Development of a purification platform process for a unique next generation antibody, the

κλ-body

• JF Depoisier, N Fouque, S Darthenay, A Carron, L Dejoint, L Di Grazia, PA Cayatte, M Alcox, G Pontini,

Y Poitevin, S Raimondi, L Bernasconi, D Schrag, N Fischer, M Kosco-Vilbois, G Elson

• Novimmune SA, 14 Chemin des Aulx, 1228 Plas-Les-Ouates, Geneva, Switzerland

• Presented at CHI-PepTalk, Palm Springs, CA, USA, 21 – 25 January 2013

1. Two common heavy chains

2. Two different light chains: kappa and lambda

Light chain-driven specificity

3. Full length IgG1 format

Fc-effector functions and stability

4. Absence of linkers or unnatural sequences

Indistinguishable from a standard IgG

κλ-body format

Abstract

• In order to exploit novel mechanisms of action and achieve superior clinical efficacy, various bispecific antibody formats have been developed over the past decade.

• NovImmune’s novel bispecific antibody format, the κλ-body, has a molecular structure similar to standard monoclonal antibodies.This poster is addressing challenges to design a platform purification process for this novel bispecific antibody format.

High level κλ–body expression

• A: κλ-bodies are expressed in CHO cells by co-transfection of the genes encoding the common heavy chain and the two

different light chains

• B and C: two different κλ-bodies (POCA and B) presented a high level of expression (following a limited cell line screening

protocol) while the κλ-body distribution was close to the theoretical maximum of 50%

• D: Seven randomly selected CHO cell lines presented a high level of genetic stability over 50 generations

• E: Robust scale-up of a semi-stable pool expressing the κλ-body POC A from 100 mL erlen to 25 L Wave bag in a chemically

defined fed-batch process

κλ-bodies are expressed at high productivity and consistency by standard CHO

expression systems

(B)

Stability study of 7 cell lines

0

10

20

30

40

(D)

50

Cell line generation number

% κ

λ -b

ody A

at

ha

rve

st

0

20

40

60 20.92% 43.72 % 35.36 %

%

38.81% 45.75% 15.44 %

0

20

40

60

κλ-body B

%

κλ t

iter

(g/L

)

mA

b t

ite

r (g

/L)

Via

ble

Cell C

on

ce

ntr

atio

n

(x1

06ce

lls/m

L)

an

d V

iab

ility (

%)

Theoretical distribution

Stable CHO cells (A)

(C) Antibody titer at harvest by fed-batch culture

0

1

2

3

0

0.5

1

1.5

2

0

20

40

60

80

(E)

0 48 96 144 192 240 288 336 384

Elapsed time (h)

Scale-up of fed-batch process

Wave bag - VCC Wave bag -

Viability Wave bag -

mAb titer

Erlen - VCC Erlen -

Viability Erlen -

mAb titer

% κκ, κλ, λλ % κκ, κλ, λλ

κλ-body A

100

Initial purification platform

• «Version 0» process – Separation of monospecific IgGs and κλ-body based on a three affinity step platform process

• A: The bispecific antibody was purified from CHO derived supernatant using a three affinity step process, consecutively involving the MabSelect SuRe, KappaSelect and LambdaFab Select resins (GE Healthcare)

• B: Non-reduced SDS PAGE analysis revealed efficient purification of the κλ-body with the removal of major process contaminants, notably the two monospecific mAbs κκ and λλ

Mabelect SuRe 2

Flow through: HCPs, DNA, etc

Free light

chains

5 Eluate:

κλ-body Mono κ

KappaSelect

Eluate:

κλ- body

6

Flow through:

Mono κ

LambdaFabSelect

Mono κ

3 Eluate:

Mono λ κλ- body

1 Supernatant

7

4

Flow through:

Mono λ

(A)

188

62

49

28

17

14

6

38

M

HC

λ LC κ LC

Abbreviations: HC: Heavy chain; κ LC: kappa light

chain; λ LC: lambda light chain;

M: Molecular weight marker

1 2 3 4 5 6 7

(kDa)

(B)

98

Purification process optimization

• Further process development on KappaSelect and LambdaFab Select resins.

• Optimization of the running conditions, including elution pH, flow rate, and loading capacity was shown to be critical in establishing robust and efficient operating conditions for the KappaSelect (A and B) and LambdaFabSelect resins (C and D).

1X 1.33X

60%

65%

70%

75%

85%

80%

90%

95%

100%

3.2X

1.6X

1X

100.0%

88.9%

81.5%

94.6%

86.5%

79.0%

% R

ecovery in

kl-b

od

y

(B)

KappaSelect

1X 1.5X

2X

60%

65%

70%

75%

80%

85%

90%

95%

100%

4X

2X

1.25X

1X

NT

90.8% 87.8%

90.1%

79.8%

100.0%

82.0%

73.4%

95.4%

83.8%

77.4%

NT

% R

ecovery in

kl-b

od

y

LambdaFabSelect

(D)

0

100

0 50 100 150 200 250 ml

Equili

bra

tio

n

Loadin

g

Washin

g

Elu

tion

F2 W aste F3 W aste

Mono κ

κλ- body

lambdaselect cycle5 20110902:10_UV lambdaselect cycle5 20110902:10_Conc lambdaselect cycle5 20110902:10_Frac tions lambdaselect cycle5 20110902:10_Logbook

200

300

400

(C)5

00

600

mAU

Equili

bra

tio

n

Loadin

g

Washin

g

Washin

g

Elu

tion

Str

ip

Mono λ

run 10 column5mlph3001:10_UV run 10 column5mlph3001:10_Conc run 10 column5mlph3001:10_Frac tions run 10 column5mlph3001:10_Logbook

0

500

1000

1500

2000

mAU

0 50 100 150 ml

F2 W aste F3 W aste F4 W aste

κλ- body

Mono κ

(A)

Mono κ

KappaSelect

LambdaFabSelect

Scale-up at 100L pilot

Representative chromatograms of ligand

affinity steps at pilot scale.

MabSelectSuRe

KappaSelect

LambdaFabSelect

Capture

HIC-HPLC analysis of unprocessed bulk

(A)

LambdaFabSelect step: •Column size: 760 mL (7cm Ø, ~20 cm height) •Loading: 20g/L IgG •Step recovery: 93% (k l-body)

(C)

NI-1501, POC1B, NI-1501-007, LambdaFab S, Cycle 2

KappaSelect step: •Column size: 380 mL (5cm Ø, ~20 cm height) •Loading: 20g/L IgG •Step recovery: 91% (k l-body)

(D) NI-1501, POC1B, NI-1501-007, Kappa S, Cycle 2

MabSelectSure step: •Column size: 805 mL (7cm Ø, ~20 cm height) •Loading: 20g/L IgG •Step recovery: 94% (k l- body)

(B) NI-1501, POC1B, NI-1501-007, MabSS, Cycle 1

Kappa and LambdaFabSelect resin can be used as

first or second step depending on predominent % of

monospecific.

Product quality analysis of purified κλ-body

Purified κλ -body demonstrated a quality profile typical of conventional monoclonal antibodies as illustrated by reduced SDS-PAGE gel (Panel A), HIC-HPLC (Panel B) and SEC-HPLC (Panel C) analyses

(B)

HIC-HPLC

κλ- body

(C)

SEC-

HPLC

(A)

Reduced SDS-PAGE

Mono κλ- Mono

κκ body λλ

Purity >99%

λ LC κ LC

HC Mono κ Mono λ

% κλ >99 % Aggregates: 1.2%

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

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00

Minutes

Monomers

Mono κ

κλ-body

Aggregates

Zoom Mono λ

Conclusions and perspectives

• κλ-bodies were shown to be readily expressed at high level (above 1g/L) using a standard CHO expression system.

• A three-affinity step purification process was shown to be successful in isolating high quality κλ-body.

• Platform purification process development allowed the determination of robust running conditions with the KappaSelect and LambdaFab Select resins.

• Scale–up at a 100L pilot scale has been successfully performed.

• Further process development have been initiated to reduce the ligand affinity step numbers whilst maximising the product recovery and process efficiency.

Take home message

The κλ-body represents the ideal bispecific format as it is indistinguishable from a standard fully human

monoclonal antibody.

It therefore has all the favorable characteristics required, such as platform manufacturability, stability, extended half-life,

effector function and low intrinsic immunogenicity, in order to become a successful class of

next generation biologics.