M. Hebben

CONFIDENTIAL

Debottlenecking Downstream Process of AAV9 Gene Therapy Vectors using Customized Chromatography

Resin BioInnovation Leaders Summit

London, Feb 11th 2015

Matthias Hebben, Ph.D. Bioprocess Development

11/02/2015

M. Hebben

CONFIDENTIAL

Genethon at a Glance

•  GenethonBioprod:GMPmanufacturingsitegrantedin2013byANSM

•  Produc@onofclinicalbatches

•  VectorplaCorms:AAVandlen@viralvectors.

•  Createdin1990byAFM(FrenchAssocia@onofMuscularDystrophies)

•  Anintegrated,nonprofitorganiza@onof220people

•  Transla@onalresearchandclinicaldevelopmentintheareaofgenetherapy

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M. Hebben

CONFIDENTIAL

In vivo and ex vivo gene therapy

Muscular Dystrophies Myotubular myopathy Eye diseases Liver-mediated gene therapy

in vivo delivery

Recombinant AAV vectors are directly injected into the target organ

Hematopoietic stem cells taken from the patient are transduced with an HIV-derived lentivector and reinfused

Primary immunodeficiencies, Hemoglobinopathies

ex vivo delivery

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M. Hebben

CONFIDENTIAL

Gene Therapy Evolution since 1980

1980 1990 2000 2010 2020 2030

Proof of concept Clinical Trials

Commercial Phase

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M. Hebben

CONFIDENTIAL

STRONG FLOW OF ACQUISITIONS AND LICENSE AGREEMENTS WITH BIG HEALTHCARE PLAYERS:

MORE THAN USD 470 M FUND RAISED IN 2012 AND 2013:

(Chatham Therapeutics)

An incredible progression in 2014!

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M. Hebben

CONFIDENTIAL

AAV Vectors

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SmallandTough!18nm,nolipid

envelope

Simple!3proteins(VP1,VP2,VP3)encodedby1

gene

TissueSpecific!Dependingonnatural

serotypes

Safe!Naturallynonpathogenic

ForeignDNApackaging!

Upto4.8kbofsinglestrandedDNAof

interest

M. Hebben

CONFIDENTIAL

Vector scAAV9-SMN1

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Transgene cassette DNA

AAV vector

M. Hebben

CONFIDENTIAL

AAV9 as a Gene Therapy Vector

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•  UniquefeaturesofAAV9:–  Topismformusclesandheart–  TropismforCNS–  CapabilitytocrossBBB

Foust et al. Nat. Biotechnol. 2009

M. Hebben

CONFIDENTIAL

AAV9 as a Gene Therapy Vector

Disease Vector Administra=onroute Authors

SMA scAAV9-SMN IV Dominguezetal.2010

MPSIIIA AAV9-SGSH IC/ICV Haurigotetal.2013

MPSIIIB AAV9-NAGLU IV Fuetal.2011

Pompe AAV9-GAA intrapleural Falketal.2013

ALS AAV9-ADAR2 IV Yamashitaetal.2013

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•  AAV9suitableforgenetherapyof:–  Neuromusculardisorders–  Lysosomalstoragediseasesaffec@ngthebrain

•  1clinicaltrialini@atedinUS:scAAV9-SMN1forGTofSMA(AveXis)

•  LargescalemanufacturinginGMPcondi@onsisneededtosupportfutureclinicaltrials

M. Hebben

CONFIDENTIAL

AAV9 Upstream Process: Transfection

Transfection of suspension cells PEI

Scale: 200L disposable bioreactors

Transfection of adherent cells Calcium Phosphate

Scale: 24 CF10 (25L)

Triple plasmid transfection

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HEK293 cells

+

M. Hebben

CONFIDENTIAL

Baculovirus genome recombination in E. coli

Generation of infectious Baculovirus in Sf9 cells

Production

2x 200L in disposable bioreactors

Bac-Rep/cap rep polh p10 pApA cap

promoter-TransgeneBac-rAAV-ITRITR ITR

rBac-rAAV rBac-Rep/Cap

Co infection

Sf9 cells

AAV9 Upstream Process: Baculovirus

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M. Hebben

CONFIDENTIAL

AAV Upstream Processes

Transfec=onofHEK293cells

Adherentcells(cellfactories)orsuspensioncells(s@rredtanks)

Versa@leprocess

Fastimplementa@onExpensive(plasmidcost)

Mainimpuri@es:

DNA/Transfec@onagent

Baculovirus/Sf9cells

Suspensioncells(s@rredtanks)

Versa@leprocessSlowimplementa@on(8months)Costdecreaseswithnumberoflots

Mainimpuri@es:

BaculovirusDNAandproteins

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M. Hebben

CONFIDENTIAL

Purification of AAV9 Vectors

•  NoindustrialfriendlymethodavailabletopurifyAAV9atlargescale

•  ImmunoaffinityAVBSepharose(GEHealthcare):–  OriginallydeveloppedforAAV1–  CrossbindingofAAV2,AAV5,AAV6,AAV8,AAV10–  NobindingofAAV9capsids

•  Needtodevelopapurifica@onprocessfromscratch

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M. Hebben

CONFIDENTIAL

AAV9 Downstream Process using IEX

Clarification

CEX

AEX AEX

Negative Mode

TFF

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SF

M. Hebben

CONFIDENTIAL

AAV9 Downstream Process using IEX

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0

20

40

60

80

100

120

Clarifica@on CEC AEC1 AEC2 TFF

%vectorrecovery

Averageyield(n=6)

AAV9 5e9VG 1e10VG

M. Hebben

CONFIDENTIAL

AAV9 Downstream Process using IEX

•  Lowyield(approximately20%)–  Impliesalargebatchsizetogeneratetherequiredamountofvector–  Impliesahighconcentra@onfactortoachievetarget@tersinclinical

doses–  Atsmallscale,thehighest@terobtainedwas1-2e12VG/mL

•  Scalabilityissues–  AfempstoscaleuprevealedtechnicalpiCalls:verynarrowpH/

temperatureopera@ngrangeaffec@ngpurityandvectorstability

•  Processnotviable?

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M. Hebben

CONFIDENTIAL

POROS®CaptureSelect™ AAV9 •  AffinityresindevelopedbyThermoFisherScien@fic•  Ligandbasedonasingle-domain[VHH]an@bodyfragment

•  VHHaffinityligandsareproducedinyeast(S.cerevisiae)inananimaloriginfreeproduc@onprocess–  ISO9001cer@fiedmanufacturingfacility(Netherlands)

12-15kDa

•  Small size:12-15 kDa fragment: ~1/10th mAb

•  Tunable specificity/affinity

CaptureSelect™ technology

Caution: For manufacturing, processing, or repacking.

M. Hebben

CONFIDENTIAL

POROS®CaptureSelect™ AAV9 •  ResinbasedonPOROS®beads

–  Polystyrene-DivinylbenzeneBackbone•  Rigid,Incompressible,Easy-to-Handle,

–  PerfusiveMedia•  LargeThroughporesUnlockBeadInterior;

HighSurfaceArea

–  50micronPar@cleSize•  ProvidesSuperiorResolu@onIndependentofScaleandFlowRate

•  Resinishighlyselec@veforAdeno-AssociatedVirusSerotype9(AAV9)

•  CaptureSelect™AAV9ligandleakageELISAkitavailable

Caution: For manufacturing, processing, or repacking.

M. Hebben

CONFIDENTIAL

Resin Selectivity

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•  No residual proteins detected

•  Same purity profile as 3 steps of IEX chromatography

M. Hebben

CONFIDENTIAL

Resin Capacity

•  Thehighertheflowrate,thehigherthecapacity!•  Capacity=1e12VG/mlofresinat450cm/husingBaculovirus-expressedAAV9•  Considering30%offullpar@clesàbindingof3e12capsidspermLofresin•  Highcapacityresultsinsmallvolumeofresin•  Smallresinvolumeresultsinhighconcentra@onfactor

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0.0E+00

5.0E+11

1.0E+12

1.5E+12

2.0E+12

0 100 200 300 400 500 600 700

AAV9

=ter(VG

/mL)

Flowrate(cm/h)

CapacityofPOROSAAV9

Eluate

FlowThrough

M. Hebben

CONFIDENTIAL

AAV9 Purification Process

Clarification Immuno Affinity Chromatography

Concentration and Formulation

Sterile Filtration

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Fill & Finish

200L 50mL

M. Hebben

CONFIDENTIAL

Process Yield

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0

20

40

60

80

100

120

POROSAAV9 TFF STERILEFILTRATION

%vectorrecovery

Vectorrecoveryatkeysteps

Batch#1

Batch#2

Batch#3

•  ≥ 80% vector recovery at each step

•  Overall process yield = 50 to 65%

•  Reproducibility at 10L and 50L scales

M. Hebben

CONFIDENTIAL

Process Efficiency to Remove DNA

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-5

-4

-3

-2

-1

0

DNAredu

c=on

(log)

BaculovirusDNAremoval

Batch#1

Batch#2

Batch#3

Total process allows reduction of 4 log of residual DNA

M. Hebben

CONFIDENTIAL

Conclusions

•  ManufacturingofAAV9vectorsfeasibleatlargescale–  Processefficiencyconfirmedat50Lscale–  Scaleupto200Lscaleinprogress

•  Immunoaffinitychromatographyallowsasimple,efficientandhighyieldprocess–  Genericproduc@onplaCormforAAV9vectors

•  Fullprocessgeneratesfinalproductaround1e13VG/mL

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M. Hebben

CONFIDENTIAL

Thanks

Généthon–  FulvioMavilio,CSO

•  BioprocessDevelopment–  MohammedRisi–  LudivineDejoint–  LaurentBortolussi–  NicolasMarceau–  ChristopheLecomte–  FrancisBoussicault–  DelphineDufour–  LaurenceGuianvarc’h

•  Analy@cs–  Chris@neLeBec–  BrunoDalle

•  R&D–  OfoMerten–  SamiaMar@n–  FedericoMingozzi

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