ADCs: Analytical and Bioanalytical Challenges

Manchester, Nov 20, 2014 (Waters ADC WS)
ADCs: analytical & bioanalytical
challenges
Alain Beck, PhD
Senior Director, Antibody Physico‐Chemistry
Centre d’Immunologie Pierre Fabre, FR
Associate Editor, mAbs

Waters ADC WS, Nov 20‐21, 2014, Manchester Alain BECK, PhD
2
Outline
(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 131
I‐tositumomab[mIgG2a]
(CD20) )[withdrawn 2014]
omalizumab(IgE‐Fc)
efalizumab(CD11a)[withdrawn, 2009]
alefacept[PrFc]
(CD2)
2002 111
In/90
Y‐ibritumomab tiuxetan[mIgG1]
(CD20)
adalimumab(TNF)
2001 alemtuzumab(CD52)
2000 gemtuzumab ozogamicin[IgG4s‐calicheamycin]
(CD33)[withdrawn 2010]
1999
1998 basiliximab(CD25)
palivizumab(RSV‐F)
infliximab(TNF)
trastuzumab(HER2)
etanercept[PrFc]
(TNF)
Technologies 1997 rituximab(CD20)
daclizumab(CD25)
Transgenic mice (10Y) 1996
1995 edrecolomab[mIgG2a]
(EpCAM)[Ger, withdrawn]
1994 abciximab[Fab]
(GPIIb)
Phage display (9Y) 1993
Humanization (11Y) 1986 muromomab[mIgG2a]
(CD3)
Chimerization (10 Y) 1984 Nobel Prize for mAbs
> 50
approved
mAbs, 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 cysteines
conjugation
 Thio-trastuzumab
K. Lin (Genentech),
Pharm Res, 2012Covalent IgGs Covalent +
Non covalent
Covalent IgGs
A B C

9
(1)
1st generation ADC reaching the market
Mylotarg® (gentuzumab ozogamicin)
(2000‐2010)

10
Mylotarg®
O
O
O
I
S
O
OH
O
HO
O
HO
O
N
H
O
HO
O
HO O
H
N
O
O
S
SN
H
O
N
O
O
NH
O
O
N
O
O
O
Calicheamycin =
(2 to 3/ IgG)
Gemtuzumab
(HzIgG4SerPro)
-Lys-NH2 (random)
Linker
-hydrazone-
Hughes B,
Nature Drug
Discovery
2010
FDA approval: 2000
Market 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
N
H
O
N
H3C
OH
O
O
O
N
O
S
O
N
O
O
N
HO
O
3.5
Ado trastuzumab emtansine (Roche/Genentech, ImmunoGen)
Wakankar A et al, Bioconj Chem 2010
Beck 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–MS
Shows 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®
N
N
H
H
N
N
H
O
O
O
O
NH
NH2O
O O N
H
N
N
N
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: 2011
EMA 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 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

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 cynomolgous
monkey 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

22
ThiomAbs (TDC)
• Damle NK, Nature Biotech 2008
• Junutula JR, Nature Biotech 2008
Genentech

23
ThiomAbs (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

24
TDC
Evaluation of ADCs and antibody fluorophore conjugates (AFCs) as model
Identification 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 faster
maleimide ring hydrolysis, which prevented drug loss through the maleimide exchange from antibody
to thiol‐reactive constituents in the plasma
Shen BK, Jutunula J et al, Nature Biotech 2012
20082012

25
TDC
• 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 succinimide
ring 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

27
Highly‐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

28
Optimized 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)
CIPF
St Julien
CRDPF
Toulouse
P&I
Gaillac
API
Pau

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 cytotoxic
payload: 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, FcRs, 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)
N
N
H
H
N
O
O
O
O
NH
NH2O
N
H
O O N
H
N
O
O
N
N
O
OCH3O OCH3O
N
H
HO
S
Maleimide Caproic acid
Maleimidocaproyl Valine Citruline
PABC Methyl
Valine
Valine Dolaisoleucine Dolaproline Norephedrine
MMAE
Attachment group
Protease cleavable
linker
MMAE
Cytotoxic drug
N
N
H
H
N
O
O
O
O
NH
NH2O
N
H
O O N
H
N
O
O
N
N
O
OCH3O OCH3O
N
H
HO
S
PABC Methyl
Valine
Valine Dolaisoleucine Dolaproline Norephedrine
MMAE
Attachment group
Protease cleavable
linker
MMAE
Cytotoxic drug
N
N
H
H
N
O
O
O
O
NH
NH2O
N
H
O O N
H
N
O
O
N
H
S
O
O
NS
PABC Methyl
Valine
Fluorochrome
N
N
H
H
N
O
O
O
O
NH
NH2O
N
H
O O N
H
N
O
O
N
H
S
O
O
NS
PABC Methyl
Valine
PABC Methyl
Valine
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‐flow
ADC profiling: panel of orthogonal methods (covalent/ non‐covalent species)
Denaturing conditions
(covalent)
Native conditions (covalent
and non covalent)
(1)
SDS-PAGE/CE-SDS
non reducing (a)
reducing (b)
(2)
RP-HPLC-MS
reducing (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
25
20
trastuzum
ab
A
FC
m
onom
ers
A
FC
m
ultim
ers
trastuzum
ab
A
FC
m
onom
ers
A
FC
m
ultim
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)
(B) SDS‐PAGE
AFCs/ADCs

37Brentuximab vedotin
3.288
3.826
4.746
5.629
6.564
8.159
9.311
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
Minutes
2.00 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)
Average
DAR = 4.0

38
2,953
4,477
5,784
6,090
6,565
8,730
10,257
AU
-0,10
0,00
0,10
0,20
0,30
0,40
0,50
0,60
0,70
0,80
0,90
Minutes
2,00 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
Minutes
2.00 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
149197
1953
147185
1052
145173
114
151208
1252
153219
488
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)
Native
MS
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
AFCs/ADCs

40


 1
0
1
0
1
)(
nLC
LC
A
An
LCDAR


 3
0
3
0
)(
n
n
Fd
Fd
A
nA
FdDAR ))()((2 FdDARLCDARDAR 
UV chromatogram at 210nm:
Fd1
Fd2 Fd3
Time
(min)
10.00 14.00 18.00 22.00 26.00
%
20
100
Ionchromatogram
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
Theoritical
MWav
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/expExperimental
MW
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 twice
in 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
L0
Fc/2 Fd0
AU
0.0
1.0e-2
2.0e-2
L0
Fc/2 Fd0
Time
10.00 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
L0
12%
Fd1
3%
Fd2
16%
Fd3
72%
Fd4
9%
L1
75%
Fc/2
L2
13%
Time
10.00 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
L0
12%
Fd1
3%
Fd2
16%
Fd3
72%
Fd4
9%
L1
75%
Fc/2
L2
13%
Av. DAR = 3.8
Av. DAR = 7.8
A
B
C
0.0
1.0e-2
2.0e-2
a ge 6 5e
L0
32%
L1
68%
Fd1
53%
Fd0
16%
Fd2
26% Fd3
5%
Fc/2
0.0
1.0e-2
2.0e-2
a ge 6 5e
L0
32%
L1
68%
Fd1
53%
Fd0
16%
Fd2
26% Fd3
5%
Fc/2
Monomeric AFC
Multimeric AFC
Trastuzumab
AFCs/ADCs

42
(A) Trastuzumab (B) Trastuzumab-DSEA
IdeS cleavage suitable for
1) mAbs (hIgG1 to 4)
2) Fc‐fusion proteins
3) AFCs, ADCs
IdeS: IgGs and AFCs
 Janin MC et al, Meth Mol Biol 2013
 Wagner 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

43
Developability
(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

45
OptimAbs/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

46
Acknowledgements
CIPF, 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)
2014
ADC
• 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

48
Xevo 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

50
Cover stories
2008 2009
2010
2011
2012
2013
2013
2015

51
alain.beck@pierre-fabre.com
2009
www.landesbioscience.com
IF: 5.275 (2012), 3.174 (2011)
Anal Chem (5.695), Anal Biochem (3.247)

52
Glycovariants
 Beck A, Wagner E, Ayoub D, Van Dorsselaer A, Cianferani S, Anal Chem 2013

53
N‐Glycoforms
 Beck A et al, Curr Pharm Biotech 2008
 Beck A & Reichert JM, mAbs 2012
 Beck A, Meth Mol Biol 988, 2013

54
Charge variants

55
Cys‐linked variants

56
Oxidized variants

57
Size variants

58
mAbs
LC/ HC
(classical)
Fab/ Fc
(functional
assays)
Fd/ LC/ Fc/2
(screening)
Mass Spec Characterization flowchart
*IgG‐degrading enz. of
Streptococcus pyogenes
(www.genovis.com)
*

59
IgGs
Analytical and structural methods
• Trastuzumab
• Rituximab
• Cetuximab
• Adalimumab
• Bevacizumab
• Native MS
• Ion Mobility‐MS
• CESI‐MS/MS
• Middle down/up
• ETD

60
Trastuzumab
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

62
ADC 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

64
ADCs 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 Non
small 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 mAb
discovered 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. Discov
Med. 2010
 Cochet O, Corbière JC, Sinclair A, Monge M, Brown A, Eschbach G. A Sustainable, Single‐Use Facility for mAbs
Production. 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. 2013
 Wagner‐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

ADCs: Analytical and Bioanalytical Challenges

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