ADCs: Analytical and Bioanalytical Challenges

Manchester, Nov 20, 2014 (Waters ADC WS)

ADCs: analytical & bioanalyticalchallenges

Alain Beck, PhDSenior Director, Antibody Physico‐Chemistry

Centre d’Immunologie Pierre Fabre, FRAssociate Editor, mAbs

Waters ADC WS, Nov 20‐21, 2014, Manchester Alain BECK, PhD

2Outline(1) Introduction• Therapeutic mAbs& related products• 1st, 2d and 3d generation ADCs

(2) CIPF/PFM• R&D: mAbs and ADCs• Analytical and structural platform

(3) Summary & Take‐home messages• OptimAbs/ OptimADCs• CIPF’s: analytical and MS network• PF/LSMBO/Waters collaboration

Beck A et al, Anal Chem 2012

2


3

(1)mAbs and ADCs


4

2015? MAA secukinumab(IL17a) dinutuximab(GD2) evolocumab[IgG2](PSK9) blinatumomab(CD19/CD3) necitumumab(EGFR)

2014 ramucirumab(VEGFR‐2) siltuximab(IL6) vedolizumab(α4β7) nivolumab[IgG4s](PD1)[Jpn] pembrolizumab[IgG4s](PD1)

2013 itolizumab(CD6)[Ind] ado trastuzumab emtansine(HER2) obinutuzumab[low Fuc](CD20) [Remsima/infliximab, EMA]

2012 mogamulizumab[low Fuc](CCR4)[Jpn] pertuzumab(HER2) ziv‐aflibercept[PrFc](VEGF) raxibacumab(antrax)

2011 brentuximab vedotin[IgG1‐vcMMAE](CD30) belatacept[PrFc](CD80/86) aflibercept[PrFc](VEGF)

belimumab(BLysS) ipilimumab(CTLA‐4)

2010 denosumab[IgG2](RANK‐L)

2009 golimumab(TNF) catumaxomab(EpCAM/CD3) ustekinumab(IL12/23) canakinumab(IL1) ofatumumab(CD20)

2008 rinolacept[PrFc](IL1) certolizumab[Fab‐PEG](TNF) romiplostim[FcPe](TPO) [Clotinab/abciximab So‐Ko]

2007 eculizumab[IgG2/4](C5) [Reditux/rituximab Ind]2006 ranibizumab[Fab](VEGFA) panitumumab[IgG2](EGFR)

2005 nimotuzumab(EGFR)[Chi] abatacept[PrFc](CD80/86) tocilizumab(IL6R)[Jpn]

2004 cetuximab(EGFR) bevacizumab(VEGFA) natalizumab[IgG4](4 integr)

2003 131I‐tositumomab[mIgG2a](CD20) )[withdrawn 2014] omalizumab(IgE‐Fc) efalizumab(CD11a)[withdrawn, 2009] alefacept[PrFc](CD2)

2002 111In/90Y‐ibritumomab tiuxetan[mIgG1](CD20) adalimumab(TNF)

2001 alemtuzumab(CD52)

2000 gemtuzumab ozogamicin[IgG4s‐calicheamycin](CD33)[withdrawn 2010]

19991998 basiliximab(CD25) palivizumab(RSV‐F) infliximab(TNF) trastuzumab(HER2) etanercept[PrFc](TNF)

Technologies 1997 rituximab(CD20) daclizumab(CD25)

Transgenic mice (10Y) 19961995 edrecolomab[mIgG2a]

(EpCAM)[Ger, withdrawn]

1994 abciximab[Fab](GPIIb)

Phage display (9Y) 1993Humanization (11Y) 1986 muromomab[mIgG2a]

(CD3)

Chimerization (10 Y) 1984 Nobel Prize for mAbs

> 50 approvedmAbs, Fabs, Fc‐fusions, ADCs, RadioICs, Bispecs[28 years](INN= International Non‐proprietary Names, WHO)

* FDA, EMA, SFDA and /or DCGI(SFDA = China FDA;

DCGI = Drugs Controller General of India)[Biosimilars mAbs]

TheramAbs*


5


6


7ADCs

Panowski S et al, mAbs 2014

(A) Architectures (B) Mechanisms of Action


8Current Conjugation Strategies

(A) Lysines conjugation Gemtuzumab ozogamicin Trastuzumab emtansine

(B) Reduced native cysteines conjugation Brentuximab vedotin

(C) Engineered cysteinesconjugation Thio-trastuzumab

K. Lin (Genentech), Pharm Res, 2012Covalent IgGs Covalent +

Non covalentCovalent IgGs

A B C


9

(1) 1st generation ADC reaching the market

Mylotarg® (gentuzumab ozogamicin)(2000‐2010)


10Mylotarg®

O

O

O

IS

O

OHO

HO

O

HO

ONH

O

HOO

HO OHN

O

OSSN

H

O

N

OO

NH

O

ONO

O

O

Calicheamycin =(2 to 3/ IgG)

Gemtuzumab(HzIgG4SerPro)

-Lys-NH2 (random)

Linker-hydrazone-

Hughes B, Nature Drug Discovery

2010FDA approval: 2000Market withdraw: 2010

Gemtuzumab ozogamicin


11

Gemtuzumab ozogamicin: highly heterogeneous

(A) nrSDS‐PAGE

Labrijn et al, Nat Biotech 2009 (Genmab)

(B) IEF & cIEF

Laeda E et al, J Chrom A 2010 (Takeda)

+ PNGase F

+ PNGase F

50% of naked IgG

Mylotarg®


12

(2) 2d generation ADCs reaching the market

(A) Kadcyla® (ado trastruzumab emtansine): 2013=> Lys, maytansinoids (SMCC/SPP‐DM1, SPDB/sulfo‐DM4), ImmunoGen=> 12 in clinical trials

(B) Adcetris® (brentuximab vedotin): 2011=> Cys, auristatins (mcMMAE, vcMMAF), Seattle Genetics=> 24 in clinical trials


13

Cl

H3CO

H3CO

NH

O

N

H3C

OH

O

O

O

NO

S

ON

O

O

NHO

O

3.5

Ado trastuzumab emtansine (Roche/Genentech, ImmunoGen)

Wakankar A et al, Bioconj Chem 2010Beck A et al, Discovery Medecine 2010

FDA/EMA approval: 2013

Kadcyla®


14

T‐DM1 manufacturing: 2 step process

Wakankar A, AAPS 2010 (Genentech)

(A) (B)

Junutula JR et al, Clin Cancer Res 2010 (Genentech)

SMCC = N-succinimidyl-4-(N-maleimidomethyl)

cyclohexane-1-carboxylate

Linker

Kadcyla®


15T‐DM1 drug distribution (PK in Cyno)

T‐DM1 in cynomolgus monkey PK study by HER2 ECD affinity capture LC–MSShows the DAR distribution shifts to lower values over time (2 min to 28 days)• Kaur S (Genentech), Bioanalysis 2013

(A) 2 min (B) 3 days

(C) 28 days


16Adcetris®

NNH

HN

NH

O

OO

O

NH

NH2O

O O N

HN

NN

O

O

O

O O

NH

OCH3 O

HO

Brentuximab(chIgG1)

-Cys-SH (Hinge)

Peptide linker-Cit-Val-

Auristatin E =(4/ IgG)

Beck A et al, Discovery Medicine, 2010

FDA approval: 2011EMA approval: 2012

Brentuximab vedotin (Seagen/Takeda)


17Brentuximab 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


18Profiling ADC positional isomers

(UV)

(UV)

Reverse Phase‐HPLC

(UV, MS)

Hydrophobic Interaction Chrom (HIC)

nrCapillary Electrophoresis‐SDS

• Le LN, Anal Chem 2012 (Genentech)

Key QC method(Ab‐vcPAB‐MMAE)

Key QC method(Ab‐mcMMAF)

Native Hinge Cys (8, c/hIgG1)

Native

DenaturingDenaturing


19STEAP1 drug distribution (PK in Cyno)

Affinity capture HIC chromatogram of 100 μg/ml anti‐STEAP1 ADC in cynomolgousmonkey in vitro plasma stability samples.Formation of new odd numbered DARs(e.g., DAR1, DAR3 and DAR5) • Kaur S (Genentech), Bioanalysis 2013

(A) T0

(B) 96 H


20

Effect of drug loading on ADC clearance(A) MMAE‐Cys‐IgG (Seattle Genetics) (B) DM1‐Lys‐IgG (ImmunoGen)

Hamblett KJ et Al, Clin Cancer Res 2004 & 2006 McDonagh CF et Al, (Seattle Genetics) Prot Eng Design & Selection 2006


21

(3) 3d generation ADCs:

clinical and pre‐clinical stage

Abzena (Polytherics), Ambrx, Catalent (Redwood), Innate, Genentech, MedImmune, Novartis, Pfizer, Novartis, Seattle Genetics, Sutro Biopharma and

many more


22ThiomAbs (TDC)

• Damle NK, Nature Biotech 2008• Junutula JR, Nature Biotech 2008 Genentech


23ThiomAbs (TDC)

• Junutula JR, Nature Biotech 2008• Junutula JR, Clin Cancer Res 2010

Site specific conjugation on HC (Cys 114): improved homogeneity and therapeutic index• LC/MS profile for (A) TMAb‐mcc‐DM1 and (B)

thioTMAb‐mpeo‐DM1• ‘L’ is a linker without drug conjugated to antibody

(A) (B)

Genentech


24TDC

Evaluation of ADCs and antibody fluorophore conjugates (AFCs) as modelIdentification of additional conjugation sites in the LC, HC (Fd) and Fc of trastuzumab • engineered site‐specific thio‐trastuzumab variants for coupling to thiol‐reactive linkers• without perturbing antibody structure and function

Based on structural modeling, selection of 3 variants (LC‐V205C, HCA114C, Fc‐S396C)

The stability and superior in vivo efficacy of the LC‐V205C conjugate may be higher due to fastermaleimide ring hydrolysis, which prevented drug loss through the maleimide exchange from antibodyto thiol‐reactive constituents in the plasmaShen BK, Jutunula J et al, Nature Biotech 2012

20082012


25TDC

• Shen BQ, Junutula JR et al, Nature Biotech 2012 (Genentech)• Jeffrey S, Senter P et al, Bioconjugate Chem 2013 (Seattle Genetics): basic amino‐acids• Lyon R, Senter P et al, Nature Biotech 2014 (Seattle Genetics): self‐hydrolyzing maleimides• Tumey N et al, Bioconj Chem 2014 (Pfizer): mild Method for succinimide Hydrolysis

(A) Maleimide exchange from the antibody conjugate(B) Hydrolysis of succinimidering in the linker=> key steps that influence conjugate stability and therapeutic activity

(A) (B)

AlexaFluor-488-Maleimide

Retro-Michael reaction Succimide ring hydrolysis

+18 Da


26

h1F6-PBD(4)

h1F6-PBD(2)

Seattle Genetics / PBD‐based ADC


27Highly‐Potent warheads

• Chari R, Angew Chem 2014

(1) PBDs: Phase I (Seagen/Spirogen), Astra‐Zeneca (ADC‐T, Spirogen)

(2) Indolino‐BDs (IGNs, ImmunoGen): Ph I in 2015


28Optimized linkers

• Polar linker active against MultiDrug‐Resistant protein 1 (MDRP1)

(1) Polar sulfo‐SPDB and mal‐PEG4‐OSu linkers (ImmunoGen)

(2) Self‐hydolyzing maleimide (Seagen)

• Diaminopropionic acid• Basic amino‐group • Adjacent to the linker• Lyon R, Senter P, Nat Biotech 2014

• Chari R, Angew Chem 2014


29Site specific conj:

Panowski S et al, mAbs 2014


30

(2)CIPF/PFM

R&D, CD‐CMO


31

R&D of mAbs and ADCs for cancer treatments

Production Facility (cGMP)

CIPF St‐Julien en Genevois (France)

Integrated R&D Center

IGF-1R/dalotuzumab

CXCR4/hz515H7

c-Met

3 Clinical stage naked mAbs

+ confidential targets, mAbs &

ADCs programs

www.cipf.com

Cochet O et al, BioProcess Int 2013

(Abbvie)


32Pierre 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


33

Identity• Intact IgG/ADC : UHPLC‐ESI‐

TOF, CE‐SDS, SDS‐PAGE• Peptide mapping: UPLC‐ESI‐

TOF (UV), Ion Trap, MALDI‐TOF

• Immuno‐identification: ELISA, immunoblots

• Identification of cytotoxicpayload: UPLC‐ESI‐TOF

• N and Ct seq.: Ion Trap (ETD), MALDI‐TOF (ISD)

Quantity• Amino Acid Analysis• Protein content: UHPLC‐UV• Extinction coefficient:

calculation, UPLC‐UV• Protein quantif. : UV, BCA• Drug to mAb ratio &

distribution: HIC, CE‐SDS, UHPLC‐MS/UV, native MS

• Free cytotoxic drug: UHPLC‐MS/UV

Purity, integrity, stability• Mass distribution: UPLC‐ESI‐

TOF, CE‐SDS, SDS‐PAGE• Charge variants/ Isoforms: IEF,

cIEF, CEX, HIC• Aggregation/ Fragmentation:

SEC‐UV, Native MS, DSC• SEC‐MALS, A4F‐UV/MALS, DLS• Glycosylation: NP‐HPLC, UHPLC‐

UV, CE‐LIF, UPLC‐ESI‐TOF, MALDI‐TOF

• Disulfide bridges: CE‐SDS, SDS‐PAGE, UPLC‐MS

• Free thiol groups: Ellman• De‐amidation: CEX, UHPLC‐MS

Functional assays/ Potency• ELISAs• BIAcore (Ag, FcRs, FcRn, C1q,

Prot. A binding)• FACS• Cytotoxicity

IgG(150 KDa) Drug

(1.5 KDa)

ADC (avDAR3)

(154.5 KDa)

ADCs: analytical & structural platform


34Antibody Fluorophore Conjugates (AFCs)

NNH

HN

O

OO

O

NH

NH2O

NH

O O NHN

O

ON

NO

OCH3O OCH3ONH

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 NHN

O

ON

NO

OCH3O OCH3ONH

HOS



PABC MethylValine

Valine Dolaisoleucine Dolaproline Norephedrine

MMAE

Attachment groupProtease cleavable

linkerMMAE

Cytotoxic drug

NNH

HN

O

OO

O

NH

NH2O

NH

O O NHN

O

ONH

SO

O NS



PABC MethylValine

Fluorochrome

NNH

HN

O

OO

O

NH

NH2O

NH

O O NHN

O

ONH

SO

O NS



PABC MethylValine



PABC MethylValine

Fluorochrome

A

B

• Dansyl Sulfonamide Ethyl Amine (DSEA)‐linker maleimide

•Model of brentuximab vedotin + most of ADCs in clinical trials

Wagner E, Janin MC, Beck A et al, mAbs 2014


35

Cys‐linked ADCs DAR, load & distribution : work‐flowADC profiling: panel of orthogonal methods (covalent/ non‐covalent species)

Denaturing conditions (covalent)

Native conditions (covalent and non covalent)

(1)

SDS-PAGE/CE-SDSnon reducing (a)

reducing (b)

(2) RP-HPLC-MSreducing (a)

non reducing (b)IdeS digestion/ red (c)

IgGZERO/ red (d)

(3)HIC

(4)

Native MS

(+/- IgGZERO)

Le LN et al, Anal Chem 2013 (Genentech) Valliere-Douglas JR et al, Anal Chem 2012/ Anal Chem 2013 (Seagen) Rosati S, Parren PW, Heck AJ et al, mAbs 2013 (Genmab) Stojko J, Debaene F, Xuan Y, Bromirski M, Van Dorsselaer A, Beck A, Cianférani S, 2014

ADC


36

(A) SEC (before purification)

• Multimeric and monomeric species• Characterized by SDS‐PAGE, CE‐SDS, HIC, Native MS, LC‐MS (IdeS digestion + red.)• Relationship between aggregation 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%

250

150

100

75

50

37

2520

trastu

zumab

AFC monomers

AFC multim

erstra

stuzu

mabAFC m

onomers

AFC multim

ers

NR R

• Non Reducing• H2L2, H2L, H2, HL, H, L + payloads• Mono vs multi: different distribution

• Reducing• Increased MW (multimers vs mono.)• higher conjugation level (multimers)Wagner E, Janin MC, Beck A et al, mAbs 2014

(B) SDS‐PAGE

AFCs/ADCs


37Brentuximab 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


38

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

• HIC• Monomers: average DAR: 4.3 • Mutimers: high loaded forms

B

AU

-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

1491971953

1471851052

145173114

1512081252

153219488

2 %

26 %

2 Payloads

22 %

40 %

10 %

0 Payload

4 Payloads

6 Payloads

8 Payloads

Av. DAR: 4.4

• Native MS (200mM NH4Ac, pH7)• Intact bivalent H2L2 structure of AFCs• De‐glycosylated (IgGZero)• Relative distribution (drug loaded species)• Direct av. DAR determination

(A) HIC (B) NativeMS


AFCs/ADCs


39

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


40

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


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


41

• 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


AFCs/ADCs


42

(A) Trastuzumab (B) Trastuzumab-DSEA

IdeS cleavage suitable for1) mAbs (hIgG1 to 4)2) Fc‐fusion proteins

3) AFCs, ADCs

IdeS: IgGs and AFCs

Janin MC et al, Meth Mol Biol 2013Wagner E, Beck A et al, mAbs 2014 Fornelli L et al, Anal Chem 2014 Boeuf A, Structural Biol., in press

Beck A et al, Anal Chem 2012 & 2013 Beck A et al, Trends Anal Chem 2013 Ayoub D et al, mAbs 2013


43Developability

(1) Sequence liabilities• Hot spots (in silico)• Chromatographic and electrophoretic profilling (size, charge, hydrophob.)• Mass spectrometry (main isoform, micro‐variants structures)• Short stress studies (pH, heat, oxidation, glycation, light, freezing/thawing)• Functional assays (Critical Quality Attributes ranking)• Lead structure optimization (OptimAbs/OptimADCs): Hinge, pI, Glc…

(2) Safety/PK/PD• Serum stability• Half‐life• Administration schedule• Immunogenicity• Off‐target activity

(3) Manufacturability• Expression yields• Purification yields• Stability (process)• Stability (long term)• Cost of goods

CIPF’s: OptimAbs/ADCs developability platforms


44

(3)Summary


45OptimAbs/ADCs• Past decade: several hundreds of papers on mAbs analytical and structural characterization• Trend was amplified the last years

• Beck A, Cianferani S et al, Anal Chem 2012/2013; Beck A, Cianferani S et al, TRAC 2013• Beck A MiMB 2013; Beck A, Cianferani S 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


46AcknowledgementsCIPF, 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)


47SYNAPT 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


48Xevo 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

Manchester, Nov 20, 2014 (Waters ADC WS)

Thank you for your attention!alain.beck@pierre‐fabre.com


50Cover stories2008 2009 2010

2011

2012

2013

2013

2015


51

[email protected]

2009www.landesbioscience.com

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


52Glycovariants

Beck A, Wagner E, Ayoub D, Van Dorsselaer A, Cianferani S, Anal Chem 2013


53N‐Glycoforms

Beck A et al, Curr Pharm Biotech 2008 Beck A & Reichert JM, mAbs 2012

Beck A et al, Anal Chem 2013 Beck A, Meth Mol Biol 988, 2013


54Charge variants



55Cys‐linked variants



56Oxidized variants



57Size variants



58mAbs

LC/ HC(classical)

Fab/ Fc(functional

assays)

Fd/ LC/ Fc/2(screening)

Mass Spec Characterization flowchart


*IgG‐degrading enz. ofStreptococcus pyogenes(www.genovis.com)

*


59IgGs


Analytical and structural methods

• Trastuzumab• Rituximab• Cetuximab• Adalimumab• Bevacizumab

• Native MS• Ion Mobility‐MS• CESI‐MS/MS•Middle down/up• ETD


60Trastuzumab

Beck A et al, Anal Chem 2012

Trastuzumab = gold standard • Originator (1998)• Sub‐cutaneous, high concentration (2013)• Synergic combination (+ pertuzumab, 2012)• Antibody‐Drug Conjugate (T‐DM1) (2013)• Biosimilars (many clinical trials in progress)• Biobetters (e.g. low fucose)• Bispecifics (e.g. HER2 x VEGFA)• Biobetters of T‐DM1 (Ambrx, Synthon, Redwood…)


61

(A) Top: ESI‐Q‐TOF and (B) Native IM‐MS(1) Trastuzumab

Beck A, Cianferani S, Van Dorsselaer A, Anal Chem 2012


62ADC catabolism

• John Lambert, WADC Frankfurt 2014 (ImmunoGen)


63

HPLC / UHPLC / MALLS / DLS / A4F / Ultracentrifugation analytique

Aggregation

Boé JF, Beck A, Carrie A, Duhau L et al. STP Pharma Pratiques 2014


64ADCs ADMET

• Gorovits B et al, Bioanalysis 2013 (American Assosiation of Pharmaceutical Scientists ADC Working Group)

=> Roadmap lab # 5: « Bioanalyse »=> CIPF: Experim. Cancerology/ Physico‐Chimystry Dpts

ADCs bioanalysis: 6 keys parameters


65

ADC PK• mAb (mouse/cyno serum): ELISA• Capture protein: 5T4 • Detection antibody: • biotinylated goat anti‐human

kappa chain (mAb assay)• biotinylated anti‐MMAF

antibody (ADC assay)• Optical density: spectrophotom. • Released payload cys‐mcMMAF, • UPLC‐MS/MS system (5500

Qtrap, C18 column• cys‐mcMMAD as the Internal

standard) • LOD 0.002 ng/ml

(plasma)• LOD 0.1 ng/ml (tumor)

Sapra P et al, Mol Cancer Ther 2013 (Pfizer)


66

ADCs: analytics and bioanalytics


67

Pierre Fabre Oncology portfolio• 1989: Navelbine, the 1st chemotherapy to provide significant improvement in Nonsmall cell lung cancer (NSCLC) treatment that led to its registration in US and Europe• 1991: Navelbine registered in Metastatic Breast cancer (mBR)• 2001: Oral Navelbine, the 1st oral chemotherapy approved in Europe in NSCLC, thereafter approved in mBC, subsequently allowing patients to be treated at home• 2003: Busilvex a bone marrow transplantation conditioning treatment, co-developed and commercialized in Europe, Turkey, Middle-East and Latin America (Otsuka License)• 2004: a partnership agreement was signed with Merck (US). It covered a 1st mAb for the treatment of cancers, including Colon, Pancreatic, and Non-small cell lung cancers• 2009: Javlor, the 1st chemotherapy approved in Europe in advanced or metastatic transitional cell carcinoma of the urothelial tract after failure of prior platinum-containing regimens• 2010: a licensing agreement was signed with Abbott (US) to develop a 2nd mAbdiscovered by the Pierre Fabre researchers• 2013: a 3rd mAb CXCR4 has entered into phase 1 using pilot batches produced at the Pierre Fabre Immunology Centre• 2014: Aurigene (India) and Pierre Fabre licensing agreement for a new cancer therapeutic in immuno-oncology an immune checkpoint modulator targeting the PD-1 pathway (AUNP12 peptide)


68

Beck A, Wurch T, Bailly C, Corvaia N. Strategies and challenges for the next generation of therapeutic mAbs Nat Rev Immunol. 2010 Beck A, Reichert JM. Editorial: ADCs: Present and future. MAbs. 2014 Beck A, Haeuw JF, Wurch T, Goetsch L, Bailly C, Corvaïa N. The next generation of ADCs comes of age. DiscovMed. 2010 Cochet O, Corbière JC, Sinclair A, Monge M, Brown A, Eschbach G. A Sustainable, Single‐Use Facility for mAbsProduction. BioProcess International, 2013 Beck A, Wagner‐Rousset E, Ayoub D, Van Dorsselaer A, Sanglier‐Cianférani S. Characterization of therapeutic mAbs and related products. Anal Chem. 2013Wagner‐Rousset E, Janin‐Bussat MC, Colas O, Excoffier M, Haeuw JF, Rilatt I, Perez M, Corvaïa N, Beck A. ADCs fast characterization by LC‐MS. MAbs. 2013 Broyer L, Goetsch L, Broussas M. Evaluation of complement‐dependent cytotoxicity using ATP measurement and C1q/C4b binding. Methods Mol Biol. 2013 (A. Beck, Editor) Broussas M, Broyer L, Goetsch L. Methods Mol Biol. 2013 (A. Beck, Editor) Beck A. Review of ADCs, Methods in Molecular Biology series: A book edited by Laurent Ducry. MAbs. 2013 Beck A, Carter PJ, Gerber HP, Lugovskoy AA, Wurch T, Junutula JR, Kontermann R, Mabry R, Ghayur T. 8th European Antibody Congress 2012: November 27‐28, 2012, Geneva. MAbs. 2013 Beck A, Senter P, Chari R. World ADC Summit Europe: Feb 21‐23, 2011; Frankfurt, Germany. MAbs. 2011 Beck A, Lambert J, Sun M, Lin K. 4th WADC: Feb 29‐Mar 1, 2012, Frankfurt, Germany. MAbs. 2012 Klinguer‐Hamour C, Strop P, Shah DK, Ducry L, Xu A, Beck A. WADC, Oct 15‐16, 2013, San Francisco. MAbs. 2014

Publications: ADCs


69

Site specific conjugation (HC‐Cys 114): improved homogeneity & therap. index• Junutula JR, Nature Biotech 2008 – Nature Biotech 2012

ThiomAbs (TDC)

Genentech


70Site specific conjugation

• Perez HL et al, DDT 2014

Science

ADCs: Analytical and Bioanalytical Challenges