Majid Mehtali - Tecanww3.tecan.com/platform/content/element/4801/Tecan...955 million chicks & 30...

1

Duck Embryonic Stem CellsMajid Mehtali

ZZüürich, September 18rich, September 18thth 20082008

2

VIVALISNantes, France

VIVALISVIVALISNantes, FranceNantes, France

Founded in 2000, ~65 FTEs, based in Nantes

Public company, Listed on NYSE-Euronext-Paris (July 2007)

Raised €46 millions

Focus on infectious diseases & therapeutic Proteins

Mission:To develop drugs to treat viral diseases & to provide innovative cell-based solutions for the industrial manufacture of viral vaccines & therapeutic proteins

22 licensees & 5 collaborations & manufacturing contracts

GMP manufacturing facility for phase I & II trials

A subsidiary of group Grimaud

Founded in 2000, ~65 FTEs, based in NantesFounded in 2000, ~65 FTEs, based in Nantes

Public company, Listed on NYSEPublic company, Listed on NYSE--EuronextEuronext--Paris Paris (July 2007)(July 2007)

Raised Raised €€46 millions46 millions

Focus on infectious diseases & therapeutic ProteinsFocus on infectious diseases & therapeutic Proteins

Mission:Mission:To develop drugs to treat viral diseases & to provide innovative cell-based solutions for the industrial manufacture of viral vaccines & therapeutic proteins

22 licensees & 5 collaborations & manufacturing 22 licensees & 5 collaborations & manufacturing contracts contracts

GMP manufacturing facility for phase I & II trialsGMP manufacturing facility for phase I & II trials

A subsidiary of group GrimaudA subsidiary of group Grimaud

VivalisCorporate Overview

3

For over 45 years, a leader in avian, rabbit & pigs genetic selection2 million birds raised permanently100 million eggs incubated/year55 million chicks & 30 millions ducklings hatched/year Sales in >60 countries

Industrial & technical know-how in animal health

World’s second largest company in its field1400 collaborators worldwideturn-over: 200 million €

GROUPE GRIMAUDVIVALISFILAVIE

GRIMAUD ITALIA

GRIMAUD BRINKMANN

GRIMAUD CHINA

GRIMAUD MALAYSIA

GROUPE GRIMAUDVIVALISFILAVIE GRIMAUD ITALIA

GRIMAUD FARMS OF CALIFORNIA

GRIMAUD CHINA

GRIMAUD MALAYSIA

HUBBARD BRASIL

HUBBARD

HUBBARD POLAND

Group GrimaudAn industrial & Financial Back-up

The EggA Breakthrough Technology

5

Building on E. Goodpasture legacy, a very wide range of viral vaccines can be produced on avian Cells

Ernest Goodpasture (1886-1960), the founder of mumps vaccine, discovered in 1933 the first practical method for developing uncontaminated viruses in chick embryos, which made possible the mass-production of vaccines for such diseases as smallpox, influenza, yellow fever, typhus, Rocky Mountain spotted fever, and other illnesses caused by agents that can be propagated only in living tissue

The Chicken Egg Production PlatformA Breakthrough Technology in the 1930’s

6

Human & Animal Vaccines produced on eggs & chicken cells

Traditional vaccines Recombinant vaccinesInfluenza virus (avian, equine, porcine) Rabies (Poxvirus)

Rabies Feline leukemia (Poxvirus)Poxvirus diseases (chicken & canarypox) Canine distemper (Poxvirus)

Newcastle Disease Virus (avian) West Nile (Poxvirus)Herpes virus (falcon, pigeon, turkey, psittacine) Equine Influenza (Poxvirus)

Infectious bursal disease (avian)Infectious bronchitis virus (avian)Marek's disease virus (avian)

Reovirus (avian)

Chicken anemia virusEncephalomyelotis virus (avian, equine)Polyomavirus type I & II (avian)

Avian adenovirus type I, II & IIIJapanese encephalitis (swine)Canine distemper VirusCanine Parainfluenza virusBovine Ibaraki virusBovine Parainfluenza virus

Veterinary infectious diseases

Traditional vaccines Recombinant vaccines

Influenza Influenza (Alphavirus)

Measles CMV (Alphavirus)

Mumps virus AIDS (Poxvirus)

Smallpox Pancreatic cancer (Poxvirus)

Rabies Melanoma cancer (Poxvirus)

Yellow fever prostate cancer (Poxvirus)

Thick-borne encephalitis Breast cancer (Poxvirus)

Thick-borne encephalitis Cervical cancer (Poxvirus)

Lung cancer (Poxvirus)Kidney cancer (Poxvirus )

Colorectal cancer (Poxvirus)

Japanese encephalitis (Poxvirus)

Human infectious diseases

The Chicken Egg Production PlatformA Breakthrough Technology in the 1930’s

7

JF Bach, NEJM. NJF Bach, NEJM. N°°437, p911437, p911--920; 2002920; 2002

The Vaccine RevolutionThe Vaccine RevolutionUnequalled impact on public healthUnequalled impact on public health

8

Hundred million fertilized eggsHundred million fertilized eggs ~400 million doses~400 million doses

Irreducible Irreducible 66Months processMonths process

ISSUESISSUES Cumbersome manufacturing processSlow reactivity & Limited capacityExposure to risks of outbreak of bird diseases & eggs penuryEgg-component allergiesQuality concernSusceptible to contaminations

(e.g. Shortage of Influenza vaccines in the US in 2004)

The Egg Production PlatformAncient & beset by major technical issues

e.ge.g. Influenza Vaccines. Influenza Vaccines

9

The Egg Production PlatformAncient & beset by major issues: 2004 Flu vaccines shortage in the US

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Avian ES Cell LineAvian ES Cell Line

VirusVirus

ProductionProduction PurificationPurification

FormulationFormulation

VACCINESVACCINESVACCINES

BENEFITSBENEFITSNo 4No 4--6 months lead times to organize egg supply6 months lead times to organize egg supplyHigher initial purityHigher initial purityClosed system: improved qualityClosed system: improved qualityFully qualified cell line: reduced sanitary hazardFully qualified cell line: reduced sanitary hazardFaster manufacturing process (~4 months)Faster manufacturing process (~4 months)Possibility of scalePossibility of scale--upup

Cell-Based Production TechnologiesA process compatible with most stringent regulations

Indefinite self-renewalGenetically StableLarge scale cultureGrowth in SuspensionAbsence of SerumHigh Production Yields

Indefinite selfIndefinite self--renewalrenewalGenetically StableGenetically StableLarge scale cultureLarge scale cultureGrowth in SuspensionGrowth in SuspensionAbsence of SerumAbsence of SerumHigh Production YieldsHigh Production Yields

11

Unique Biological Properties of Stem CellsStem Cell Therapies

MuscularMuscular cellscells

InnerInner CellCell MassMass

Totipotent Totipotent embryonicembryonicstem stem cellscells

Unlimited regenerative capacityIndefinite self-renewalGenetically Stable




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Full documentation of the whole process (from animals to EBx® cells)Full documentation of the whole process Full documentation of the whole process (from animals to EBx(from animals to EBx®® cells)cells)

EMBRYO ISOLATIONEMBRYO ISOLATION EMBRYONIC STEM CELLSEMBRYONIC STEM CELLS

••IMMORTALIMMORTAL••GENETICALLY STABLEGENETICALLY STABLE

STEP I STEP I (PATENTS(PATENTS))

The EBx® PlatformExploiting the unique biological properties of ES cells

EBxEBx®® CELLSCELLS

••LARGE SCALELARGE SCALE••SUSPENSIONSUSPENSION••NO SERUMNO SERUM

PHASE IIPHASE II

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Phase IIsolation of Avian Embryonic Stem CellsIsolation of Avian Embryonic Stem Cells

1 day chicks1 day chicks19 weeks chicken19 weeks chicken

Derivation of avian EBx cell linesPhase I: isolation of avian ES cell

ES cellsES cellsES cells

EGGSEGGS

EMBRYO ISOLATIONEMBRYO ISOLATION

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Embryo(black feathers)

EmbryoEmbryo(black feathers)(black feathers)

Isolation &Isolation &

expansionexpansion

ES cellsES cellsES cells

InjectionInjectionEmbryo

(white feathers)EmbryoEmbryo

(white feathers)(white feathers)

ChimeraChimeraChimera

HatchingHatchingDONORDONOR RECIPIENTRECIPIENT CHIMERACHIMERA

B H L S GB H L S G

B: blood; H: heart; H: liver; S: spleen; G: gonads; M: muscle

Germ Line transmissionGerm Line transmissionGerm Line transmission


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EMBRYONIC STEM CELLSEMBRYONIC STEM CELLSCHICKENCHICKEN

The Avian EBx® PlatformAll chicken strain harbor endogenous retroviral particles

EMBRYOEMBRYOENDOGENOUS ALVENDOGENOUS ALV

Selection of avian species with no endogenous retroviral particlesSelection of avian species with no endogenous retroviral particlSelection of avian species with no endogenous retroviral particleses

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DUCKSDUCKS

No reports on endogenousretroviral particles

No reports on endogenousretroviral particles

EGGS EMBRYO

Embryos collection

22,000 Eggs22,000 Eggs22,000 Eggs

DUCK ES CELLS

CRITICAL PARAMETERSCell Culture MediumCell Growth factorsFeeder CellsTemperatureCell Dissociation Agents…

CRITICAL PARAMETERSCRITICAL PARAMETERSCell Culture MediumCell Culture MediumCell Growth factorsCell Growth factorsFeeder CellsFeeder CellsTemperatureTemperatureCell Dissociation AgentsCell Dissociation Agents……

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Phase IIDerivation of EBx CellsDerivation of EBx Cells

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Full documentation of the whole process (from animals to EBx® cells)Full documentation of the whole process Full documentation of the whole process (from animals to EBx(from animals to EBx®® cells)cells)

EMBRYO ISOLATIONEMBRYO ISOLATION EMBRYONIC STEM CELLSEMBRYONIC STEM CELLS

••IMMORTALIMMORTAL••GENETICALLY STABLEGENETICALLY STABLE

STEP I STEP I (PATENTS(PATENTS))

The EBx® PlatformExploiting the unique biological properties of ES cells

EBxEBx®® CELLSCELLS

••LARGE SCALELARGE SCALE••SUSPENSIONSUSPENSION••NO SERUMNO SERUM

PHASE IIPHASE II

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EBx CELLS: maintenances of ES cells featuresEBx CELLS: maintenances of ES cells featuresSmall cells (~7µm)Large nucleus & nucleolusHighly metabolically activeCytoplasm rich in ribosomes & mitochondria

Derivation of duck EB66 cellsUltrastructure similar to ES cells

Mouse ES cellsMouse ES cellsMouse ES cellsDuck EB66Duck EB66Duck EB66Chicken EB14Chicken EB14Chicken EB14

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EB66

EB66

EB66

CH

OC

HO

CH

O

SSEA-1SSEASSEA--11 EMA-1EMAEMA--11Cont.Cont.Cont.

Phase II: Derivation of duck EBx® cellsExpression of ES-cells specific markers

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0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

2

ChickenChickenEB14EB14

MDCKMDCKcellscells

Absorbance (A450Absorbance (A450--A655)A655)

T. WinslowT. WinslowDuckDuckEB66EB66

ChickChickFibroblastsFibroblasts

CHO CHO cellscells

Derivation of duck EB66 cellsTelomerase promotes immortality & genetic stability

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EBx® cells are genetically stableKarytoping analysis; presence of macro- & microchromosomes

CHICKENEB14 CELLS

DUCKEB66 CELLS

MCB p160

EPC p184

MCB p130

EPC p154

DIPLOID DIPLOID DIPLOID DIPLOID

Analysis performed by Bioreliance (UK) on 50 metaphasesAnalysis performed by Bioreliance (UK) on 50 metaphases

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Unusual metabolic profile of duck EBx cellsNo accumulation of GlutamateNo Consomption of Glutamine, but progressive accumulationNo accumulation of NH4+n but consomptionRapid consomption of GlucoseAccumulation of Lactate

Unusual metabolic profile of duck EBx cellsUnusual metabolic profile of duck EBx cellsNo accumulation of GlutamateNo accumulation of GlutamateNo Consomption of Glutamine, but progressive accumulationNo Consomption of Glutamine, but progressive accumulationNo accumulation of NH4+n but consomptionNo accumulation of NH4+n but consomptionRapid consomption of GlucoseRapid consomption of GlucoseAccumulation of LactateAccumulation of Lactate

Expansion of duck EB66 CellsDevelopment of custom cell growth medium

0,00

0,50

1,00

1,50

2,00

2,50

3,00

3,50

4,00

0 1 2 3 4 5 6 7 8 9 10Time (Days)

[glu

c]g/

L - [

gln]

mm

ol/L

- [N

H4+]

mm

ol/L

0

1

2

3

4

5

6

7

8

[lac]

g/L

- [gl

u]m

mol

/L

Gluc g/L Gln mmol/L NH4+ mmol/LLac g/L Glu mmol/L

0,00

0,50

1,00

1,50

2,00

2,50

3,00

3,50

4,00

0 1 2 3 4 5 6 7 8 9 10Time (Days)

[glu

c]g/

L - [

gln]

mm

ol/L

- [N

H4+]

mm

ol/L

0

1

2

3

4

5

6

7

8

[lac]

g/L

- [gl

u]m

mol

/L

Gluc g/L Gln mmol/L NH4+ mmol/LLac g/L Glu mmol/L

25SPINNER FLASKSSPINNER FLASKSSPINNER FLASKS

ROLLER BOTTLESROLLER BOTTLESROLLER BOTTLES

BIOREACTORS(up to now: 50L100L: ongoing)

BIOREACTORSBIOREACTORS(up to now: 50L(up to now: 50L100L: ongoing)100L: ongoing)

SHAKEN FLASKSSHAKEN FLASKSSHAKEN FLASKS

WAVE BIOREACTORWAVE BIOREACTORWAVE BIOREACTOR

Industrial production process in SFMCollaboration with SAFC-Biosciences (KS, USA)

ERLENMEYERSERLENMEYERSERLENMEYERS

26

0

5

10

15

20

25

30

Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7

Cell density in 10Cell density in 106 6 cells/mlcells/mlDisposable bioreactorDisposable bioreactor

EB66 cells have the potential to reach very high cell densities

Cell Growth in Stirred-Tank BioreactorsSplit ratio: up to 1/201/20; Cell densities: 10 to 30 million/ml in batch culture

27

PRODUCTION OF VIRAL VACCINESPRODUCTION OF VIRAL VACCINES

Influenza Vaccines

Recombinant Poxvirus-based Vaccines

Measles Vaccine

Veterinary vaccines

The EBx® PlatformA wide spectrum of applications

28

Poxvirus VaccinesA wide spectrum of applications: Emerging Vaccines

COMPANIES INDICATIONS STAGE (Production on CEF)

Smallpox, 2nd generation Phase IIISmallpox, 3nd generation Phase I

HIV/AIDS Phase IIColorectal cancer Phase II

Melanoma Phase IISmallpox, 2nd generation Preclinical

Dengue Phase IJapanese Encephalitis Phase I

Smallpox, 3rd generation Phase IISmallpox, 2nd generation Phase I

HIV/AIDS Preclinical

Oxford BioMedica / sanofi-pasteur Colorectal cancer Phase II

Transgene/Roche Cervical cancer Phase IILung cancer Phase II

Prostate cancer Phase IIKidney cancer Phase II

Colorectal cancer Phase IRabies (cats) MarketedRabies (dogs) Marketed

Feline leukemia MarketedCanine distemper Marketed

West Nile MarketedEquine influenza Marketed

Acambis/Baxter

Merial

sanofi-pasteur

Bavarian Nordic

Transgene

COMPANIES INDICATIONS STAGE (Production on CEF)

Smallpox, 2nd generation Phase IIISmallpox, 3nd generation Phase I

HIV/AIDS Phase IIColorectal cancer Phase II

Melanoma Phase IISmallpox, 2nd generation Preclinical

Dengue Phase IJapanese Encephalitis Phase I

Smallpox, 3rd generation Phase IISmallpox, 2nd generation Phase I

HIV/AIDS Preclinical

Oxford BioMedica / sanofi-pasteur Colorectal cancer Phase II

Transgene/Roche Cervical cancer Phase IILung cancer Phase II

Prostate cancer Phase IIKidney cancer Phase II

Colorectal cancer Phase IRabies (cats) MarketedRabies (dogs) Marketed

Feline leukemia MarketedCanine distemper Marketed

West Nile MarketedEquine influenza Marketed

Acambis/Baxter

Merial

sanofi-pasteur

Bavarian Nordic

Transgene

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The EBx® PlatformA wide spectrum of applications: Emerging Vaccines

4

5

6

7

8

9

10

1h 2 4 h 4 8 h 72 h 9 6 h

EB66 CellsEB66 CellsEB66 Cells

Concentration of poxviruses in LogTCIDConcentration of poxviruses in LogTCID5050/ml/ml 24h24h 48h48h

72h72h 96h96h

Best Medium for Influenza Virus ProductionBest Medium for Influenza Virus Production

NO VIRUS REPLICATIONNO VIRUS REPLICATION

30

36h36h36h 96h96h96h 192h192h192h

Production of MVA-GFP on EBx CellsVirus propagation is enhanced in presence of cell clumpingVirus propagation is enhanced in presence of cell clumping

Cell to cell contact necessary for efficient viral propagationCell to cell contact necessary for efficient viral propagation

31

AA BB CC

FF EE DD

Development of custom-serum free media for all vaccine applications

Production of PoxVirusesInfluence of Medium Formulation on cell clumping

32

BBAA

24h24h

48h48h

72h72h

96h96h

120h120h

Production of MVA-GFP on EBx CellsInfluence of media formulations on virus production & clumpingInfluence of media formulations on virus production & clumping

144h144h

CC

NO CLUMPINGNO CLUMPING

FF GG HHDD EE

CLUMPINGCLUMPING

33

Production in a 100L BIOREACTOR~ 100,000 purified vaccine doses (108TCID50/dose)

(Productivity of 108.5TCID50/ml & 30% recovery)

Production in a 100L BIOREACTORProduction in a 100L BIOREACTOR~ 100,000 purified vaccine doses ~ 100,000 purified vaccine doses (10(1088TCIDTCID5050/dose)/dose)

(Productivity of 10(Productivity of 108.58.5TCIDTCID5050/ml & 30% recovery)/ml & 30% recovery)

PoxvirusesViral production in stirred-tank bioreactors (2L & 20L)

4

5

6

7

8

9

10

1h 2 4 h 4 8 h 72 h 9 6 h

Bio 72Bio 62Bio 60Bio 71Bio 63

EB66 CellsEB66 CellsEB66 CellsConcentration of poxviruses in LogTCIDConcentration of poxviruses in LogTCID5050/ml/ml

Day Day --2 pi2 pi Day 1 piDay 1 pi

Day 3 piDay 3 pi Day 4 piDay 4 pi

Day 2 piDay 2 pi

Production of MVA-GFP on duck EBx cellsCell morphologies

Day 5 piDay 5 pi

x40x40

35Source: Lister VV strain-JM Crance & D. Garin, CRSSA, Grenoble

EBx®-based vaccines are as efficient as egg-based vaccines

Emerging vaccines: Poxvirus-based vaccinesAn excellent vaccine efficiency

0

25

50

75

100

D ay0

D ay2

D ay4

D ay6

D ay8

D ay10

D ay12

D ay14

D ay16

D ay18

D ay20

Egg-based VaccineEBx-based VaccineNon-Vaccinated

% Live animalsEBxEBx®® VaccineVaccine

EggEgg--Based VaccineBased Vaccine

CHALLENGECHALLENGECOWPOXCOWPOX

(I.N. at 10(I.N. at 106.36.3

PFU/mouse)PFU/mouse)

SMALLPOX VACCINESMALLPOX VACCINE

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Production of Influenza Viruses on duck EBx cells

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Avian Influenza ReceptorSIA (α 2-3) Gal

Avian Influenza ReceptorAvian Influenza ReceptorSIA (SIA (αα 22--3) Gal3) Gal

Human Influenza ReceptorSIA (α 2-6) Gal

Human Influenza ReceptorHuman Influenza ReceptorSIA (SIA (αα 22--6) Gal6) Gal

Cell Surface receptorsExpression of avian (α2-3 Gal) & human (α2-6 Gal) receptors

85

0

coun

t100 101 102 103

FITC

>96.7 %>96.7 %

100 101 102 103

75

0

coun

t

FITC

1.0

98.7 %98.7 %

Duck EBx CellsDuck EBx CellsDuck EBx Cells

38

72h

72h

96h

96h

120h

120h

144h

144h

AA

Production of Influenza VirusesInfluence of Medium Formulation on HA production

HA Antigen productionHA Antigen production

POXVIRUSPOXVIRUSMEDIUMMEDIUM

72h

72h

96h

96h

120h

120h

144h

144h

72h

72h

96h

96h

120h

120h

144h

144h

72h

72h

96h

96h

120h

120h

144h

144h

72h

72h

96h

96h

120h

120h

144h

144h

72h

72h

96h

96h

120h

120h

144h

144h

72h

72h

96h

96h

120h

120h

144h

144h

Days post inf.Days post inf.

72h

72h

96h

96h

120h

120h

144h

144h

BB CC DD EE FF GG HH

MEDIA FOR EXTRACELLULAR VIRUSESMEDIA FOR EXTRACELLULAR VIRUSES

High Viral Yields & No ClumpingHigh Viral Yields & No Clumping

39D ay 0 D ay 1 D ay 2 D ay 3 D ay 4 D ay 5 D ay 6 D ay 7 D ay 8

A / H1N 1/ B eijing / 2 6 2 / 9 5B / Jiang su/ 10 / 2 0 0 3A / H3 N 2 / N ew- Y o rk/ 55/ 2 0 0 4A / H1N 1/ B eijing / 2 6 2 / 9 5B / yamanashi/ 16 6 / 9 8B / Jiang su/ 10 / 2 0 0 3A / H3 N 2 / W isco nsin/ 6 7/ 2 0 0 5B / Y amanashi/ 16 6 / 8 8A / N ew C aled o nia/ 2 0 / 9 9B / Y amanashi/ 16 6 / 8 8

Concentration of HA antigens in Concentration of HA antigens in µµg/mlg/ml

Influenza VirusesAntigen production in stirred-tank bioreactors (2L & 20L)

4010µg HA ANTIGEN (IM)1010µµg g HA ANTIGEN HA ANTIGEN (IM)(IM)

EBxEBx®®--based Vaccinebased Vaccine

EggEgg--based Vaccinebased Vaccine

0

0,2

0,4

0,6

0,8

1

1,2

1,4

Concentration in anti-HA antibodies(ELISA Signal OD493)

Pre-vaccination Post-vaccination(week5)

EBx® VaccineEgg Vaccine

Influenza VirusesIn vivo immunogenicity similar to egg-produced HA

41

Production of MeaslesEfficient replication of Measle-GFP virus

24h24h 48h48h 72h72h

96h96h 120h120h 144h144h

Measles-GFPMeasles-GFP

2

3

4

5

6

7

8

24h 48h 72h 96h 120h 144h 168h 192h

EB66-AEB66-B

VERO

TCID50/mL (log10)TCID50/mL (log10)

42

TCID50/mL (log10)TCID50/mL (log10)

2

3

4

5

6

7

8

9

0 1 2 3 4 5 6

Avian Newcastle Disease VirusAvian Newcastle Disease Virus

Production of NDVEfficient replication of NDV virus on duck EBx cells

43

Human & Animal Vaccines produced on eggs & chicken cells





Reovirus (avian)

Chicken anemia virusEncephalomyelotis virus (avian, equine)Polyomavirus type I & II (avian)

Avian adenovirus type I, II & IIIJapanese encephalitis (swine)Canine distemper VirusCanine Parainfluenza virusBovine Ibaraki virusBovine Parainfluenza virus








Yellow fever prostate cancer (Poxvirus)

Thick-borne encephalitis Breast cancer (Poxvirus)






Virus that can propagate on EBx® cellsSource: Company

Not tested yet







Yellow fever prostate cancer (Poxvirus)Thick-borne encephalitis Breast cancer (Poxvirus)










Reovirus (avian)Chicken anemia virusEncephalomyelotis virus (avian, equine)Polyomavirus type I & II (avian)Avian adenovirus type I, II & IIIJapanese encephalitis (swine)Canine distemper VirusCanine Parainfluenza virus

Bovine Ibaraki virusBovine Parainfluenza virus


The EBx® PlatformA wide spectrum of applications

44

The EBx® PlatformEfficient DNA engineering

Suspension EBx cellsSuspension EBx cells Adherent EBx cellsAdherent EBx cells

4545

The EBx cell substrateAn emerging platform for the production of Therapeutic

Proteins

Milliard US $Milliard US $

Monoclonal AntibodiesMonoclonal AntibodiesA major & fast growing marketA major & fast growing market

20

11,4

0

5

10

15

20

25

Antibodies Vaccines

Market in 2006Market in 2006Market in 2006

20

30,3

0

5

10

15

20

25

30

35

2006 2011

Milliard US $ (CAGR: 14,2%)Milliard US $ (CAGR: 14,2%)

Monoclonal AntibodiesMarket Growth

Monoclonal AntibodiesMonoclonal AntibodiesMarket GrowthMarket Growth

47Source : Walsh (2003) Source : Walsh (2003)

Monoclonal AntibodiesMonoclonal AntibodiesCHO is the standard cell lineCHO is the standard cell line

48

Glycosylation profile in different speciesAvian proteins display low fucose content

50

60

70

80

90

100

110

Human Rhesus Mouse Rat Avian

Natural Fucose content of IgG in %Natural Fucose content of IgG in %

Raju et al, glycobiology, 10, 477, 2000.Raju et al, glycobiology, 10, 477, 2000.

Low fucose enhances the antibody-dependent cell cytotoxicity

(ADCC)

Low fucose enhances the antibodyLow fucose enhances the antibody--dependent cell cytotoxicitydependent cell cytotoxicity

(ADCC)(ADCC)

49

0

20

40

60

80

100

Human Rhesus Mouse Rat Avian0

20

40

60

80

100

Human Rhesus Mouse Rat Avian0

20

40

60

80

100

Human Rhesus Mouse Avian

Bisecting GlcNacBisecting GlcNacBisecting GlcNac Terminal GalTerminal GalTerminal GalNANA & NGNANANA & NGNANANA & NGNA

Raju et al, glycobiology, 10, 477, 2000.Raju et al, glycobiology, 10, 477, 2000.

NANANANA NGNANGNA

Glycosylation profile in different speciesAvian proteins display low fucose content

50

Avian EBx®Avian EBxAvian EBx®®

DNADNADNA

The EBx® PlatformAn Emerging Platform for Protein production

Antibodies with enhancedAntibodies with enhancedADCC activityADCC activity

51

EB66-Vector Backbone2323 bpKm/Neo

Chimeric Intron

SV40 late polyA

HSV TK polyA

PROMOTER

PROMOTER

SV40early Prom

Bact Prom

MC

SM

CS

GENEhIgG1hIgG4GFP

Antibody production on EB66 cellsOptimization of expression vectors

Vector backbone & regulatory sequences

PROMOTERCMVSV40-Actin

mPGKRSV-LTR

eIF4EF1 /HTLV

CAGENS1

ENS1mut

52

0

1

2

3

4

5

6

7

8

RSVproRSVpro--IgG1IgG1

0

1

2

3

4

5

6

7

8eIF1/HTLVproeIF1/HTLVpro--IgG1IgG1

0

1

2

3

4

5

6

7

8CAGproCAGpro--IgG1IgG1

Antibody production on EBx cellsIsolation of stable IgG-expressing clones

53

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18

20

Day0

Day1

Day2

Day3

Day4

Day5

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Day8

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Day12

Day13

0

50

100

150

200

250

300Cell densityIgG

Cell density in 10Cell density in 106 6 cells/mlcells/ml IgG conc. In mg/LIgG conc. In mg/L

Antibody production on EBx cellsIgG production in stirred tank bioreactors

54

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60

70

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100

p137 p142 p152 p137 p142 p152

% Total IgG1% Total IgG1

Total IgG w/o FucoseTotal IgG w/o Fucose Total IgG with FucoseTotal IgG with Fucose

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50

60

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IgG-HER2 IgG-HER2 IgG-IgE IgG-HER2 IgG-HER2 IgG-IgE

% Total IgG1% Total IgG1

EBx CELLS CHO CELLS

Shields et al J. BioL Chem. 277, p26733Shields et al J. BioL Chem. 277, p26733--26740, 2002 26740, 2002 (Genentech)(Genentech)(Clone ID7)

Total IgG w/o FucoseTotal IgG w/o Fucose Total IgG with FucoseTotal IgG with Fucose

Production of antibodies on EBx® cellsEBx antibodies display low fucose content

55

Biological Activity of EB66-IgG1Activation of NK Cells (Analysis of IFNγ secreting cells)

0

5

10

15% % IFNIFNγγ Positive NK Positive NK CellsCells

NKNKFreshly ThawedFreshly Thawed

NK +NK +5U IL25U IL2


0

5

10

15

20

25

30

35

40

45% % IFNIFNγγ Positive NK Positive NK CellssCellss

NKNKFreshly ThawedFreshly Thawed



TumorTumor + Anti+ Anti--TumorTumor IgG1 (25IgG1 (25µµg; g; EBxEBx))TumorTumor + Anti+ Anti--TumorTumor IgG1 (25IgG1 (25µµg; CHO)g; CHO)

TumorTumor

DONOR 1DONOR 1DONOR 1 DONOR 2DONOR 2DONOR 2

TumorTumor + Anti+ Anti--TumorTumor IgG1 (25IgG1 (25µµg; g; EBxEBx))TumorTumor + Anti+ Anti--TumorTumor IgG1 (25IgG1 (25µµg; CHO)g; CHO)

TumorTumor

56

0

10

20

30

40

50

60

1,E-12 1,E-11 1,E-10 1,E-09 1,E-08

IgG1 WT1D9

1F111H1

2B9

% of Cell Death % of Cell Death (Chromium Release)(Chromium Release)

Monoclonal Antibody from Pharma XMonoclonal Antibody from Pharma X

CHO-IgG1CHO-IgG1EBx-IgG1EBx-IgG1

>20x

Concentration in MConcentration in M

Production of antibodies on EBx® cellsEBx antibodies display low fucose & enhanced ADCC activity

5757

The EBx cell substrateSanitary & regulatory status

58

Pre MCBPre MCBPre MCB

WORKING CELL BANKWORKING CELL BANKWORKING CELL BANK

MASTER CELL BANKMASTER CELL BANKMASTER CELL BANK

R&D CELL BANKR&D CELL BANKR&D CELL BANK

END OF PRODUCTIONCELL BANK

END OF PRODUCTIONEND OF PRODUCTIONCELL BANKCELL BANK

PRODUCTION LEVEL (10,000L)PRODUCTION LEVEL (10,000L)PRODUCTION LEVEL (10,000L)

BIOSAFETYTESTING

BIOSAFETYBIOSAFETYTESTINGTESTING BIOLOGICAL

MASTER FILEBIOLOGICALBIOLOGICALMASTER FILEMASTER FILE

Sanitary & Regulatory StatusOverview of the GMP banking strategy

59

The EBx® PlatformBiologic Master File (BMF) filed with the U.S. FDA in June 2008

60

EBx® cells display unique technical, industrial & regulatory features

> Derivation from Embryonic Stem Cells with no genetic, viral or chemical modifications

> Genetically stable and diploïd

> Benefits from 70 year of historical background on vaccines production in eggs

> Avian embryonic stem cells raise no ethical or religious issues

> Industrial scale-up: growth to high cell densities in bioreactors, as suspension cells, in serum-free medium

> Full documentation & traceability; cGMP Master Cell Banks available

> Biological Master File filed with the US FDA

> Strategic partnership with SAFC-Biosciences (USA) to offer a custom GMP serum-free medium

> Broad virus susceptibility: efficient production of a wide range of human and animal viral vaccines

> Efficient DNA engineering & recombinant protein production

> Monoclonal antibodies expressed in EBx cells display low fucose content and enhanced ADCC activity

> Endorsed by the industry

The EBx® PlatformA breakthrough technology in vaccines manufacturing

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27 PARTNERSHIPS WITH 22 CUSTOMERS

> 10 commercial licenses on EBx® platformSanofi-Pasteur Recombinant vaccines for HIV and cancersSanofi-Pasteur UndisclosedGSK Influenza vaccine (seasonal and pandemic)CSL Influenza vaccine (seasonal and pandemic)Nobilon (Schering-Plough) Influenza vaccine (seasonal and pandemic)Merial Canarypox based vaccines for pets diseases Virbac BiologicalsSAFC-Biosciences Cell culture mediaGeovax Recombinant HIV VaccinesIntervet Veterinary vaccines

> 10 research licenses on EBx® platformNovartis Vaccines Kaketsuken (2 distinct licences)Bavarian Nordic Fort-Dodge (Wyeth) sanofi-aventis3 human undisclosed 1 veterinary undisclosed

> 7 ongoing research collaborations & pilot biomanufacturing contracts

The EBx® Cell TechnologyEndorsed by the industry

63

Les pièges techniques, tu éviteras

THANKS TOTHANKS TO

Fabienne GuehenneuxFabienne GuehenneuxArnaud LeonArnaud LeonPatrick ChampionPatrick ChampionAnneAnne--Laure DavidLaure DavidRichard CasseronRichard CasseronAudrey BoncompainAudrey Boncompain

Brice MadelineBrice MadelineSandrine AspaSandrine AspaOlivier NerriereOlivier NerriereStStééphane Olivierphane OlivierCCéédric Brillondric BrillonCCééline Bredaline Breda

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