BILS 2015 Genethon M. Hebben
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Transcript of BILS 2015 Genethon M. Hebben
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
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
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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
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
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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
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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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