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1
Emerging Mass Spectrometry based methods
for therapeutic mAbs, biosimilars and ADCs
deep structural characterization.
Elsa Wagner-Rousset, PhD
Centre d’Immunologie Pierre Fabre, FRANCE
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 2
Outline
• ADCs : Present and Future
• Case studies from the ADC’s analytical toolbox
• LC-MS following IdeS digestion
• Native MS & Ion Mobility MS
• Biosimilarity assessment of mAbs
• CESI-MS/MS
• Summary
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 3
• 2 ADCs recently approved by FDA and EMA :
- brentuximab vedotin (Adcetris®, Seattle Genetics)
- ado-trastuzumab emtansine (Kadcyla®, Genentech):
• > 30 in clinical trials
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 4
Antibody Drug Conjugate structure
* Panowski et al., mAbs 6:1, 34-45, 2014.
A cytotoxic drug is conjugated to a monoclonal antibody by means of a select linker
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 5
Delivery of drugs by ADCs to cancer cells
* Panowski et al., mAbs 6:1, 34-45, 2014.
1. ADC selective binding to a cell-surface tumor antigen
2. Internalization of the ADC-antigen complex
3. Traffic to lysosomal compartments : degradation
4. Releasing of the active cytotoxic drug inside the cell > cell death
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 6
ADC’s analytical characterization
• Challenging when compared to unconjugated mAbs.
3 levels of complexity:
1. mAb heterogeneity
2. linker & payload
3. Conjugation chemistry
N-term. blocking(Gln/Glu =>
PyroGlu: -17/-18 Da)
C-term. clipping (- Lys: -128 Da)C-term. amidation (OH => NH2: -1 Da)
GlycosylationHeterogeneity
(Fab & Fc :+1445, 1607, 1769 Da…)
Disulfide shufflingFree thiol groups
Cysteinylation (+119 Da)Thioether (-32 Da)
Oxidation (Met-O, Trp-O: +16 Da)Deamidation (Asn => Asp: +1 Da)
Isomerization (Asp => IsoAsp: 0 Da)Succinimide (Asn/Asp => Suc: -17 Da)
Glycation (Lys-Glc: +162 Da)
Dimers, aggregatesIgG fragments (H2L, H2, HL, H, L)
Misfolded forms
CH3
CH2
CH1
VH
CL
VL
Hi
IgG1 = H2L2(148 000-149 000 Da)
* Beck et al., Anal Chem, 2013 , 15;85(2):715-36.
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 7
ADC’s analytical characterization
2. Linker & payloads
* P.D. Senter, Current Opinion in Chem. Biol., 2009; 13:1-10.
* Wakankar et al., mAbs, 2011 ; 3:2; 161-172.
• Contain labile bonds leading to drug release.
• Stability issue
• Other optimized combination of drug/linker to come.
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 8
A B C
ADC’s analytical characterization
3. Conjugation chemistry
* Lin et al., Pharm Res, 2012 ;29(9):2354-66.
• (A) Trastuzumab emtansin
• (B) Brentuximab vedotin
• (C) Thio-trastuzumab
• ADC production using A) or B) results in high heterogeneity in DAR and
location of conjugation site
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 9
Brentuximab vedotin manufacturing
Wakankar A et al, AAPS, 2010 (Genentech)
Le LN et al, Anal Chem, 2012 (Genentech)
Valliere-Douglas JR et al, Anal Chem 2014 (Seagen)
• Mixture of covalent/ non-covalent IgGs
• Need of specific analytical methods
• (1) “Denaturing” = non-covalent interchain
bonds (L-H, H-H) are disrupted
• (2) “Native” = non covalent interchain bonds
(L-H, H-H) are maintained
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 10
The ADC’s analytical tool box
DAR
Drug Load
Profile
Un-conjugated
mAb
Conjugation sites
Drug related
impurities
Higher order
structure
Denaturing methods•SDS-PAGE
•CE-SDS
•Peptide mapping
•LC-MS (+/- Red ; IdeS)
Native methods•UV
•HIC
•SEC
•Native MS
•Ion mobility MS
Nature Reviews, A. Mullard, 12, 2013.
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 12
Antibody Fluorophore Conjugates (AFCs)
NNH
HN
O
OO
O
NH
NH2O
NH
O O N
HN
O
O
NN
O
OCH3O OCH3O
NH
HOS
Maleimide Caproic acid
Maleimidocaproyl Valine Citruline
PABC MethylValine
Valine Dolaisoleucine Dolaproline Norephedrine
MMAE
Attachment groupProtease cleavable
linkerMMAE
Cytotoxic drug
NNH
HN
O
OO
O
NH
NH2O
NH
O O N
HN
O
O
NN
O
OCH3O OCH3O
NH
HOS
Maleimide Caproic acid
Maleimidocaproyl Valine Citruline
PABC MethylValine
Valine Dolaisoleucine Dolaproline Norephedrine
MMAE
Attachment groupProtease cleavable
linkerMMAE
Cytotoxic drug
NNH
HN
O
OO
O
NH
NH2O
NH
O O N
HN
O
O
NH
SO
ONS
Maleimide Caproic acid
Maleimidocaproyl Valine Citruline
PABC MethylValine
Fluorochrome
NNH
HN
O
OO
O
NH
NH2O
NH
O O N
HN
O
O
NH
SO
ONS
Maleimide Caproic acid
Maleimidocaproyl Valine Citruline
PABC MethylValine
Maleimide Caproic acid
Maleimidocaproyl Valine Citruline
PABC MethylValine
Fluorochrome
A
B
Non toxic Dansyl Sulfonamide Ethyl Amine (DSEA)-linker maleimide:
• designed and synthetized to mimic Brentuximab vedotin and most of ADCs in
clinical trials
• conjugated on interchain cysteines of trastuzumab
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 13
Size-exclusion chromatography (SEC)
• Trastuzumab-mc_DSEA displayed 2 populations by SEC, multimeric and
monomeric species isolated and characterized by :
• SDS-PAGE
• CE-SDS
• HIC
• Native Mass Spectrometry
• LC-MS after IdeS digestion and reduction
• Relationship between aggregation of the AFC and average Dye to Antibody
Ratio (DAR)?
Monomers
Multimers
20.0
10.0
0.0
30.0
40.0
50.0
60.0
mAU
0.0 50.0 100 150 200 250 300 mL
69.5%
30.5%
Monomers
Multimers
20.0
10.0
0.0
30.0
40.0
50.0
60.0
mAU
0.0 50.0 100 150 200 250 300 mL
69.5%
30.5%
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 14
SDS-PAGE
• Non Reducing
• H2L, H2, HL, H, L + payloads
• Monomeric versus multimeric profiles : different payload distributions
• Reducing
• Increased MW (multimers versus monomers)
• Higher conjugation level for multimers
250
150
100
75
50
37
2520
tras tu
zumab
AFC mon
o mer
s
AFC mult
imer
stra
s tuzu
mabAFC m
ono m
ers
AFC mult
imer
s
NR R
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 15
2,95
3
4,47
7
5,78
4
6,09
0
6,56
5
8,73
0
10,2
57
AU
-0,10
0,00
0,10
0,20
0,30
0,40
0,50
0,60
0,70
0,80
0,90
Minutes2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00 10,00 11,00 12,00
Hydrophobic Interaction Chromatography (HIC)
• Monomeric trastuzumab-mc_DSEA• homogeneous HIC distribution centered on D4
• Mutimeric fraction• contains high loaded forms
BA
U
-0.05
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
Minutes2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
Monomeric
0PL
2PL 4PL
6PL
8PL
Av. DAR: 4.3
Multimeric
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 16
Native MS
• Native desalting conditions (200mM NH4Ac, pH7), maintain intact the bivalent structure of AFC allowing :
• Relative distribution of drug loaded species
• Direct av. DAR determination
1491971953
1471851052
145173114
1512081252
153219488
2 %
26 %
2 Payloads
22 %
40 %
10 %
0 Payload
4 Payloads
6 Payloads
8 Payloads
Av. DAR: 4.4
« monomeric » trastuzumab-mc_DSEA
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 17
IdeS for ADC/AFC characterization
AFC mixture(150 kDa)
LC(25 kDa)
Fc/2(25 kDa)
1) IdeS
2) DTT
Fd(25 kDa)
Fd0 Fd1 Fd2 Fd3
L0 L1
IdeS• Immunoglobulin-degrading enzyme of Streptococcus pyogenes
• FabRICATORTM (www.genovis.com)
• 3 fragments of ≈25kDa providing LC and MS resolution
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 18
LC-UV-MS Trastuzumab (IdeS + reduction)
A
Time10.00 20.00 30.00 40.00
AU
0.0
5.0e-3
1.0e-2
1.5e-2
2.0e-2
2.5e-2 Range: 1.6e-2
17.5011.28
6.37
13.62
mass25000 25200 25400 25600 25800
%
0
100 4.03e425237
2521825091
25399
2525525560
mass23200 23400 23600 23800
%
0
100 6.53e423443
23462
mass25000 25200 25400 25600 25800
%
0
100 4.84e425383
25364 25401
B Fc/2 at 11.3 min LC at 13.6 minC Fd at 17.5 minD
• 3 fragments identified according to their MS profiles :
• Fc/2 carrying glycosylation : G0F, G1F + G0 and G2F (CHO prod.)
• Light Chain
• Fd
1) Non-conjugated mAb
Fc/2
L0
Fd0
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 19
Time10.00 20.00 30.00
AU
0.0
5.0e-3
1.0e-2
1.5e-2
2.0e-2
2.5e-2Range: 2.65e-211.48
16.64
14.25
20.52
25.8921.34
30.00
mass25000 25250 25500 25750
%
0
100 2.82e425235
2521625089
25397
2525525418 25559
mass25000 25250 25500 25750
%
0
100 2.82e425235
2521625089
25397
2525525418 25559
mass23200 23400 23600 23800
%
0
100 3.68e423442
23463
mass23200 23400 23600 23800
%
0
100 3.68e423442
23463
mass24000 24250 24500 24750 25000
%
0
100 3.80e424447
24468
mass24000 24250 24500 24750 25000
%
0
100 3.80e424447
24468
mass25000 25250 25500 25750
%0
100 1.92e425383
25362 25404
mass25000 25250 25500 25750
%0
100 1.92e425383
25362 25404
mass26000 26250 26500 26750
%
0
100 2.83e426388
26368 26408
mass26000 26250 26500 26750
%
0
100 2.83e426388
26368 26408
mass27000 27250 27500 27750
%
0
100 1.18e427392
27373 27413
mass27000 27250 27500 27750
%
0
100 1.18e427392
27373 27413
mass28000 28250 28500 28750
%
0
100 3.17e328397
2837828418
28455
mass28000 28250 28500 28750
%
0
100 3.17e328397
2837828418
28455
Fc/2 at 11.5 min
L0 at 14.3 min
L1 at 16.6 min
Fd1 at 20.5 min
Fd0 at 18.1 min
Fd2 at 25.9 min
Fd3 at 30.0 min
BA
C
D E
F
G
H
• Un-conjugated & loaded IdeS fragments well separated
• Fd1 and Fd2 split in 2 peaks : positional isomers
• The number of payloads fits with the number of possible free thiol
residues oxidized during AFC preparation : interchain cys residues
LC-UV-MS AFC (IdeS + reduction)2. Monomeric fraction
Fc/2
L0Fd0
L1Fd1
Fd2
Fd3
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 20
Time10.00 20.00 30.00 40.00
AU
0.0
5.0e-3
1.0e-2
1.5e-2
2.0e-2
2.5e-2 Range: 2.127e-2
11.47
5.00
16.34
14.06
29.28
25.4219.70 34.51
mass27000 27200 27400 27600 27800
%
0
100 1.07e427393
27371 27416
mass25000 25200 25400 25600 25800
%
0
100 9.60e425236
2521425089
25398
252582556125420
mass28000 28200 28400 28600
%
0
100 3.38e428398
2837828418
mass29200 29400 29600 29800
%
0
100 4.84e329402
29422
mass25400 25600 25800 26000 26200 26400
%
0
100 7.42e325452
25472
26387
mass24000 24200 24400 24600 24800 25000
%
0
100 8.62e424448
24469
mass23200 23400 23600 23800
%
0
100 2.54e423443
23465
Fc/2 at 11.5 min
L0 at 14.1 min
L1 at 16.3 min Fd4 at 34.5 min
BA
C
D E
F
G
HL2 & Fd1 at 19.7-21.1 min
Fd3 at 29.3 min
Fd2 at 24.5-25.4 min
• Qualitatively & quantitatively different from monomeric fraction
• Highly loaded fragments
• Higher conjugation rate than expected
• L2 and Fd4 reveal extra-conjugation sites
LC-UV-MS AFC (IdeS + reduction)3. Multimeric fraction
Fc/2
L0Fd0
L1
Fd1Fd2
Fd3
Fd4
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 21
• DAR (LC) = Σ[nALn / ΣALC]
• DAR (Fd) = Σ[nAFdn / ΣAFd]
• Av.DAR= 2 x [DAR (LC) + DAR (Fd)]
AFC : Average DAR
DAR is more than doubled in the multimeric fraction
10.00 15.00 20.00 25.00 30.00 35.00
AU
0.0
1.0e-2
2.0e-2
L0Fc/2 Fd0
10.00 15.00 20.00 25.00 30.00 35.00
AU
0.0
1.0e-2
2.0e-2
L0Fc/2 Fd0
Time10.00 15.00 20.00 25.00 30.00 35.00
AU
0.0
1.0e-2
2.0e-2
LCT-13-0911-OC 2: Diode Array Range: 2.105e-2
L012%
Fd13%
Fd216%
Fd372%
Fd49%
L175%
Fc/2
L213%
Time10.00 15.00 20.00 25.00 30.00 35.00
AU
0.0
1.0e-2
2.0e-2
LCT-13-0911-OC 2: Diode Array Range: 2.105e-2
L012%
Fd13%
Fd216%
Fd372%
Fd49%
L175%
Fc/2
L213%
Av. DAR = 3.8
Av. DAR = 7.8
A
B
C
10.00 15.00 20.00 25.00 30.00 35.00
0.0
1.0e-2
2.0e-2
Range: 2.645e-2
L032%
L168%
Fd153%
Fd016%
Fd226% Fd3
5%
Fc/2
10.00 15.00 20.00 25.00 30.00 35.00
0.0
1.0e-2
2.0e-2
Range: 2.645e-2
L032%
L168%
Fd153%
Fd016%
Fd226% Fd3
5%
Fc/2
Monomeric AFC
Multimeric AFC
Trastuzumab
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 22
Case study 1 - conclusions
• IdeS for characterization of AFC/ADCs by LC-
UV-MS
• Sub-unit analysis reduces sample complexity
• Fast sample preparation
• 25 kDa fragments easily ionized and analyzed by ESI-TOF-MS
• Payload distribution and DAR
• Monitor variants and PTMs
• C-ter lysine truncation, pyroglutamation, oxidation, degradation
• Fc/2 > N-glycosylation
• Highly conjugated mAb tend to aggregation
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 23
Case Study 2 : Native MS of ADCs
Native MS gives information on assemblies maintained
by noncovalent interactions
ESI-MSinstrument
3000 3500 m/z
Gas phaseSolution
Instrumental setting
optimizations
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
Sample preparation
= desalting step
- Buffer exchange
- Volatile aqueous buffer
- Ammonium buffer
(0-1M) with controled pH
* Cianferani S., ASMS, 2014.
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 24
1008040 6020 120 140 Mass (kDa)
145,910 Da
123,500 Da101,072 Da
75,586 Da
25,042 Da53,177 Da
• Non-covalent interactions are
maintained in the gas phase
• ADC is detected as an intact
molecule
• DAR can be assessed
6000 7000 m/z1000 3000 500040002000
6000 7000 m/z1000 3000 500040002000
Native ConditionsIgGZero treated ADC @ 5 µM in 150 mM AcONH4 pH7.5
Denaturing conditionsIgGZero treated ADC @ 2 µM in H2O:ACN:FA (50:50:1)
Native MS for cysteine -linked ADCs
37+
15+
37+
• Non-covalent interactions are
disrupted
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 25
Comparison of HIC & Native MS
Minutes3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
D0
D2 D4
D6
D8
∑
∑=
8
0
8
0
n
n
DAR
DAR
A
nADAR = 4.0
D0
D2
D4
D6
D8
D826+
D425+
D024+
D625+
D224+
b)
1008040 6020 120 mass (kDa)D2
D424+
D224+D6
25+
D023+
D825+ D0
D2 D4
D6D8
d)
e)
1.HIC
3.Native MS
(deglycosylated)
2.Native MS
* Vallière-Douglass J.F., Anal Chem, 2012.
* Debaene F. et al., Anal Chem 2014; in press.
6.1%
25.2% 34.8%
24.0%
8.3%
6.4%
40.8% 38.5%
9.4%5.0%
6.4%
26.7%
34.5%
21.9%
7.2%
Brentuximab Vedotin :
Drug load distribution and average DAR
are identical when calculated from HIC or
native MS after deglycosylation
DAR = 4.0
DAR = 3.3
DAR = 4.0
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 26
Native MS
146 152 mass (kDa)145 150148147 151149 153
D0
D1
D3
D2
D4
Av. DAR = 1.8
Advantages of native MS over HICExample of a hinge cys-linked ADC
HIC
2 3 4 5 67
8 9 10 11 min.
D0
D2?
D4?D1?
Av. DAR = 2.0• Broad peaks
• Ambigous DAR assessment
• Odd DAR not expected
• Unambigous DAR assessment
• Confidence in average DAR
determination
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 27
Brentuximab Vedotin
Off line coupling HIC & Native MS of ADC
Tim
e (m
in.)
34
56
78
910
D0
D4
D2
D6
D8
Mass (kDa)145 150 155
145904 ± 2 DaD0
148539 ± 1 DaD2
151173 ± 1 DaD4
153815 ± 2 DaD6
156457 ± 3 Da
D8
a) b)
•HIC peak collection and
subsequent fractions analyzed
by native MS
• Drug load confirmation
•Native MS of ADC as support
for HIC method development.
* Debaene F. et al., Anal Chem 2014, in press.
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 28
Ions separation takes place according to Ion Mobility
• Drift times can be related to collisional cross sections (CCS)
• CCS : projection of the area of complexes measured in the gas phase
• Information on ion gas phase conformation
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
Combining Ion Mobility and Mass Spec
* Cianferani S., ASMS, 2014.
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 29
What’s Ion Mobility able to perform for ADCs?
* Debaene F. et al., Anal Chem 2014, in press.
• Native IM-MS :
• ADC heterogeneity in drug binding is observed on native IM-MS plots
Deglycosylated Parent mAbO25596FD E.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
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 30
What’s Ion Mobility able to perform for ADCs?
* Debaene F. et al., Anal Chem 2014, in press.
• Native IM-MS :
• DAR are separated from each other in the IM cell
• Relative quantitation for average DAR determination
• Collision cross section determination
Deglycosylated Parent mAb Deglycosylated ADC
16 17 18 19 tD (ms)
6700
6600
6500
6400
m/z
23+
22+
16 17 18 19 tD (ms)
6700
6600
6500
6400
m/z
DO 23+
D0 22+
D2 23+
D6 24+
D4 23+
D8 24+
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 31
Average DAR from semi quantitative native MS and IM-MS
MaxEnt1 deconvolution
Quantitation based on ion
intensities
n=8
D0
D2D4
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
Minutes3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
D0
D2 D4
D6
D8
Native MS
HIC
Average DAR determination and drug-load profiles obtained
from native IM-MS are in good agreement with HIC & native MS data
146 Mass (kDa)150148 154152 158156
D4
D2
D6
D8D0
Native IM-MS
* Debaene F. et al., Anal Chem 2014, in press.
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 32
Native IM-MS of intact cysteine-linked ADC
• Drug binding induces constant and reproducible ∆∆∆∆tD and ∆∆∆∆CCS differences
• Very slight conformational changes are induced on the parent mAb upon drug binding
Parent mAb ADC
- DO D2 D4 D6 D8
Drift Time 24+
(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
∆(drift time) (ms) - - + 0.7 + 0.7 + 0.7 +0.7
CCS 24+ (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
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)66
67
68
69
70
71
72
73
74
75
76
77
22 23 24 25 26 27 28 29
DAR8
DAR6
DAR4
DAR2
DAR0
Charge states
Measured CCS (nm 2)
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 33
Case study 3 : biosimilarity using CESI-MS/MS
Sheathless Capillary Electrophoresis – tandem mass spectrometry (CESI-MS/MS)
• CE-ESI-MS Coupling :
• CE is a miniaturized technique performing ultra-low flow rates
• Decreasing the flow allows for increased sensitivity in the ESI-MS*
« Ultra-low flow » CESI-MS
• MS/MS : structural informations
*Wilm, Mann International Journal of Mass Spectrometry 1994, 136, 167–180
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 34
CESI-MS: mAbs characterization workflow
In-solution tryptic digestionAnalysis by
t-ITP CESI-MS/MS
Amino acid sequence
characterization
PTMs hot spots
characterization
Glycosylations
(structure)
CESI8000 coupled to 5600 TripleTOF MS
Primary structure characterization workflow based on bottom-up proteomics strategy
• Single shot (200 fmoles of digest)
• Peptide mapping including amino acid sequence coverage with 1 enzyme
(Trypsin)
• Characterization of PTM hot spots and glycosylation
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 35
CESI-MS/MS of trastuzumab
• Sequence coverage of trastuzumab by CESI-MS/MS
100% sequence coverage achieved in a single injection through
only purely tryptic unmodified peptides
Gahoual R. et al., Anal. Chem., 2014 (86), 9074-9081
variable domain
complementarity determining region
constant domain
identified peptides
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 36
Systematically >90% of the y/b ions could be retrieved from the CESI-MS/MS data
Amino acid sequence characterization (trastuzumab)
CESI-MS/MS of trastuzumab
Gahoual R. et al., Anal. Chem., 2014 (86), 9074-9081
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 37
MS/MS spectrum of digested peptides LT04
APK
(m/z 315.2039 ; 2+)
MS/MS spectrum of digested peptides HT15
DYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKP
SNTK
(63 amino acids ; m/z 1119.898 ; 6+)
Implementation of CE allows separation and successful detection of a larger variety of peptides
than classical RP-LC
CESI-MS/MS of trastuzumab
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 38
PTMs hot spots
N-terminal glutamic acid cyclization characterization
• CE mechanism separates peptide with N-terminal glutamic acid cyclization (1.8%) from the unmodified
peptide (98.2%)
Results suggest partial
modification of sample
Favorable conditions to estimate
sample modification level
HT01
pyroglu - HT01
Extracted ion electropherograms of peptides HT01 and modified HT01
CESI-MS/MS of trastuzumab
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 39
Methionine oxidation
• Methionine oxidation causes peptide mass shift (+15.99 Da) leading to the separation of the
modified peptide in CZE
confirmed by MS/MS spectra
Gahoual R. et al., Anal. Chem., 2014 (86), 9074-9081
Methionine (M)
Methioninesulfoxide (oxiM)
EIEs and MS/MS spectra of peptides HT21 (intact and modified)
CESI-MS/MS of trastuzumab
PTMs hot spots
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 40
Asparagine (N) Aspartic acid (deaN)
• Deamidation (+ 0.98 Da) involves mobility change in CZE enabling the separation of the unmodified peptide
CE separation of deamidated peptides eases the
identification of the modification by MS
EIEs and MS/MS spectra of peptides LT04 (intact and modified)
Asparagine deamidation
Gahoual R. et al., Anal. Chem., 2014 (86), 9074-9081
CESI-MS/MS of trastuzumab
PTMs hot spots
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 41
Aspartic acid isomerization
CE separation prior to MS analysis allows in this particular case to include
aspartic acid isomerization in the overall characterization workflow
HT23 (-D283-)
HT23 (-isoD283-)
EIEs and MS/MS spectra of peptides HT23 (intact and modified)
CESI-MS/MS of trastuzumab
PTMs hot spots
Gahoual R. et al., Anal. Chem., 2014 (86), 9074-9081
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 42
Trastuzumab versus biosimilar
• Complete sequence coverage obtained for
trastuzumab
• Biosimilar candidate sequence could be
successfully identified except HC K217
Suggesting an amino acid substitution
between the two samples
Gahoual R. et al., MAbs, 2014, 6:6, 1464-1473.
Amino acid sequence similarity
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 43
Glycoforms characterization
• Fc/2 glycosylation site characterization
Heterogenous glycoforms could
be identified
Difference in glycoforms
distribution could be observed
Cetuximab possess two different N-
glycosylation sites
Significant number of glycans could
be characterized
Cetuximab versus biosimilar
Gahoual R. et al., MAbs, 2014, 6:6, 1464-1473.
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 44
• Fd glycosylation site characterization
Glycoforms exhibited by the candidate biosimilar are significantly different from cetuximab
Rejected as biosimilar� Capped gal-α1,3-gal glycans significantly reduced
� 30 % of glycans contains N-acetylneuraminic acid
Cetuximab versus biosimilar
Glycoforms characterization
Gahoual R. et al., MAbs, 2014, 6:6, 1464-1473.
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 45
Conclusions
• For mAbs & ADCs, development & analytical characterization are challenging
• High heterogeneous products
• Mass spectrometry has a golden place in the analytical pipeline of
biopharmaceutical companies
• Analysis key points
• Sample preparation (ex: IdeS digestion & Reduction)
• Hyphenation of Capillary Electrophoresis
• Emergent MS methods are becoming trend
• Native MS
• Payload distribution - av. DAR determination of « hinge cys-linked » ADCs
• Binding stoichiometries mAb/Ag
• Ion Mobility-MS
• Conformational differences between DAR allow separation
• Orthogonal method to determine av. DAR
Titre
CASSS AT Europe 2015, E. Wagner-Rousset 46
THANK YOU
Alain BECK
Elsa WAGNER-ROUSSET
Marie-Claire JANIN-BUSSAT
Christine KLINGUER-HAMOUR
Olivier COLAS
Laura MOREL-CHEVILLET
Mélissa EXCOFFIER
Thierry CHAMPION
Sarah CIANFERANI
François DEBAENE
Julien MARCOUX
Guillaume TERRAL
Johann STOJKO
Alain VAN DORSSELAER
Emmanuelle LEIZE-WAGNER
Yannis FRANCOIS
Rabah GAHOUAL
Michael BIACCHI
Armelle CHARRIE
Pierre ALBRECHT
Noelle POTIER
Cécile PERRET
Na-Thi NGUYEN
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