Post on 15-Apr-2017
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
11/02/2015
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
11/02/2015
M. Hebben
CONFIDENTIAL
Gene Therapy Evolution since 1980
1980 1990 2000 2010 2020 2030
Proof of concept Clinical Trials
Commercial Phase
11/02/2015
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!
11/02/2015
M. Hebben
CONFIDENTIAL
AAV Vectors
11/02/2015
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
11/02/2015
Transgene cassette DNA
AAV vector
M. Hebben
CONFIDENTIAL
AAV9 as a Gene Therapy Vector
11/02/2015
• 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
11/02/2015
• 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
11/02/2015
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
11/02/2015
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
11/02/2015
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?
11/02/2015
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
11/02/2015
• 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
11/02/2015
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
11/02/2015
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
11/02/2015
-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
11/02/2015
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
11/02/2015