Quartier du Rieu - Impasse des Chênes Rouges - 30319 Alès cedex - FrancePhone: +33 (0)4 66 56 40 80 - Fax: +33 (0)4 66 56 40 89

E-mail: LFB-Biomanufacturing@lfb.frwww.lfbbiomanufacturing.com

EMABling® Antibody Production Platform

An industrial solution for the production of therapeutic

antibodies with high cytotoxic activity

B I O M A N U F A C T U R I N G

EMABling®: A fully integrated development platform for the production of highly active, glyco-engineered monoclonal antibodies (mAbs) with high antibody-dependent cellular cytotoxicity (ADCC)

For more than 20 years, LFB has been focusing its research effort on the study of structure-function relationship of antibodies. Its studies on the molecular basis of IgG interaction with the receptors for the Fc portion of IgG (FcRs) has made it possible to develop antibodies with enhanced affinity to FcRIIIa (CD16) and thus high ADCC activity, both correlated to a glycosylation pattern characterized by a low fucose content*. Based on these studies, LFB has developed EMABling®, a technological platform for the production of antibodies with enhanced cytotoxicity activity.

* covered notably by US 7,931,895 , US 8,685,725 B2 and EP 1272527 patents, owned by LFB SA.

The EMABling® technology improves the efficacy of the mAbs by enhancing cellular functions suchas ADCC and phagocytosis.

Glyco-engineering provides significant improvements over existing products or addresses unmet medical needs in such areas as cancer. It also represents a good life cycle management tool for Biopharma companies to extend IP protection of their marketed antibodies with short-term patent expiry (biobetter mAbs).

Cytotoxic activity of EMABling® mAb

Glyco-engineered mAbs with improved antibody-dependent cell-mediated cytotoxicity (ADCC) are now reaching the market. Two EMABling® antibodies have entered clinical development including:

• LFB-R593 (Roledumab), a fully human anti-rhesus D (RhD) mAb in clinical phase II, for the prevention of feto-maternal allo-immunization in RhD-negative women, as a substitute for human polyclonal anti-RhD immunoglobulins

• LFB-R603 (Ublituximab), a monoclonal antibody directed against CD20 in clinical phase II, for the treatment of hematology disorders (licensed to TG Therapeutics, Inc.).

This demonstrates that EMABling® technology to improve cytotoxicity of mAbs is an efficient tool for optimizing therapeutic mAbs.

Proven clinical efficacy of glyco-engineered EMABling mAbs

GlcNac

ManFuc Gal Sialic AcidGlycan

Effector cell

IL2

Target

CD16

Mab

Perforin/granzymes

ADCC(NK CELLS)

Phagocytosis(Macrophages)

anti-Rhesus D EMABling

anti-Rhesus Dpolyclonal

anti-Rhesus D CHO

EMABling® Cell line development (CLD) platform

LFB proprietary optimized expression vector

YB2/0-E cell line

• LFB Proprietary signal peptide

• Proprietary cassette vector enabling cloning of Ig variable regions or full length heavy and light chains

• Optimized electroporation conditions giving the best transfection efficiency and recovery rate

• Royalty-free for our customers

The YB2/0 cell line is derived from a non Ig-secreting hybridoma (no rat Ig heavy or light chain) that naturally produces low amount of FucosylTransferase FUT8 leading to low-fucose levels of the expressed therapeutic antibodies.The YB2/0-EMABling® (YB2/0-E) cell line was derived from YB2/0 cells by adaptation to agitation and suspension culture conditions in a chemically defined, animal-derived component-free (ADCF) medium, called EMABPro1. The cell line has been fully characterized according to ICH guidelines and is compatible with the GMP production of recombinant therapeutic molecules.

IP: Patents

Optimized Expression vectors

IP: Know-how

CLD Process Selection for high

producers

Validated PSS

Cloning

Gene Amplification

Transfection

YB2/0-EMABPro1

+ + ➡

IP: Patents & Know-how

EMABling mAb High ADCC activity

> 1 g/l

IP: Know-how and R&D/commercial rights

Host Cell Line YB2/0-EEMABPro1 medium

Res T

Us HC TU

LC TU

Dhfr TU

LFB Expression vector

LFB Expression vector

An optimized screening process has been established to select clones suitable for an industrial development. After the transfection, several thousand clones are screened using the high throughput screening ClonePix™ system. The clones are then amplified and finally tested in ambr™ microscale bioreactors in Fed-Batch mode to select the top 10-12 clones which will then be gene amplified.

It is important to note that the whole CLD is performed using the same optimized serum-free EMABPro1 medium.

ClonePix FL™ and ambr™ are trade marks of Genetix and TAP Biosystems, respectively.

PROJECT A PROJECT B

Examples of clone selection in generic fed batch mode

CLD selection process for highly producing clones

Final IgG titer for two different antibodies (unrelated targets): 12 clones / project were assessed for response to Fed-batch mode. Most of clones respond to generic Fed-batch process (red bars) by increasing titer up to more than 3 times, compared to batch mode (blue bars), and showed titers above 1 g/L.

ClonePix FL ™

ambr™

1210

2 3 4 5 6 7 8 9 10 11

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400

1500

Batch mode Fed-batch mode

IgG

tite

r (m

g/L)

Batch mode Fed-batch mode

1210

2 3 4 5 6 7 8 9 10 11

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400

1500

IgG

tite

r (m

g/L)

15 L15 mL 50 L

50 L 250 L 1000 L 2000 L (soon)

A key parameter is the predictibility of the scaling-up of batch manufacturing from Pilot to larger GMP-scale.

At LFB BIOMANUFACTURING we have established a USP strategy using the 50L disposable bioreactor as a perfect tool for establishing the industrial cell culture parameters.

GMP MANUFACTURING

GLYCOSYLATION PROFILE

• % fucose of Ab measured in supernatant (in-house ELISA)

• Complete glycosylation profile on selected purified antibodies (High-performance capillary electrophoresis (HPCE) with laser-induced native fluorescence (LIF) detection or mass spectrometry (MS))

BIOLOGICAL ACTIVITY

• CD16 binding (target-independent ELISA assay)

• Effector cell-based assays (target-dependent assays to be developed for each new target)

1 Cytokine secretion

2 ADCC

3 Phagocytosis (ADPC)

STANDARD ANALYTICAL TECHNIQUES FOR BIOLOGICS AVAILABLE

Analytic capabilities to study structure and function of EMABling® antibodies

USP PLATFORM – From Lab-scale to large-scale manufacturing

Effector cell

IL2

1Cytokine secretion

Jurkat CD16 2ADCC

(NK CELLS)

PMA

Target

CD16

Mab

3Phagocytosis

(Macrophages)

Perforin/granzymes

PROCESSDEVELOPMENT(non-GMP)

OC

TOBE

R 2

014

- D

esig

n &

layo

ut :

Pasa

ya

Quartier du Rieu - Impasse des Chênes Rouges - 30319 Alès cedex - FrancePhone: +33 (0)4 66 56 40 80 - Fax: +33 (0)4 66 56 40 89

E-mail: LFB-Biomanufacturing@lfb.frwww.lfbbiomanufacturing.com

B I O M A N U F A C T U R I N G

EMABling® Technology Key Advantages

• A fully integrated development program for the production of glyco-engineered, biological activity-improved mAbs

• Suitable for poorly expressed antigen targets on the cell surface

• Generation of stable clones directly adapted to Fed-batch conditions in an ADCF optimized medium

• Dedicated QC tests and potency assays

• Proven scalability for industrial development

• Demonstrated clinical efficacy of EMABling® antibodies

Access to EMABling® Technology: two complementary services

Generation of research-grade purified mAbs from stable pools

• Production of stable pools of recombinant YB2/0-E cells

• Rapid delivery of sufficient product for early-stage functional assays

• Generation of recombinant CHO version to perform comparison studies

• No licence agreement required

• Intermediate step of full cell line development (generation of Intermediate Cell Banks): optimization of risk and cost management

Full cell line development

• Generation of stable clones adapted to fed-batch conditions in an in-house ADCF medium

• Proven scalability

• Production of GMP Master and Working Cell Banks

• High productivity (> 1g/L)

• License agreement required