Centre de Produc[on de Vecteurs (CPV) Research grade vector core



Centre de Produc[on de Vecteurs (CPV) Research grade vector core
-­‐ 2008 -­‐ 2006 Centre de Produc,on de Vecteurs (CPV) Research grade vector core & Development Ac,vi,es Véronique Blouin -­‐ UMR1089 – 28/03/2013 Gen2Bio-­‐ Nantes Laboratoire Thérapie Génique (INSERM U1089)
(26 pers)
Transfert de gènes in vivo
- rétine
(essai clinique)
- cerveau
- muscle
- immunologie
- pharmacologie
Valider les études pré-cliniques
chez le petit et le gros animal
CPV: Centre de Produc,on de Vecteurs Plateforme de
(17 pers)
(11 pers)
vecteurs viraux grade
Outils et procédés de
1- production
2- purification
3- contrôles qualité
vecteurs viraux
Essais cliniques (I/II)
Plateforme de production
vecteurs cliniques en 2009
Les étapes où nous pouvons intervenir dans un projet de thérapie génique? (1) Iden,fier le gène (2) Développer et produire le vecteur-­‐médicament (3) Tester le traitement chez l’animal (rongeur, chat, chien) (efficacité et innocuité) Gène médicament (6) Essais cliniques (5) Produc,on de vecteurs de grade clinique (GMP) (4) Etudes Précliniques chez le primate (Toxicité, innocuité) 3 Centre de Produc,on Vecteur (CPV) Missions & objec,fs “Vector Core” grade Recherche
Produire et caractériser les lots de vecteurs viraux de grade recherche et pré-­‐clinique à pe,te et moyenne échelle pour des équipes académiques ou privés dans le but de permeZre: -­‐ Etudes de Faisabilité -­‐ Preuve de concept in vitro & in vivo (pe,t animal et faibles cohortes gros animal) -­‐ Etudes pré-­‐cliniques gros animal Améliorer les process de produc,on grade recherche des vecteurs viraux (augmenta,on ,tres, qualité , pureté…) Développement : vecteurs grade Clinique
Développer et améliorer les process de produc,on dans le but de les transférer sur plateforme de vecteurs cliniques (ABG or Généthon) selon les recommanda,ons GMP : -­‐ Design ou,ls innovants: améliorer qualité et/ou ,tre des vecteurs rAAV -­‐ Tester des nouveaux/innovants supports cellules et
process de produc,on pour vecteur viral -­‐ Iden,fica,on/test de supports nouveaux/innovants pour purifica,on AAVr -­‐ Développement tests analy,ques (CQ) -­‐ Développement de process GMP-­‐compa,bles pour produc,on pe,te/moyenne échelle de vecteurs viraux (AAVr & AdénoV) Viral vector produc,ons for preclinical applica,ons Services Recombinant Adeno-­‐Associated Virus produc,on -­‐ Hybrid vectors (ITR AAV2), Serotypes 1, 2, 4, 5, 6, 7,8, 9 ,rh 10 -­‐ single strand and self-­‐complementary rAAV
Recombinant Adenovirus produc,on (Ad5) -­‐ Produc,on by homologous recombina,on or amplifica,on +/-­‐ plaquing Recombinant len,virus produc,on Service stopped in 2012 -­‐ HIV 2nd and 3rd genera,on ( VSVg envelop)
Vector plasmid constructs -­‐ strategy, plasmid design, molecular cloning, vector plasmid amplifica,on for produc,on Detec,on of an,-­‐ AAV (or Adv) cellular factors into serum samples Immortaliza,on / primary cells transduc,on Serotypes producQon: AAV -­‐1 to AAVrh10 AAV2 AAV5 AAV8 6 Research grade viral vector requests from the last 10 years Viral vector batches orders since 2003 to 2012 500 22 450 11 400 11 350 14 20 300 250 152 379 386 133 148 134 98 259 70 45 48 55 41 2009 2010 2011 2012 0 2003 2004 2005 2006 2007 2008 Since 2008: amount of rAAV vectors orders con,nue to increase 2012 : 440 viral vectors batches 88% of rAAV AAV Adv 315 194 164 100 50 144 92 111 LV 15 181 200 150 13 23 8 Production and purification
of rAAV vectors: research grade
Classical method of manufacturing: Transient transfec,on ITR-2
pAAV Triple transfection
Double transfection
Rep-2 cap-X
E4 Rep-2 cap-X
pDG pAd pRC E2 E4 VA HEK293 cells
(E1 gene)
E2 VA Purifica,on of research grade rAAV vectors Harvest
Cell pellet
EXTRACTION Detergent + benzonase treatment CLARIFICATION Supernatant
CentrifugaQon PEG precipitation
PURIFICATION CsCl density gradient (X2) – swing rotor Purified rAAV
Buffer exchange (dialysis) 175 kDa
83 kDa
62 kDa
48 kDa
empty 33 kDa
full 25 kDa
SDS-­‐PAGE + Silver staining Snd gradient (SW 41) first gradient (SW 28) Characteriza,on of research grade rAAV vector ✓  Quan,fica,on of vector genome: ,ter is expressed as (vg/ml) QuanQtaQve PCR with vector specific primers/probe (poly A or ITR-­‐2) ✓  Quan,fica,on of infec,ous par,cles: ,ter is expressed as (ip/ml) InfecQon of HeLa RC cells & adenovirus, Transgene probe ✓ For large animal applica,on: Endotoxin free DNA preparaQon used sterile filtraQon Purity on rAAV prep Development and improvement of producQon processes for Clinical grade vectors An alternaQve to transient transfecQon : Development of A549-­‐based rAAV (5,8,9) producer cell lines, characteriza,on and produc,on of rAAV in bioreactor Establishment and characteriza,on of a generic producer cell line through targeted integra,on of rAAV vector: From stable packaging cell lines available: genera,on of producer cell clones A549 Rep2Cap5 rep 2! cap X!
rep 2! cap X!
A549 Rep2Cap8 A549 Rep2Cap9 A549/rep-cap cells!
A549 producer cells From producer cell line: One infec,on step to produce rAAV Requirement for clinical produc,on: ✓ 1 MCB (+ 1 WCB) for the AAV producer cell line ✓ 1 MVS (+1 WVS) for the Ad helper virus ✓1 WCB for the HEK293 cells used for Ad produc,on Acquisi,on of the Sf9/baculovirus rAAV produc,on system : transfer the technology developed by Généthon First Goal: availability of a small scale manufacturing process of rAAV vectors Dual system (wt): from R. Kotin "è 2 recombinant baculovirus"
1.  Bac rep2-Capx express !
!√ AAV structural of the corresponding serotype (x = 8, 9, rh10) !
√ Rep78 & Rep52 involved in DNA replication and packaging of serotype-2!
Bac-rAAV express!
!√ Transgene cassette with ITR-2!
Cap ORF p10 polh
Rep 78/52 pA transgene Sf9 Insect cells infec,on (MOI: 0,1) Suspension Sf9 cell
culture: in spinner "
rAAV par,cles Acquisi,on of the Sf9/baculovirus rAAV produc,on system : §  Perspec,ves: •  Transfer of the SF9/baculovirus technology in 2 to 10 liters bioreactors for producQon and characterizaQon of rAAV serotype 8 and 9. Including adaptaQon of the purificaQon process to the rAAV of gene interest •  Provide rAAV-­‐bulk to start development of GMP purificaQon process based on IEX •  Process scale-­‐up to 10 liters including QC development, implementaQon of the technology in the GMP facility corresponding to process adaptaQon to fulfil the GMP requirements 2 Biostat B Twin MO 2L, 10L: for recombinant baculovirus batches & rAAV producQon Development of the produc,on for adenoviral vectors based on the use of the iCELLis fixed-­‐bed bioreactor system Pre-­‐clinical grade small-­‐scale USP and DSP development using the 1m2 iCELLis Nano Bioreactor ✓ Manufacturing and vector produc,on steps : Up Stream Process (USP) • Selec,on of the iCELLis Disposable Fixed-­‐Bed Reactor technology • Cells are seeded at a very low density and grow in the fixed bed • High cell densiQes can be reached in a small bioreactor volume • Parameters are measured and controlled: pH, DO, agitaQon speed • PotenQally high volumetric producQviQes • Compact, simple, single use and scalable system è iCELLis™Nano (scale down unit): a plug and play system for feasibility studies and small-­‐scale virus produc,on. NaOH Media in
T probe Vent DO & pH probes Sampling Media Air/
CO2/ out O2 Cap ch component
iCELLis Nano for R&D-­‐ from 0,53 to 5,35m2 y
Sampling iCELLis 200 for manufacturing – up to 200m2 iCELLis 1000 for manufacturing -­‐ up to 1000m2 g
Proprietary and Confidential ©2010 ATMI, Inc. All Rights Reserved.
Media Impeller in Fixed-­‐
bed Development of the produc,on for adenoviral vectors based on the use of the iCELLis fixed-­‐bed bioreactor system Pre-­‐clinical grade small-­‐scale USP and DSP development using the 1m2 iCELLis Nano Bioreactor ✓ Purifica,on process development: Down Stream Process (DSP) Iden,fica,on of the DSP supports and sequence 1. Depth FiltraQon 2. (anionic IEX) Column 3. TFF 4. Sterilizing FiltraQon (0,2 µm) – formulaQon Defini,on of the purifica,on parameters • DefiniQon of binding and eluQon condiQons (NaCl concentraQon) on IEX column • SaturaQon test on the column amount of vector genome injected and binding recovery on the column è to specify capacity of the column • Capacity test of the depth filter for large scale sizing Scalable purification processes for clinical-grade rAAV
Cells + supernatant
Cells disrupQon: mechanical (high-­‐pressure) or chemical (detergent) Filtration
Formulation through tangential flow-filtration
(buffer exchange + concentration or dilution)
Sterile filtration
Quality Control, Analy,cal methods Development • 
Development of quality control for process « valida,on » and new produc,on tools : AAV packaging cell line, AAV baculovirus system, AAV transfec,on, Adenovirus Seed lot produc,on –  OpQmizaQon of exisQng controls (total protein and DNA measurement during the downsteam and upsteam steps of producQon…) –  Development of new assays (residual proteins contaminaQon and residual DNA quanQficaQon in the purified bulks, new methods of vector quanQficaQon…) –  Development of in vitro Assays: InfecQous genome (ig), replicaQve competent AAV (rcAAV) –  Improvement of our knowledge on Quality and Safety aspects of parQcles (analysis between the different producQon methods) •  EncapsidaQon of non expected DNA genes in AAV parQcles, •  AnalyQcal chromatography, •  Analysis of the size of residual DNA •  CollaboraQon with Genethon to beoer characterize our product (empty/full capsids with ultracentrifugaQon method for example) Collabora,on with Ansm Produc,on des lots vecteurs viraux cliniques Atlan,c Bio-­‐GMP (ABG) St Herblain Ø  The rAAV2/4.hrpe65 clinical batch vector has been produced : •  Lot ACL rpe65.31082010SP2cg •  30 ml at 6.10e10 vg/ml •  AsepQcally filled in 60 doses of 500 µL : –  24 paQents doses (9 paQents x 2 doses + 6 back-­‐up doses) –  24 QC doses –  12 library doses 20 Principales Etapes PrésentaQon du programme et obtenQon du financement AFM Janvier-­‐mai 2007 Soumission dossier Orphan Drug / obtenQon de la désignaQon Juin 2007 / 22 octobre 2007 (ACL) et 14 novembre 2007 (RP) Présoumission Afssaps 24 mai 2007 ProducQon du lot préclinique juillet 2007 – juin 2008 Etude tox et biodi réglementaire Septembre 2008 – Février 2010 PrésentaQon et obtenQon du financement PHRC Décembre 2008 – mai 2009 ProducQon du lot clinique Juin 2009 – janvier 2011 Soumission Afssaps 26 janvier 2011 21 CPV Team Philippe Moullier: scientific director
Research grade Vector Core
Katell Harrouet
Solène Volant
Linda Poiraudeau
Armelle Cassard
Karine Pavageau
Nathalie Caillé
Dominique Aubert
Christine Audrain
Lucie Menard**
Françoise Balter
** Vector core & Development
Development for Clinical grade vectors
Achille François
Sylvie Saleun
Elodie Autrusseau
Estelle Toublanc
Christophe Chevé
Nicole Brument
Aline Roblin
Maylis Pennors
Aurélien Jacob
Florian Seigneuret*
Cécile Robin
Marie Enga*
Sandy Douthe*
Mélanie Martin*
Emilie Lecomte
Frédéric Broucque**
Christophe Darmon*
*ABG in part

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