Implementation ofImplementation of Single Use Systems in the Process Development of a rec-h Cytokine & Transfer

to GMP Production.

October 26, 2011. 

Iann Rancé Ph DIann Rancé – Ph‐DDirector Process Development – Cytheris

Cytheris SA – Technopolis – 175 rue Jean‐Jacques Rousseau  – 92138 Issy‐les‐Moulineaux Cédex – France  – www.Cytheris.com

Topical Outline

• Cytheris  &  human Interleukin‐7y• hIL‐7 Process Development & Scale Up

• Implementation of SiUs Systems in hIL‐7 Process• Performance Comparison & Advantages

• Conclusionso ‐ ‐ o ‐ ‐ oo  o  o

Disclaimer: • Products / Vendors are named & compared in presentation. • Related conclusions apply only for Cytheris’ specific process & molecule

2

• Related conclusions apply only for Cytheris  specific process & molecule.• General conclusions should not be drawn!

3Single-Use Technology Seminar, First Hotel, Copenhagen, Denmark, 25-26th October 2011

CorporateCorporate BackgroundBackground

Cytokine Therapy for the Immune System (1999)(1999)

A project driven company targeting the immune t & ifi ll i d fi i isystem & more specifically immune deficiencies

Worldwide exclusive license for lead rec.IL-7

VC Investment: 43 M€, (3 rounds)

Staff: 33

Headquartered Issy / Moulineaux, France Process7

IP & PC 5

Regul. 3

Adm & Fin 5

4

Subsidiary in Rockville, Maryland - USA7

Clinic 13

Single-Use Technology Seminar, First Hotel, Copenhagen, Denmark, 25-26th October 2011

Why is ILWhy is IL--7 interesting ?7 interesting ?

IL 7IL-7: A non redundant critical growth factor for T cells

5

Launch Dev.pt. of IL-7 for Modulationof Immune Function in Multiple Diseases Launch Dev.pt. of IL-7 for Modulationof Immune Function in Multiple Diseases pp

Clinical Centers / Phase II trials:

France USA

Physiology & diseases Application :

Endogenous production by thymic stromal France USAItaly CanadaSouth Africa TaiwanSwitzerland Spain

Endogenous production by thymic stromalcells, intestinal & other epithelial cells

Active at low dose (secreted & soluble)Involved in all stages of maturation &

UKg

proliferation of T Lymphocytes

PLURIPOTENT CYTOKINE :

OncologyChemo/RadioTher.HIV

HCV

OncologyTherapeutic

VaccinesIdiopathic Lymphopenia

ILIL--77

6

>12 trials ongoing+ Non-sponsored studies

TuberculosisBone Marrow &

Stem Cell Transplant

hILhIL--7 structural 7 structural featuresfeaturesG

1 11 21 31 41 51 61DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDL

G GO81 101 111 121 131 141 151

HLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH

Amino Acids 152

Disulfide bridges C C C C C CDisulfide bridges C1-C92 C34-C129 C47-C141

Glycosylation sites N70 N91 N116 O(T110)

MW ~22 kDa

pI 4,3<pI<8,5

7

Only the most glycosylated proteins must be purified: Glyco quality >> Yield

8Single-Use Technology Seminar, First Hotel, Copenhagen, Denmark, 25-26th October 2011

rec-hIL-7 batches, scales & releaserec-hIL-7 batches, scales & release

IL-7 Development batches (Home)

Jan Dec

IL-7 GMP batches (CMO) 2003 2004 2005 2006 2007 …

2006 2008Jan Dec

2010scale 1-5 L 5-10L 20 L 120 L 20LUse Dev Tox & Dev Dev

Batch E01 G01 J01 J02Prod.Scale 200L 600L

Analytics (in process & release)Formulation & stress studies

Analytical package comprises:Physico-chemical tests

9

Formulation & stress studiesBack-up Stability studies Purity & identity

Potency assay

ProcessProcess schemescheme

PrecultureCulture

ClarificationWCBWCB

Preculture2L

20L

Clarification

… L

recCHOcell line

Purification

In Process Analytics

10

FillingBDS & DP Release

DownstreamDownstream ProcessProcess StepsSteps

Clarified Harvest

Anion Exchange = FT mode. Prepurify IL-7

TFF/ UF Concentration to reduce volume

Cation Exchange = Capture/Elution, Purify IL-7 & remove aggregatesy & gg g

HIC, Hydrophobic Interaction = Capture/Elution.Sort glycoforms through ° sialylation & remove aggregates.

Planova 20N Removal potential virus

11

UF / DF. Formulation & Concentration of API

12Single-Use Technology Seminar, First Hotel, Copenhagen, Denmark, 25-26th October 2011

Implementation of SiUs systemsImplementation of SiUs systems

Development batches (Home)

GMP batches (CMO) 2003 2004 2005 2006 2007 …

2006 2008 2010scale 1-5 L 5-10L 20 L 120 L 20LUse Dev Tox & Dev Dev

Batch E01 G01 J01 J02Prod.Scale 200L 600LUPS

UPS UPS

200L

DSP

UPS

UPSDSP UPS

DSP

13

UPSDSP UPS

DSP

ProcessProcess schemescheme withwith SiUsSiUs

PrecultureCulture

ClarificationWCBWCB

Preculture2L

20L

600L Clarification

60L

Filters

recCHOcell line

Filters

PurificationWave

Membrane & Filters

SUB 1000L

14

FillingSUB 1000L

ProcessProcess schemescheme withwith SiUsSiUs

PrecultureCulture

ClarificationWCBWCB

Preculture2L

20L

Clarification

60L

FiltersFiltersFaster

operation

PurificationWave

Easy,

Membrane SUB 1000L

y,Faster T.Ov.

more flexible

Easy,

15

Filling& FiltersEasy,

Faster T.Ov.more flexible

Cheap,Warehouse

DSProcessDSProcess withwith SiUsSiUs

1) QMA (AE )SiUs Mbne AEx

2) TFF

1) QMA, (AEx) SiUs Mbne AEx

SiUs Mbne Replacement/

combination3) QFF, (AEx)

4) C ti E h

combination

4) Cation Exch.

5) HIC

6) Planova

16

7) UF/DFSiUs Mbne

DSP, DSP, SiUsSiUs AdvantagesAdvantages

1) QMA (AE )SiUs Mbne AExEasy plug in

2) TFF

1) QMA, (AEx) SiUs Mbne AEx

SiUs Mbne

Easy plug in, Extrem. fastWarehouse

3) Anion Exchange

4) C ti E h

Cheap,Warehouse

4) Cation Exchange

5) HIC

6) Planova

17

7) UF/DFSiUs MbneCheap,Warehouse

18Single-Use Technology Seminar, First Hotel, Copenhagen, Denmark, 25-26th October 2011

1) Culture: StSt vs. SUBioR

o Amplification from WCB. Inoculate BR with 0.3 mC/mL

o Custom Culture Medium + Glucose + AoA feedo Custom Culture Medium + Glucose + AoA feed.

o Fed batch mode.

o pO2 regulation: 40%

300L ABEC StSt BRvs.

1000L SUB Hyclone

o T° shift @ Day 3, Harvest @ Day 12

C. Viability, 99-100%

C Density 6 2 4 C/ LTT°°SS

200L 200L StStStSt

C. Density, 6.2-7.4mC/mL

19CONFIDENTIAL DATA

1 2 2A 3 4 4A 5 5A 6 6A 7 7A 8 8A 9 9 10 10A 11 11A 12 12A

Days 600L SUB2600L SUB2600L SUB1600L SUB1

1) IL-7 Glycoforms in Culture SN

End of culture featuresIL-7 Productivity: 200L SS BR = 110 – 150 mg/mL, 600L SiUs BR = 120 – 160 mg/mL

pI 5 6 7 8 9

y g , g

IL-7 Glyco Quality: 2D PAGE analysis on D12 Supernatent (Coom.B. Stain)

MW

IL-7

System pI 5-6 pI 6-7 pI 7-8 pI 8-9

20

y p p p p200L SS BR 15%15% 26%26% 39%39% 20%20%

600L SUB 25%25% 31%31% 38%38% 6%6%

2) DSP: resin AEx vs. Mbne AExo Crude Clarified harvest (CCH) load = 10 mg/ml device

o Step is FT mode. No capture of IL-7Mustang Q XT (Pall)

vs.

A) CCH B) CCH C) CCH

o Step is FT mode. No capture of IL 7

o TFF Concentration to reduce volume

vs.Q Seph. Fast Flow (GE)

A) CCH B) CCH C) CCH

4x4x

QFF

TFF/ UF TFF/ UF

Mustang QTFF/ UF, 22-24x

Mustang Q

TFF/ UF6x

TFF/ UF22-24x QFF

21

End DSP End DSP End DSP

2) DSP: resin AEx vs. Mbne AEx

o Crude Clarified harvest (CCH) loadedMustang Q

o FT is collected. No capture of IL-7

o Assay elimination of CHO CP: TP contento Check IL-7 content by specific ELISA

Mustang Qvs.

QFF

B Mustang Q C Must. Q FT,A QFF B Mustang Q FT

TP 70-80 %

IL 7 97 100%

C Must. Q FT, then, QFF FT

TP 65-70 %IL-7 95-98%

A QFF FT

TP 78-85 %IL 7 97 100% IL-7 97-100% IL-7 95-98%

Combination was found superior to each single system

IL-7 97-100%

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Initial replacement strategy was not retained

2) DSP: resin AEx vs. Mbne AEx

o Tabulated results for viral validation study (Texcell SA)

Virus/ Step QFF CEx HIC Nanofiltration R total

MLV Run A = 2.55Run B = 2.39 11

R A 2 07

A)

Reo3 Run A = 2.07Run B = 3.52 13

MVM Run A ≈ 0Run B = 0.98 8

Virus / Step QMA QFF CEx HIC Nanofiltration R total

MLV Run A > 4.52Run B > 4.57

Run A > 4.12Run B > 4.05 18

C)

Reo3 Run A > 4.78Run B > 5.13

Run A > 5.6 Run B > 5.7 20

MVM Run A > 4.81Run B > 5.17

Run A > 5.99Run B > 6.23 20

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Combination was also found superior for virus removal

3) DSP: compare TFF Mbnes

o SiUs vs. conventional TFF Membranes

Ease of use Millipore Ease of use

Storage/ imobilization of warehouse space

Pricing

Milliporevs.

Tangen X

Conventional MilliporeInitial TFF Pellicon II, XL

PLCGC 2 5 2

SiUs Tangen XInitial TFF SiUs TFF lowPLCGC, 2.5m2

Final TFF Pellicon II Utracel, 0.5m2

( 2 €

Initial TFF SiUs TFF low binding, 2.5m2

Final TFF SiUs TFF low binding, 0.5m2

Price (2.5 m2

unit)5900 €

(8500 USD)

Storage Wet, cool place

Price (2.5 m2

unit)1400 €

(2000 USD)

Storage dispose

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Recommend. Circulate once a year (better ev. 6M)

g pRecommend. SiUs

25Single-Use Technology Seminar, First Hotel, Copenhagen, Denmark, 25-26th October 2011

SiUsSiUs systemssystems

Downscale testing

Vendors provide scale down systems for easy comparative test & implementation. For IL-7:

- Biostat CultiBag RM (wave) vs Biostat A plus (glass BR)Biostat CultiBag RM (wave) vs. Biostat A plus (glass BR) assay & validate improved oxygenation/positive effect on glyco.

- Cuno Zeta+ encapsulatedvs. Zeta+ EXT.- Mustang Q 1coin (1ml) or XT 5 (5 ml) vs. XT5000,

stacking configuration tests

- 0.1 m2 SiUs Novasep Mbnes vs. 2.5 m2

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- Planova 20N, 0.001 m2 (down to 10 ml API) vs. 0.3 m2

SiUsSiUs systemssystems

Take home message (CYT experience)

Advantages / Drawbacks are process dependent. Do not generalize properties !

More flexibility upon development. Easier to accommodate to exotic processes.

Ease of use (plug & use)Ease of use (plug & use)

No warehouse immobilization (= reduced costs). Budget gap upon purchase (not always!)purchase (not always!).

Cheaper in moderate scales and low number of runs per year.

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Might no longer be the case in larger scale and multiple runs iterated.

up to 300 patients treated (Q3 2011)

28Single-Use Technology Seminar, First Hotel, Copenhagen, Denmark, 25-26th October 2011

IL-7 Oncology results

(mean ± SD 4 patients @ 3 10 30 & 60 g/kg)(mean ± SD 4 patients @ 3, 10, 30 & 60 g/kg)

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Long Lasting Changes in CD4 and CD8 T Cells (HIV Patients)CD8 T Cells (HIV Patients)

Effects up to w52 following 3 SC injections @ w0,1,2

1000

1200

1400

2000

2500

400

600

800

1000

1000

1500

CD

4 /m

m3

CD

8 /m

m3

**

0

200

0 28 56 84 112 140 1680

500

0 28 56 84 112 140 168252 365 252 365

*Wilcoxon test  W12P=0.001, CYT107 10g/kg,  n=7 P 0 0007 CYT107 20 /k 8

**Wilcoxon test  W52P=0.002 , CYT107 10g/kg,  n=7 P 0 003 CYT107 20 /k 8

Days Days

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P=0.0007, CYT107  20 g/kg, n=8P=0.002, CYT107 30 µg/Kg, n=6 Placebo, n=6 

P=0.003, CYT107  20 g/kg, n=8P=0.004, CYT107 30 µg/Kg, n=6  

irance@cytheris.comirance@cytheris.comwww Cytheris comwww Cytheris comwww. Cytheris.comwww. Cytheris.com

31Issy les Moulineaux