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WCBP, Jan 28, 2015, Washington
State‐of‐the art Mass Spectrometry Methods for mAbs, Biosimilars, pAbs,Bispecifics and ADCs
characterization
Alain Beck, PhDSr. Director, Antibody Physico‐ChemistryCentre d’Immunologie Pierre Fabre, FR
Associate Editor, mAbs, USA
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
2Outline 2
(1) mAbs and biosimilars• State‐of‐the art MS methods• Top, middle, bottom levels
(2) mAbs mixtures and “rpAbs”• Orbitrap (EMR)• Electron Transfer Dissociation (ETD)
(3) Bispecific antibodies and Ab/Ag • Ion Mobility‐MS, native MS
(4) ADCs/AFCs• HR‐MS, native MS, Ion Mobility‐MS• Positional isomers
(5) Summary and take homes messages• OptimAbs, OptimADCs platforms Beck A et al, Anal Chem 2012
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
4
Cutting‐Edge MS of Originator, Biosimilar and Biobetter mAbs
Special feature: Head comparison of trastuzumab and cetuximab with corresponding biosimilar and biobetter candidates
• Native MS and Ion‐Mobility MS at different levels (Top, Middle and Bottom)
• Middle ‐up and ‐down strategies • Hydrogen/deuterium exchange ‐MS• CESI‐MS/MS• Electron Transfer Dissociation (ETD)• Top Down‐Sequencing (TDS)• High Resolution MS (HR‐MS)• Beck A, Debaene F, Diemer H, Wagner‐
Rousset E, Colas O, Van Dorsselaer A and Cianférani A, J Mass Spec 2015 (Feb)
JMS Feature
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
5
Ions separation takes place according to Ion Mobility
Drift times can be related to collisional cross sections (CCS) Information on ion gas phase conformation
Ion Mobility cell2+ 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
IM‐MS
Debaene F, Wagner E, Beck A, Cianferani S et al, Anal Chem 2014
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
6
originator biosimilar
IM‐MS: trastuzumab vs biosimilar candidate
(A) Trastuzumab
1 or 2 non glycosylated HC
(B) Trastuzumab‐B
Beck A, Debaene F, Cianférani A et al. J Mass Spec 2015
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
7
Sheathless Capillary Electrophoresis – tandem mass spectrometry (CESI‐MS/MS)
• Bottom‐up proteomic like approach• Single shot (100 fmoles of digest) • Peptide mapping 100% sequence coverage with 1 enzyme (trypsin)• Precise characterize of PTM hot spots• Structural confirmation of major and minor N‐glycoforms
Gahoual R, Burr A, Busnel JM, Beck A, François Y, Leize‐Wagner E et al, mAbs 2013
Trastuzumab
Bottom: peptide maps by CESI‐MS and MS/MS
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
8CESI‐MS
mAbs full primary sequence/PTMs in a single inject.
Gahoual R, Busnel JM, Beck A, François Y, Leize‐Wagner E. Anal Chem 2014
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
9CESI‐MSTrastuzumab glyco-profilling by CESI-MS/MS
Gahoual R, Busnel JM, Beck A, François Y, Leize‐Wagner E. Anal Chem 2014
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
10CESI‐MSTrastuzumab: native vs de-amidated peptides
Gahoual R, Busnel JM, Beck A, François Y, Leize‐Wagner E. Anal Chem 2014
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
11CESI‐MSTrastuzumab: Asp and IsoAsp CE separation
Gahoual R, Busnel JM, Beck A, François Y, Leize‐Wagner E. Anal Chem 2014
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
13rpAbsRecombinant pAbs structural assesment
Fransen TP et al Biotech Biochem 2011 (Symphogen): mixture of 2 to 25 Abs
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
14pAbsNative MS of a mixture of 11 N‐deglyc. mAbs
Xuan Y, Debaene D, Stojko J, Beck A, Van Dorsselaer A, Cianférani S, Bromiski M
Thermo 2014 (LSMBO), Application Note 597
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
15ETDMiddle‐down with Electron Transfer Dissociation (ETD)
Fornelli L, Ayoub D, Aizikov, K, Beck A, Tsybin Y, Anal Chem 2014
**
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
16eBUPExtended Bottom‐Up Proteomics Using Sap9 for Analysis of mAbs
Srzentić K, Fornelli L, Laskay U, Beck A, Ayoub D, Tsybin Y, Anal Chem 2014
Secreted Aspartic Protease 9
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
17eBUPExtended Bottom‐Up Proteomics Using Sap9 for Analysis of mAbs
Srzentić K, Fornelli L, Laskay U, Beck A, Ayoub D, Tsybin Y, Anal Chem 2014
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
18eBUPExtended Bottom‐Up Proteomics Using Sap9 for Analysis of mAbs
Srzentić K, Fornelli L, Laskay U, Beck A, Ayoub D, Tsybin Y, Anal Chem 2014
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
20
In vivo, IgG4 can exchange half molecules in a dynamic process In vitro, the addition of GSH is sufficient to induce FAE
S228 and R409 are crucial for FAE
Their mutation leading to in vitro and in vivo FAE blocking
+ GSH
+
IgG4 natalizumab
IgG4 Hz6F4-2v3
Junctional Adhesion Molecule-A (JAM-A)
Alpha-4 integrinTysabri® (Biogen Idec)
BsAbs
Van der Neut Kolfschoten M et al, Science 2007 (Genmab) Labrijn AF et al, Nat Biotech 2009 (Genmab) Debaene F, Wagner E, van Dorsselaer A, Beck A, Cianferani S et al, Anal Chem 2013
HzIgG4wt Fab‐Arm Exchange (FAE): BsAbs
BsAbs
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
21
1510 30205 25 tD (ms)0
6000
5500
6500
7000
m/z
+25
+24
+23
+26
+27
24+
natalizumab
bispecificmAb
6F4-2
tD (ms)
0
22 242018
20.5 ± 0.2
21.1 ± 0.2
21.9 ± 0.2
CCS (nm²) MW(Da)
natalizumab 69.6 ± 0.4 148980
Bispecific 66.9 ± 0.1 148300
6F4-2 65.6 ± 0.3 147628
• All 3 mAbs revealed very close gas phase conformations• bsAb has a CCS intermediate to individual constitutive IgG4s
All charge states of bsAb are separated
BsAbsNative & Ion Mobility MS: purified BsAb (CEX)
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
22
t = 0
t = 4 h
t = 24h
6200 6400 660060005800 6800 m/z
+23+24+25
Time (min.)
0 200 400 600 800 1000 1200 1400
Nor
mal
ized
Inte
nsity
(%)
0
10
20
30
40
50
natalizumabbsAb6F4-2
Apparent rate constant:
4.66 x 10-5 s-1*
Apparition of a 3rd population after 4h corresponding to bsAb
Time resolved native MS to monitor IgG4 FAEBsAbs
Debaene F, Wagner E, van Dorsselaer A, Beck A, Cianferani S et al, Anal Chem 2013
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
23
bsAb alone
bsAb+ 2 eq. JAM-A(Antigen)
200 225150 175 Mass (kDa)
bsAb+ 8 eq. JAM-A
• bsAb binds up to 2 Ag molecules
• Confirmation thatJAM-A bindspreferentially as a dimer to Hz6F4-2
• Debaene F. Anal Chem 2013
148302 ± 1 Da
172369 ± 3 Da
196444 ± 8 Da
195772 ± 3 Da244072 ± 3 Da
Control Hz6F4-2 S228P+ 8 eq. JAM-A
Atmanene C et al, Anal Chem 2009
Native MS of bsAb/Ag complexes
BsAbs
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
26Brentuximab vedotin manufacturing
Sun M, Senter P et al Bioconj Chem 2005 (Seagen)Wakankar A, AAPS 2010 (Genentech)Le LN, Anal Chem 2012 (Genentech)Valliere‐Douglas JR et al, Anal Chem 2014 (Seagen)
Mixture of covalent/ non‐covalent IgGsNeed 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
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
27The ADC’s analytical tool boxDAR
Drug LoadProfile
Un‐conjugatedmAb
Conjugation sites
Drug relatedimpurities
Higher orderstructure
Denaturing methods•n/rSDS‐PAGE•n/rCE‐SDS
•Peptide mapping•LC‐MS (+/‐ Red ; IdeS)
Native methods•UV•HIC•SEC
•Native MS•Ion mobility MS
Beck A, mAbs 2014
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
28
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 Wagner E, Janin MC, Beck A et al, mAbs 2014
AFCs/ADCs
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
29
1
0
1
01
)(
nLC
LC
A
AnLCDAR
3
0
3
0)(
n
n
Fd
Fd
A
nAFdDAR ))()((2 FdDARLCDARDAR
UV chromatogram at 210nm:
Fd1Fd2 Fd3
Time (min)
10.00 14.00 18.00 22.00 26.00
%
20
100
Ion
chro
mat
ogra
m
LC0
Fd0
Fc/2
LC1
avDAR = 3.8
Wagner E, Janin MC, Beck A et al, mAbs 2014
AFCs/ADCs
IdeS for ADC/AFC: LC‐ESI‐TOF
Fd3 : 28 391.57
Fd2 : 27 386.57
Fd2 : 27 386.57
Fd1 : 26 381.57
Fd1 : 26 381.57
TheoriticalMWav
1.8828 393.45 +/‐ 0.15
1.7727 388.34 +/‐ 0.20
1.5926 383.16 +/‐ 0.39
1.6927 388.26 +/‐ 0.17
E
D
C
B
1.6326 383.20 +/‐ 0.31A
Δth/expExperimentalMW
Peak
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
30
• DAR (LC) = Σ[nALCn / ΣALC]
• DAR (Fd) = Σ[nAFdn / ΣAFd]
• Av.DAR= 2 x [DAR (LC) + DAR (Fd)]
Average DAR: momomer vs multimers
• The average DAR is twicein the multimeric fraction• No Fc conjugation• Payload distribution and average DAR• Correlation: number of conjugated payloads and trends to aggregation
AU
0.0
1.0e-2
2.0e-2
L0Fc/2 Fd0
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
0.0
1.0e-2
2.0e-2
a ge 6 5e
L032%
L168%
Fd153%
Fd016%
Fd226% Fd3
5%
Fc/2
0.0
1.0e-2
2.0e-2
a ge 6 5e
L032%
L168%
Fd153%
Fd016%
Fd226% Fd3
5%
Fc/2
Monomeric AFC
Multimeric AFC
Trastuzumab
Wagner E, Janin MC, Beck A et al, mAbs 2014
AFCs/ADCs
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
31
tu Vedotin IdeS reduit methodeOC
Fd1a71%
Fd1b29%
Fd2a17%
Fd2b83%
or
L15 : Cys220Cys 226Cys 229L16: Paylaod
Brentu Vedotin IdeS reduit methodeOC
Time10.00 15.00 20.00 25.00 30.00 35.00 40.00
%
13
Fc/212.0
LC14.7
LC119.6
Fd22.6
Fd1a26.2
Fd1b27.4 Fd2a
32.5
Fd2b33.9 Fd3
39.7
Fd1 Fd2
brentuximab vedotin
• Positional isomers (Fd1 and Fd2)
• Fd1 : positional isomers with 1 drug are preferentially linked to HC L15• Fd2 : positional isomers with 2 drugs are preferentially linked to HC L16
Janin MC, Dillenbourg M, Corvaïa, N, Beck A, Klinguer C, J Chrom B 2015
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
32Brentuximab vedotin
3.28
8
3.82
6
4.74
6
5.62
9
6.56
4
8.15
9
9.31
1
AU
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
Minutes2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
HIC peak identification: denaturing vs native Mass Spec
Hamblett KJ et al, Cancer Res 2004 (Seagen)
AverageDAR = 4.0
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
33
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 maintained in the gas phase
• ADC detected as an “intact” IgG
• Drug distribution (% of isomers)
• Amount of naked IgG (D0)
• Average DAR calculation6000 7000 m/z1000 3000 500040002000
6000 7000 m/z1000 3000 500040002000
(B) Native ConditionsIgGZero treated ADC, 5 µM in 150 mM AcONH4 pH7.5
(A) Denaturing conditionsIgGZero treated ADC, 2 µM in H2O:ACN:FA (50:50:1)
Denaturing vs Native MS
37+
15+
37+
• Non‐covalent interactions disrupted
• Not suitable for structure confirmation and QC
Debaene F, Wagner E, Beck A, Cianferani S et al, Anal Chem 2014
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
34
(B) Native MS
146 152 mass (kDa)145 150148147 151149 153
D0
D1
D3
D2
D4
Av. DAR = 1.8
(A) HIC
2 3 4 5 6 7 8 9 10 11 min.
D0
D2?
D4?D1?
Av. DAR = 2.0• Broad peaks (hydrophobic drug)• Ambigous DAR assessment• Odd DAR ?• No confidence in average DAR calculation
• Unambigous structure assesment• Confidence in average DAR calculation
HIC vs Native MS
Debaene F, Wagner E, Beck A, Cianferani S et al, Anal Chem 2014
ADC (av DAR 2 theo): structure, Drug distribution and avDAR
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
35
O23564FD E.raw : 1
Brentuximab vedotin (de‐glycosylated): Ion‐Mobility Mass Spec
Fraction HIC : DAR2
24+
25+
Fraction HIC : DAR4
24+
25+
Fraction HIC : DAR6
24+
25+
Fraction HIC : DAR8
24+
25+
Fraction HIC : DAR0
24+
25+
6500
6000
IM‐MS: multiple species separation
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
36
O23564FDE.raw : 1
O23661FDE.raw : 1
(A) Brentuximab vedotin (Glycosylated):
(B) Brentuximab vedotin (De‐glycosylated):
24+
24+
IM‐MS: glycosylation structural impactCharge state
Drift Time (ms)Brentuximab(G1F/G0F)
Drift Time (ms)Brentuximab
Deglycosylé (IgZ)
DAR 025+ 13.4 ± 0.1 12.9 ± 0.1
24+ 14.7 ± 0.1 14.3 ± 0.1
DAR 225+ 13.9 ± 0.1 13.5 ± 0.1
24+ 15.4 ± 0.1 14.9 ± 0.1
DAR 425+ 14.5 ± 0.1 14.0 ± 0.1
24+ 16.0 ± 0.1 15.6 ± 0.1
DAR 625+ 15.2 ± 0.1 14.7 ± 0.1
24+ 16.7 ± 0.1 16.3 ± 0.1
DAR 825+ 15.8 ± 0.1 15.4 ± 0.1
24+ 17.4 ± 0.1 17.0 ± 0.1
DAR 0 DAR 2 DAR 4 DAR 6 DAR 8
ADC Glyco Dégly Glyco Dégly Glyco Dégly Glyco Dégly Glyco Dégly
CCSnm²
70.6±2.5
70.1±2.6
71.2±2.5
70.7±2.4
71.8±2.3
71.4±2.4
72.5±2.3
72.1±2.3
73.2±2.3
72.8±2.4
Glycosylation: ~ 1.5% in mass, ~ 0.70% in CCSDrug Conjugation x2: ~ 1.6% in mass, ~ 0.85% in CCSConformational changes linked both to
• glycosylation• drug conjugation
can be assesses by Ion Mobility Mass Spec
10 11 12 13 14 15 16 17 18 19 Dt (ms)
DAR0
DAR2
DAR4
DAR6
DAR8
10 11 12 13 14 15 16 17 18 19 Dt (ms)
DAR0
DAR2
DAR4
DAR6
DAR8
Debaene F et al, Anal Chem 2014
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
38OptimAbs/ADCs• Past decade: several hundreds of papers on mAbs analytical and structural characterization• Trend was amplified the last years
• Beck A et al, Anal Chem 2012 and 2013; Beck A et al, Trends in Anal Chem 2013• Beck A MiMB 2013; Beck A et al, J Mass Spec 2015
• Multiple liquid chromatography, electrophoresis and MS methods • Used at all stages of mAbs discovery, preclinical and clinical development• Selection of the best antibody‐producing clone (with the right glyco‐profile)• Full structural characterization (research leads + clinical candidates)
• Combination of liquid chromatography, electrophoresis and MS• Used for identification of “hot spots”• Deleterious for stability, PK and for pharmacology properties• Early use in the R&D process of MS methods (“Developability”)
• Helps to optimize the structure of next generation mAbs (OptimAbs)• Reduced chemistry CMC liabilities and better drug‐like properties
• All these methods are mandatory for• Comparability assays, formulation, process scale‐up and transfer• To define critical quality attributes (CQA) in a quality by design approach (QbD)
• All these routine and emerging methods also help evaluate • More sophisticated and potent antibody derivatives:
• ADCs (OptimADCs), bi‐ and multispecific antibodies• Controlled mixture of recombinant oligoclonal antibodies,• High affinity protein scaffolds
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
39AcknowledgementsCIPF, St‐Julien‐en‐Genevois, FR
• E. Wagner, O. Colas, L. Morel‐Chevillet• MC. Janin, M. Excoffier, C. Klinguer• T. Champion, D. Ayoub, A. Boeuf• M. Dillenbourg M. Duhamel, G. Terrral• L. Tonini, S. Genton, A. Bourmeaud• M. Rompais, M. Trauchessec, C. Huillet• L. Goetsch, JF. Haeuw, M. Tesar and coll.• O. Cochet, S. Lauthier and coll.• N. Corvaïa
CRDPF, Toulouse, FR• M. Perez, C. Bailly, JF. Boe and coll.
CRPF, Castres, FR• I. Rilatt and coll.
API, Pau, FR• franck.pavan@pierre‐fabre.com
Plantes et Medecines, Gaillac, FR• herve.limouzin@pierre‐fabre.com
LSMBO, University of Strasbourg, FR• S. Cianferani, F. Debaene• H. Diemer, JM. Strub, G. Terral• C. Carapito, C. Atmanene• J. Stojko, C. Schaeffer, J. Marcoux• A. Van Dorsselaer(> 20 years collaboration, 25 papers)
Waters, FR, UK, US (LC‐MS prototypes)• D. Petit, A. Fabre, F. Delsene• W. Chen, A. Millar, P. Boyce• L. Denbigh, H. Haesebaert• D. Lascoux, JM. Casanova, C. Siroit(> 15 years collaboration)
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
40CIPF’s structural networkLSMBO, University of Strasbourg, FR*Promise Advanced Therapeutics, FR*
• D. Lebert, G. Picard, V. BrunPXTherapeutics, Grenoble, FR*
• N. Mouz, W. LowCEA, Grenoble, FR*
• M. Jaquinod, J. GarinBioPark Agim (CNRS), Archamps, FR*
• S. Voisin, K. Arafah, AS. Navionis, P. BuletEMBL, ESRF, Grenoble, FR*
• J. Marquez, E. GordonLyonBiopole & Alsace Biovalley, FR*Quality Assistance, BE (Biophysics)
• G. Vanvyncht, A. Delobel, K. Goose, N. DrauxGenovis, SW (Enzymes)
• S. Fredriksson, S. Olauson, F. OlssonLNA, FR (National Metrology Lab)
• V. DelatourCovalix, Zurich, CH
• A. Nazabal
LSMIS, Univ. of Strasbourg, FR (CESI‐MS)• Y. François, R. Gahoual, E. Leize‐Wagner
EPFL, BMSL, Lausanne, CH (Top/Mid Down)• Y. Tsybin, L. Fornelli, D. Ayoub
University of Geneva, CH (Chromatography)• D. Guillarme, S. Fekete, S. Rudaz, JL. Veuthey
University of Marseille, FR (MALDI‐Imaging)• D. Lafitte, R. Ait‐belkacem
Gustavus Adolphus College, US (2D‐LC)• D. Stoll
University of Lyon, FR (2D‐LC)• S. Heinish
ABSciex, FR, USA (CESI‐MS, MS prototypes)• JM. Busnel, H. Dewald, M. Anselme
Agilent, FR (MS prototypes)• S. Ville‐Renon, F. Petot, L. Arnaud
Bruker, GE, FR (MS prototypes)• W. Jabs, A. Resemann, C. Evans, D. Suckau
Thermo, GE (MS prototypes)• Y. Xuan, M. Bromirski
Shimadzu, FR, UK, US (MS prototypes)• T. Legoupil
Waters, FR/UK/US (MS prototypes)
*
WCBP, Jan 28, 2015, Washington
Thank you for your attention!alain.beck@pierre‐fabre.com
www.cipf.com
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
42
2009www.landesbioscience.com
IF: 5.275 (2012), 3.174 (2011) Anal Chem (5.695), Anal Biochem (3.247)
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
43Rituximab
(A) Top:MWexp – MWcalc (eg. IMGT data base):
+54 +/- 2 Da (IgG)(not linked to Glc)
(B) Middle:IdeS/TCEP:
+28 Da (Fd)(Fc/2 and LC OK)
(C) Bottom:Peptide mapping:
V119 is an A
Correct sequence assessment
Beck A, Van Dorsselaer A, Cianferani S et al, Trends in Anal Chem 2013
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
44
1) IdeS
2) DTT
Janin‐Bussat MC, Beck A, Methods in Mol Biol 988 (Beck A ed.) 2013 Ayoub D, Jabs W, Resemann A, Suckau D, Beck A et al, mAbs 2013
2 N‐glycosylation sites: each Heavy ChainCetuximab
76 cetuximab‐treated patients: 25 hypersensitivity reaction IgE against cetuximab found in pretreatment samplesIgE specific to a Gal‐‐1,3‐Gal epitope (Cetuximab, SP2/0)
Zhou Q et al, Anal Biochem 2007 Chung C et al, NEJM 2008 Sundaram S et al, mAbs 2011
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
45
Primary structure assessment and glyco‐profillingCombination of
• Intact, middle‐up/ down• Bottom‐up• ESI and MALDI mass spectrometry
Correct primary structure assessment• Light Chain Ct:
• ‐RGA_ (IMGT, Drug bank): not correct• ‐RGEC: corrected
Extensive Fc and Fd glyco‐profiling• Fc/2: 11 glycoforms• Fd: 20 glycoforms
Ayoub D, Jabs W, Resemann A, Suckau D, Beck A et al, mAbs 2013
Sep/Oct 2013
Cetuximab
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
46
Monitoring therapeutic mAbs in brain tumor by MALDI MSI
Cover story
• Imaging method coupled to protein identification using matrix‐assisted laser desorption/ionization MS
• In Source Decay fragmentation (ISD)
• Monitoring of bevacizumab (anti‐VEGFA) distribution within the brain structures of mice
• especially within the tumor• without any labeling• palivizumab (anti‐RSV) negative
control• Ait‐Belkacem R, Berenguer C, Villard C,
Ouafik L, Figarella‐Branger D, Beck A, Chinot O, Lafitte D. Monitoring therapeutic mAbs in brain tumor. mAbs2014, Nov/Dec
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
47CIPF/LSMBO mAbs papers (1/2)Beck A, Debaene F, Diemer H, Wagner E, Colas O, Van Dorsselaer A and Cianférani S. Cutting‐edge
Mass Spectrometry Characterization of Originator, Biosimilar and Biobetter Antibodies. J Mass Spec, 2015 (invited special feature and cover story).
Boeuf A, Debaene F, Ayoub A, Diemer H, Wagner‐Rousset E, Van Dorsselaer A, Cianférani S, Beck A. Mass Spectrometry based strategies for therapeutic antibodies extensive characterization and optimization (OptimAbs). Structural Biology in Drug Discovery: Methods, Techniques, and Practices, edited by Jean‐Paul Renaud, John Wiley & Sons, 2015, in press.
Debaene F, Boeuf A, Wagner‐Rousset E, Ayoub D, Colas O, Corvaïa N, Van Dorsselaer A, Beck A, Cianferani S. MS and IM‐MS for ADC Characterization. Anal Chem, 2014, 86, 10674.
Xuan Y, Debaene F, Stojko J, Beck A, Van Dorsselaer A, Cianférani S and Bromiski M. Monoclonal Antibody and Related Product Characterization Under Native Conditions Using a Benchtop Mass Spectrometer. Thermo Scientific Application Note 2014.
Beck A, Wagner‐Rousset E, Ayoub D, Van Dorsselaer A, Sanglier‐Cianférani S. Characterization of Therapeutic Antibodies and Related Products. Anal Chem. 2013, 85(2):715‐36.
Beck A, Dimer H, Ayoub D, Debaene F, Wagner‐Rousset E, Carapito D, Van Dorsselaer A, Sanglier‐Cianférani S. Analytical characterization of biosimilar antibodies and Fc‐fusion proteins. Trends in Anal Chem. 2013, 48, 81‐95.
Debaene F, Wagner‐Rousset E, Colas O, Ayoub D, Corvaïa N, Van Dorsselaer A, Beck A, Cianférani S. Time Resolved Native Ion‐Mobility Mass Spectrometry to Monitor Dynamics of IgG4 Fab Arm Exchange and “Bispecific” Monoclonal Antibody Formation. Anal Chem. 2013, 85, 9785.
Beck A. Ed.; Glycosylation Engineering of Biopharmaceuticals, Methods in Molecular Biology. Vol 988, 355 p. Springer: 2013 (www.springer.com/).
Wagner‐Rousset, E.; Schaeffer‐Reiss, C.; Bednarczyk, A.; Corvaïa, N.; Van Dorsselaer, A.; Beck A. NanoLC Chips MS/MS for the Characterization of N‐Glycopeptides Generated from Trypsin Digestion of a Monoclonal Antibody. Glycosylation Engineering of Biopharmaceuticals, Methods Mol. Biol.Vol 988. Beck A. Ed.; Springer: 2013; Chapter 6.
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
48CIPF/LSMBO mAbs papers (2/2)Atmanene, C.; Wagner‐Rousset, E.; Corvaïa, N.; Van Dorsselaer, A.; Beck A.; Sanglier‐Cianferani, S.
Noncovalent Mass Spectrometry for the Characterization of Antibody/Antigen Complexes. Glycosylation Engineering of Biopharmaceuticals, Methods Mol. Biol. Vol 988. Beck A., Ed.; Springer: 2013; Chapter 16.
MC. Janin‐Bussat, JM. Strub, E. Wagner‐Rousset, O. Colas, C. Klinguer‐Hamour, N. Corvaia, A. Van Dorssealer & A. Beck. Antibody Characterization by Mass Spectrometry. Antibody Engineering, Springer Lab manual, Chap. 39, Vol 1, 2d Edition. R. Konterman & S. Dübel (Eds), 2010, 613.
MC. Janin‐Bussat, E. Wagner‐Rousset, C. Klinguer‐Hamour, N. Corvaïa, A. Van Dorssealer & A. Beck. Antibody Glycan Characterization. Antibody Engineering, Springer Lab manual, Chap. 40, Vol 1, 2d Edition. R. Konterman & S. Dübel (Eds), 2010, 635.
C. Atmanene, E. Wagner‐Rousset, M. Malissard, B. Chol, A. Robert, N. Corvaïa, A. Van Dorsselaer, A. Beck & S. Sanglier‐Cianferani. Extending Mass Spectrometry contribution to therapeutic monoclonal antibody lead selection: characterization of immune complexes using non‐covalent ESI‐MS. 2009, Anal Chem 2009, 81, 6347.
A. Beck, E. Wagner‐Rousset, MC. Bussat, M. Lokteff, C. Klinguer‐Hamour, JF. Haeuw, L. Goetsch, T. Wurch, A. Van Dorsselaer & N Corvaïa. Trends in glycosylation, glycoanalysis and glycoengineering of therapeutic antibodies and Fc‐fusion proteins. Special issue on Therapeutic Antibodies, Fc‐fusion proteins and novel protein scaffolds, Guest Editor A. Beck, Curr. Pharm. Biotech. 2008, 9, 482.
E. Wagner‐Rousset, A. Bednarczyk, MC. Bussat, O. Colas, N. Corvaïa, C. Schaeffer, A. Van Dorsselaer & A. Beck. The way forward, enhanced characterization of therapeutic antibody glycosylation:: comparison of three level mass spectrometry‐based strategies. J Chrom B 2008, 872, 23.
A. Beck, C. Klinguer‐Hamour, MC. Bussat, T. Champion, JF. Haeuw, L. Goetsch, T. Wurch, M. Sugawara, A. Milon, A Van Dorsselaer, T. Nguyen & N. Corvaïa. Peptides as tools and drugs for immunotherapies. J. Peptide Science – NP2D 2006 special issue, 2007, 13, 588.
A. Beck, MC. Bussat, N. Zorn, V. Robillard, C. Klinguer‐Hamour, S. Chenu, L. Goetsch, N. Corvaïa, A. Van Dorsselaer & JF. Haeuw. Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti‐Insulin growth factor–1 receptor antibodies produced in CHO and NS0 cells. J. Chrom. B, (2005), 819, 203‐218.
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
49
100% sequence recovery + extensive PTMs characterization of trastuzumab vs biosimilar, cetuximab vs biobetter, bevacizumab, palivizumab, panitumumab…
• Gahoual R, Burr A, Busnel JM, Kuhn L, Hammann P, Beck A, Francois Y, Leize‐Wagner E.Rapid and multi‐level characterization of trastuzumab using sheathless CE‐MS/MS. mAbs2013
• Gahoual R, Biacchi M, Chicher J, Kuhn F, Hammann P, Beck A, François Y, Leize‐Wagner E.mAbs biosimilarity assessment using transient isotachophoresis CE‐MS/MS. mAbs 2014
• Biacchi M, Bhajun R, Saïd N, Beck A, François Y, Leize‐Wagner E. Analysis of mAb by a novel CE‐UV/ MALDI‐MS interface isotachophoresis preconcentration‐capillary zone electrophoresis‐tandem MS. Electrophoresis 2014
• Gahoual R, Busnel JM, Beck A, François Y, Leize‐Wagner E. Full protein antibody primary structure and micro‐variant assessment in a single injection using transient isotachophoresis and sheathless CE‐MS/MS. Anal Chem 2014
• Santos M, Bush D, Dewald H, Viner R, Beck A, Heemskerk A, Karger B, Ivanov A. Middle‐down and bottom‐up characterization of trastuzumab with sheathless CESI‐MS coupled to the Orbitraps MS. ASMS 2014
=> CESI‐MS/MS Workshop (AB‐Sciex, Y. François, E. Leize), Sep 30, 2014, Strasbourg
CESI‐MS (LSMIS collab)
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
50
(A) Size Exclusion Chromatography (SEC)(1) Fekete S, Beck A, Veuthey JL, Guillarme D. Theory and practice of SEC for the analysis of proteins aggregates. J Pharm Biomed Anal 2014
(B) Cationic Exchange Chromatography (CEX)(2) Fekete S, Beck A, Fekete J, Guillarme D. Method development for the separation of mAbscharge variants in CEX, part I: Salt gradient approach. J Pharm Biomed Anal 2015(3) Fekete S, Beck A, Fekete J, Guillarme D. Method development for the separation of mAbscharge variants in CEX, part II: pH gradient approach. J Pharm Biomed Anal, 2015(4) Fekete S, Beck A, Veuthey JL, Guillarme D. Ion‐exchange chromatography for the characterization of biopharmaceuticals. J Pharm Biomed Anal 2015, submitted(5) Fekete S, Beck A, Guillarme D. Characterization of cationic exchanger stationary phases appliedfor the separation of therapeutic monoclonal antibodies. J Pharm Biomed Anal 2015, submitted
(C) Reverse‐Phase High Performance Chromatography (RP‐HPLC)(6) Fekete S, Beck A, Wagner E, Vuigner K, Guillarme D. Adsorption and recovery issues of mAbs in RP‐HPLC. J Sep Sci 2015(7) Bobály B, Beck A, Fekete J, Guillarme D, Fekete S. Systematic evaluation of mobile phase additives for the LC‐MS characterization of therapeutic proteins. Talanta 2015
(D) In progress: HIC, 2D‐LC of mAbs and related products
Liquid ChromatographyCollaborationLaboratory of Analytical Pharmaceutical ChemistryS. Fekete, S. Rudaz, JL Veuthey, D. Guillarme
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
51Cover stories2008 2009 2010
2011
2012
2013
2013
2014
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
53SYNAPT G2‐Si HDMS (Waters)
2014ADC
• SynaptTM G2‐Si HDMS : Q‐TOF with resolution up to 50 000• Denat/ Native MS of mAbs/ADCs (D loading/distr., D0, avDAR), peptide mapping, proteomics• Ion Mobility (shape & size), ETD (Asp/IsoAsp, labile PTMs, disulfide cross‐linking)
• AcquityTM UPLC H‐class Bio, 2D with PDA detector: orthogonal separations (CEX+RP, HIC+RP, RP+RP…) • TriVersa NanoMateTM Advion : In chip‐based electrospray ionization techics
• Nano‐infusion of low sample amounts with robustness and sensitivity, fraction collection
TriVersa
NanoMateTM
Advion
AcquityTM UPLC H‐class Bio,
2D, PDA
SynaptTM G2‐Si
HDMS, ETD
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
54Xevo TQ‐S (Waters)
2014 ADC
•XevoTM TQ‐S : Triple quadrupole MS for quantification(1) High sensitive quantification of residual payloads (ADC batches)(2) Controls and cleaning validation (ADCs labs)(3) In vitro/vivo stability sudies of payloads in plasma (ADCs, mAbs)
• AcquityTMUPLC I‐class, 2D•Well suited for complex samples, orthogonal dimensions of separations• eg. Residual drug in ADCs batches
AcquityTM UPLC I‐class, 2D, TUV detector
XevoTM TQ‐S
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
55
R&D of mAbs and ADCs for cancer treatments
Production Facility (cGMP)
CIPF
Integrated R&D Center
IGF-1R/dalotuzumab 1st hz mAb
(licenced to MERCK)
CXCR4/hz515H7
3rd hz mAb (Pierre Fabre)
c-Met 2nd hz mAb (licenced to ABBOTT)
Clinical Stage naked mAbs
www.cipf.com
ADCsConfidential
F58003 (Plat.)
mAbsConfidential
F58000 (Plat.)
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
56Pierre Fabre: R & D & Production
mAbs/ADCs R&D (CIPF), SMDs/Pharma Dev (CRDPF), SMDs Dev. (P&I), SMDs/mAbs/ADCs Fill & Finish (API)
CIPFSt Julien
CRDPFToulouse
P&IGaillac
APIPau
WCBP 2015 (CASSS) – Jan 28, 2015 – Washington, DC Alain BECK, PhD
57
mAbs worshops LyonBiopole: OptimAbs (2010)