600 6700 6800 6900
m/z
6200 6400 660060005800 6800 m/z
+23+24+25
Native MS can serve for online monitoring
of FAE and bsAb formation
2D Graph 4
f = 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
NormalizedIntensity(%)
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.

21. 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
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. 1. Native MS and IM-MS
for reference mAb analysis
22
2. Native MS and IM-MS
for bsAb formation
3. Native MS and IM-MS
for ADC formation

23. ADCs are next generation empowered therapeutic mAbs
 ADCs are tripartite molecules, made by the chemical conjugation of cytotoxins to mAbs
Cytotoxic drugLinker
*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

24. 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

25. 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

26. 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)

27. HIC chromatography : the gold standard for Cys-linked ADC
Minutes3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
D4D2
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

28. ec-2013Brentuximab IgZero Viva5 replicat 4 5uM AcNH4 150mM pH7.4 80v 6mbar
m/z
0 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500
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
1008040 6020 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)

29.  Full scan mass spectrum
Native MS is mandatory for cysteine-linked ADC analysis
29
Native conditions
IgGZero treated ADC @ 5 µM in 150 mM AcONH4 pH 7.5
ec-2013Brentuximab IgZero Viva5replicat 45uMAcNH4150mMpH7.480v6mbar
m/z
00 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 75006000 7000 m/z1000 3000 500040002000
Polymer
contamination
free LC + 1
payload
Intact ADC signals
Clarification/simplification of native mass spectra due
to fewer charges
(increased sensitivity)

30.  MaxEnt deconvoluted mass spectrum
Native MS is mandatory for cysteine-linked ADC analysis
30
Native conditions
IgGZero treated ADC @ 5 µM in 150 mM AcONH4 pH 7.5
mass
144000 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)
150148 154152 158156
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

31.  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
= 4.0DAR
Comparison between HIC and Native MS for brentuximab vedotin
= 3.9DAR

32. 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
Minutes3.004.005.006.007.008.009.0010.0011.00
D0
1
3
D4
D2
2
D6
4
D8
5
2014-09-19 Bretux IgZero HIC Fraction1 3uM AcNH4 150mM pH7.4 6mbar 180v
mass
140000 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
mass
140000 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
mass
140000 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
mass
140000 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 155145 Mass (kDa)
1
3
2
5
2014-09-24 Bretux IgZero HIC Fraction6 3uM AcNH4 150mM pH7.4 6mbar 180v
mass
140000 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

33. 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
%
100 146 152 mass (kDa)145 150148147 151149 153
D0
D1
D3
D2
D4
= 2.0DAR
 Native MS profile
 HIC profile
- Broad HIC peaks
- Difficulty to unambiguously assess
drug binding
- No odd drug load expected
= 1.8DAR
- 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’?

34. 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

35. 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
35
SynaptG2, 3µM,
AcNH4 150mM pH7,4
Vc=80v, Pi=6mbar, Bias25,
WH=40V, WV=923m/s,
He=130mL/min, N2= 45mL/min

36. 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+
36
SynaptG2, 3µM,
AcNH4 150mM pH7,4
Vc=80v, Pi=6mbar, Bias25,
WH=40V, WV=923m/s,
He=130mL/min, N2= 45mL/min

37. Native IM-MS of intact cysteine-linked ADC
resolving power of IM cell
tD/tD at FWHM : 16.4 ± 0.8
D0
14.2
D2
14.9
D4
15.6
D6
16.3
D8
17.0
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

38. 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 ± 0.1DAR
(triplicates)
38
NormalizedIntensity(%)
Charge States
22 24 26 28
0
20
40
60
80
100 D0: 14%
D2: 27%
D4: 30%
D8: 10%
D6: 19%

39. Minutes3.00 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 ± 0.1DAR
= 3.7 ± 0.1DAR
= 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
mass
144000 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)150148 154152 158156
D4
D2
D6
D8D0
Native IM-MS
39
Debaene F. et al. Anal Chem. 2014, 86(21):10674-83.

40. Take home message
40From 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.)

41. 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

42. 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