process development of bi -specific antibodies€¦ · prepare bi-specific antibodies • cmc...

Janssen Research & Development

Process Development of Bi-Specific Antibodies

Mark ChiuOn behalf of Janssen Research & Development & Genmab


Bispecific Antibody Technology

• Application of controlled Fab arm exchange to prepare Bi-specific antibodies

• CMC considerations for the development, characterizations, and manufacturing

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Rationale For EGFR/cMet Therapeutic Combination

Aberrant EGFR signaling is frequent driver of human cancer

cMet pathway activation frequently mediates resistance to anti-EGFR therapy Amplification of MET gene Increased cMet protein Stromal ligand secretion

Current combination approaches do not cover wide spectrum of resistance mechanisms

EGFR/cMet DuoBody approach will target multiple mechanisms of resistance

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EGF, TGFα, AREG, others

Janssen Research & Development 3

c-Met: A challenge to target effectivelyBivalent c-Met antibodies are agonistic

P P

Cell proliferationTumor development

PP

PM

• HGF receptor• Monovalent antibody format required to prevent agonism

After Genmab


P P

Cell proliferationTumor development

PP

PM

EGFRc-Met

• HGF receptor• Monovalent antibody format required to prevent agonism

• DuoBody cMet x EGFR• Overcome acquired resistance• Synergy: enhanced efficacy by dual targeting• Increased specificity

After Genmab

c-Met: A challenge to target effectivelyBivalent c-Met antibodies are agonistic


Genmab DuoBody Ab Technology

• DuoBody Abs are made by controlled Fab arm exchange of matched CH3 mutations

• K409R and F405L are destabilizing mutations in the CH3 interface

• The complementary mutations favor heterodimerization

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Resolution of Parentals from Bispecifics

6

Alternative chromatographic techniques to resolve bispecific from parentals

AU

-0.05

0.00

0.05

0.10

0.15

0.20

Minutes30.0 35.0 40.0 45.0 50.0 55.0 60.0

35.6

68

34.3

52

43.7

74

51.9

21

AU

-0.050

-0.040

-0.030

-0.020

-0.010

0.000

0.010

0.020

Minutes30.0 35.0 40.0 45.0 50.0 55.0 60.0

92%

3%5%

Bispecific DuoBody

Parental K409R

CEX

AU

-0.05

0.00

0.05

0.10

0.15

0.20

Minutes30.0 35.0 40.0 45.0 50.0 55.0 60.0

50.7

85

52.5

45

Parental F405L

Elution time (min)

HIC

92%

3% 5%97.8%1.2%

1%


DuoBody Generation is a Robust Process

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• Parental mAbs generated separately with downstream in vitro Fab arm recombination

• Bispecific (heterodimer) formation is typically greater than > 90%

0

10

20

30

40

50

60

70

80

90

100

F405L

DuoBody

K409R


EGFR x cMet : from the controlled Fab arm exchange of EGFR and cMet

EGFR binding

cMET binding

cMET mAb Bispecific Ab

RedOx

2-MEA

EGFR

cMET

2-MEA Reduced EGFR / cMet

EGFR / cMet

Reduction

UF/DF to remove MEA

OxidationEGFRxcMet

EGFR mAb

2-MEA= 2-Mercaptoethylamine

Low fucose

Low fucose


API Risks Based on Non-Platform DuoBody ProcessExpected Purication Scheme (Stage 4+)

Low pH VIN

VIN storage at -40°C

CNTO9809 DPC

CNTO4782 DPC

Low pH VIN

Thaw & Pool

UF/DF 1(Oxidation)

Capto S (B&E)

Capto Adhere (Flow-Through)

Viru

s Filt

ratio

n (P

lano

va)

UF/DF 2

Reduction

Formuled Bulk

Thaw & Pool

Platform

New Stages – Fab Arm Exchange

Platform

VIN (viral inactivation)• Address aggregation

observed at low pH• VIN process

mitigation influences scope of remaining steps

FAE (Fab Arm Exchange)• Minimize amount of 2-

MEA required (process contaminant, difficult to clear)

• Minimize free parental Abs (homodimers)

CaptoS• Identify operating

ranges to separate parental Abs from Bispecific Abs

CNTO G

CNTO F


Non-platform

Upstream

Downstream

Platform

FAE

Platform

Off Platform media

Platform Stage pushed forward

Lower yield with VIN after parental Ab DPC

Shift the stage for VIN step

In addition to mAb Process Purification process, need to remove each parental speciesand 2-MEA

Separation steps need to do more and more testing is required

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CNTO C

CNTO D


Another BsAbPlatform + FAE Upstream

Downstream

Platform

FAE

Platform

CNTO A

CNTO B

Capto SCEX

(B/E)

CaptoAdhere

(FT)

Platform process is adapted to meet cFAE

step without further modifications

In the end…the molecule rules!

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Potential to reduce 16 disulfide bond in an IgG1 scaffold, not just the ones you are interested in….

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2 - MEA (cysteamine hydrochloride)

• Mild reducing agent• Supplier: Sigma (SAFC), 98.0 % purity• Derived through chemical synthesis, certified non-animal origin• Country of manufacture: Switzerland / China• Detection method is accurate and precise to 1 µM• LOQ predicted to be 1-2 µM sufficient for release development• Hygroscopic: Special handling required (store 4-8 oC, bring to RT to

open, limited reuse of opened containers)• Weakly mutagenic - need to show removal and explore alternative mild

reducing agents as appropriate (1-10 µg/day dose limit)

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2-MEA (Cysteamine) Stability

~ 8 % decrease over 14 days ~ 4 % decrease over 14 days

50 mM Sodium Phosphate/150 mM NaCl pH 6.7 50 mM Sodium Acetate pH 5.0

2-MEA does not degrade significantly over 14 days post solution preparation, regardless of storage temperature

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Confirmation of BsAb PreparationHIC assay after 2-MEA (80 mM) x (5 h), 20oC, pH 6, > 90% purity

EGFR cMET

BsAb

EGFR cMET


Small Scale Fab Arm Exchange – cFAE

• 96 well plate• 10 kDa MWCO to remove 2-MEA and oxidize • Bispecific IgG measured by analytical HIC- HPLC assay• Conditions Screened

– pH 6.0 and 7.5– 50, 60, 70, 80, 90, 100 mM 2-MEA– 2, 4, 5, & 6 h time points– Temp 16°C and 26°C


BsAb cFAE Contour Plots

2-MEA StabilityOptimal Operating Space

cFAE Conditions Optimal Operating Space

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Predicted Disulfide Bond Structure of BsAb

SSS

S

EGFR cMET

EGFR x cMet BsAb

VL

CH1

CL

VH

CH2

CH3

BsAb structure has 16 disulfide bonds

CH2

CH3

CH1

VH

VL

CLConfirmation of cFAE process control

• Free Thiol Analysis -- detect/quantify unpaired Cysteine

• Fab/Fc Mass Analysis -- detect LC swapping

• Non-reduced Peptide Map -- detect disulfide-bonded peptide pairs


Free Thiol Analysis of BsAb and Parental Abs

Sample Mean Free SH / Protein

BsAb 0.26

EGFR 0.19

cMet 0.13

• Use HPLC method based on fluorescent dye labeling of free thiols (DACM, N-(7-Dimethylamino-4-Methylcoumarin-3-yl))Maleimide)

• Observed free thiol values for BsAb and the parental Abs EGFR and cMetwere typical for commercial IgG products


Fab, Fc Mass Analysis use to detect LC Swapping

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Cleave(HNE)

Cleave(HNE)

BsAb

Fab EGFR Fab cMet Fc BsAb

If present, LC swapping will be detected due to new Fab species


Fab, Fc Mass Analysis of BsAb and Parental Abs

Peak ID Theoretical Mass (Da)

Measured Mass (Da)

BsAb EGFR cMet

Fc BsAb 52795 52795 ND ND

Fc cMet 52857 ND ND 52857

Fc EGFR 52733 ND 52733 ND

Fab EGFR 48092 48092 48092 ND

Fab cMet 47357 47357 ND 47357

• Fab EGFR and Fab cMet are readily separated and identified by RP-HPLC/ESI-MS

• BsAb yields large peaks for both Fab cMet and Fab EGFR; no peaks or masses corresponding to LC swapping were detected

• Analysis of BsAb yielded a major peak for Fc BsAb; no species corresponding to Fc EGFR or Fc cMet were detected

• Other peaks are observed in RP-HPLC/ESI-MS as a result of nonspecific cleavage by human neutrophil elastase


BsAb Technology Summary

• Twice the fun, Twice the specificity , Twice the work …until we platform bispecific Ab technologies!

– More complex characterization to ensure structure/function– New steps to develop (FAE, Purification, VIN, Hold Steps)

• Bispecific Antibody Technologies will help address unmet needs in complex disease areas by attacking multiple targets an engaging our own body mechanisms to fight disease

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Questions?


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