alain beck, phd director, antibody physico chemistry d ... · wcbp, jan 28, 2015, washington...

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


3

(1)mAbs & biosimilars


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


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


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


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


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


9CESI‐MSTrastuzumab glyco-profilling by CESI-MS/MS



10CESI‐MSTrastuzumab: native vs de-amidated peptides



11CESI‐MSTrastuzumab: Asp and IsoAsp CE separation



12

(2)mAbs mixtures

« rpAbs »


13rpAbsRecombinant pAbs structural assesment

Fransen TP et al Biotech Biochem 2011 (Symphogen): mixture of 2 to 25 Abs


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


15ETDMiddle‐down with Electron Transfer Dissociation (ETD)

Fornelli L, Ayoub D, Aizikov, K, Beck A, Tsybin Y, Anal Chem 2014

**


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


19

(3)Bispecific mAbs& Mass Spec


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


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)


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


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


24

(4)ADCs &

Mass Spec


25


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


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


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


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


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


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


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


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



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


ADC (av DAR 2 theo): structure, Drug distribution and avDAR


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


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


37

(5)Summary


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


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)


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


42

[email protected]

2009www.landesbioscience.com

IF: 5.275 (2012), 3.174 (2011) Anal Chem (5.695), Anal Biochem (3.247)


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


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


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


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


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.


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.


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)


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


51Cover stories2008 2009 2010

2011

2012

2013

2013

2014


52Cover stories

2014

2015


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


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


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.)


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


57

mAbs worshops LyonBiopole: OptimAbs (2010)


58

From OptimAbs to MabDesign (LyonBiopole) (2014)

Démonstrateur CIMTECH

LabexIGO Laboratoire de SM

Bio-Organique.

alain beck, phd director, antibody physico chemistry d ... · wcbp, jan 28, 2015, washington...

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