lentiviral vector production core indiana university vector production facility principal...
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Lentiviral Vector Production Core
Indiana University Vector Production Facility
Principal Investigator: Ken Cornetta, M. D. Co-Principal Investigator : Lakshmi Sastry, Ph.D. Co-Principal Investigator : Daniela Bischof, Ph.D.
Philosophy
• Focus on bringing forth novel improvements in integrating vectors
• Work aimed at Phase I/II products
• Major aim is to serve academic community
• In addition to production, focus on developing new release testing (viral vector specific)
• Prior experience with retroviral vector production through the now defunct National Gene Vector Laboratory program
• Currently maintain > 50 SOP related to vector production, certification and facility organization
• DMF for lentiviral vectors filed with FDA• Audit by BCG for retro production in 2000• Audit by BCG in 2005 for lenti production• Audit by FDA in 2003 without major deficiencies
Infrastructure
Organization Chart
Production Team
Supervisor
Certification LaboratorySupervisor
Molecular DiagnosticsSupervisor
IU VPF Manager Administrator
Management
Technicians Technicians Technicians
Erol CetinokQA Specialist
Rafat Abonour, M.D.Director, IUSCC Clinical Research Office
Stephen Willaims, M.D.IU Simon Cancer Center Director
Quality Assurance
Legend1: Biological safety cabinets2: Incubators3: Refrigerators4: -20°C Freezers5: -70°C Freezers6: Storage racks Lab benches or shelves Sinks Pass through autoclave Clean room pass over line
R4-029 Floor Plan
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Funded in part by a NCRR Construction grant
Institutions Receiving VectorInstitutions Receiving Vector
• Case Western ReserveCase Western Reserve• Cincinnati Children’s HospitalCincinnati Children’s Hospital• Columbia UniversityColumbia University• Dana-FarberDana-Farber• Fred Hutchinson Fred Hutchinson • Indiana UniversityIndiana University• LA Children's HospitalLA Children's Hospital• MD AndersonMD Anderson• New England DeaconessNew England Deaconess• NIHNIH• Stanford UniversityStanford University• University of MichiganUniversity of Michigan• University of WashingtonUniversity of Washington• Washington UniversityWashington University
Retroviral Vectors Lentiviral Vectors• University of WisconsinUniversity of Wisconsin• University of WashingtonUniversity of Washington
Producing Lentiviral Vectors
LENTIVIRAL VECTORSLENTIVIRAL VECTORS• Vector integrates Vector integrates
• Gene transfer rate can exceed 90% Gene transfer rate can exceed 90%
• Less dependent on cell cycle Less dependent on cell cycle
• Vectors (VSVG psedotyped) can be Vectors (VSVG psedotyped) can be concentrated to high titerconcentrated to high titer
• Possible risk of insertional mutagenesisPossible risk of insertional mutagenesis
• Possible risk of replication competent virusPossible risk of replication competent virus
ProPro
ConCon
Types of Lentiviral Vectors
1. HIV-1/HIV-2, FIV, SIV, EIAV
2. HIV-1 vectors most commonly used
3. Process developed for HIV-1 vector production
CMV gag RRE polyApro pol
RSV REVpRev
5’LTR gag SIN-3’LTRpsi
RRE CMV GFPTransfer vector
Packaging Construct
CMV VSV-Gß-globin intron
pMD.G
5’LTR pro pol
vif
vpr
vpuEnv
tatrev
3’LTR
nef
psiHIV
Lentiviral Vector Plasmids
gag
Production of LentivirusProduction of Lentivirus
CollectCollectSupernatantSupernatant
Hollow Fiber Tangential Flow FiltrationHollow Fiber Tangential Flow Filtration
Continue concentration100-200 fold
Perform Diafiltration 6 volume exchange
Add Benzonase
Concentration by Ultrafiltration to 2 Liters
Conduct Transient Transfection10 liters, serum free
Store overnight at 4 CHarvest second 10 liters day 2
CaPO4
*Serum free Media
Change
20 Liter Production
*16 to 24 hours post transfection
Final Product (Vial + Certify)
20 Liter Production Runs20 Liter Production Runs
200-400 fold concentration with recovery of IU 80%
Vector CertificationVector Certification
Master Cell Bank
Production Run
Filter and Vial
Certification
Certification
Sterility, Mycoplasma, In vitro, In vivo, bovine, porcine, Cell Identify, Human viruses
Sterility, Mycoplasma, RCL, Titer, Endotoxin, SV40/E1A transfer Vector integrity
RELEASE TESTING
MCB
SterilityMycoplasmaIn Vitro viral assayIn Vivo viral assayBovine virusesPorcine virusesHuman virusesCell identity
Vector Supernatant
SterilityMycoplasmaEndotoxinIn Vitro viral assayVector Insert StabilityTransfer of E1A, SV40RCL (supernatant)RCL (co-culture)P24 TiterInfectious TiterTransgene expression
Core Services for GTRP InvestigatorsProduce clinical-grade lentiviral vectors for use in heart, lung, and
blood clinical studies
• Provide pilot runs for pre-clinical evaluation prior to production of large- scale vector runs
• Generate vectors under cGMP using envelopes and media tailored to the investigators needs
• Assist in release testing to certify vectors for clinical use
• Assist investigator with FDA required documentation
Factors Influencing Quality of Lentiviral Vectors
• Assessed by Potency, Safety and Stability
• Influenced by production and processing methods
Physical titers predict1 Infectious particles per 1000 Virions
Kahl et al. J. Virol 78: 1421, 2004
Vectors contain transduction inhibitors or defective particles
1.00E+05
1.00E+06
1.00E+07
1.00E+08
1.00E+09
1.00E+10
1.00E+11
VSV-G RRV
Infectious TiterPhysical Titer (p24)RNA Molecules/mL
•HIV-1 Vectors not Uniform-Vector particles of Expected Size (80-140 nm)-Smaller particles (30-50 nM)-Aggregates
HIV-1 Vector HIV-1(CSCGW/VSVG) (R7-GFP)
50 nm
D
500 nm100 nm
Transmission Electron Microscopy (TEM) of HIV-1 Vectors
Concentration of Vectors Removes Smaller Particles
Vector/beads ---- Stain ---- Particle size distribution (Count ~ 200 particles/5 grid spaces)
aggregates not quantitated
Pseudotyping Envelope Influences HIV-1 Vector Composition
POSTERFactors Influencing Lentiviral Vector
Quality-Dynamic Light Scattering (DLS) Analysis of HIV-1 Vectors
(Formulation/Storage)
Challenges/Future Goals
• Increase scale of production
• Packaging cell lines
• Simplification of RCL testing
• Continued development of release testing
IU CollaboratorsIU CollaboratorsKaren Pollok, Ph.D.Karen Pollok, Ph.D.Laura Haneline, Ph.D.Laura Haneline, Ph.D.Christie Orschell, Ph.DChristie Orschell, Ph.DEddy Srour, Ph.D. Eddy Srour, Ph.D. Vince Gatone, Ph.D.Vince Gatone, Ph.D.Mike Vasko, Ph.D.Mike Vasko, Ph.D.
CollaboratorsCollaboratorsDavid Sanders, PurdueDavid Sanders, PurduePhil Zoltick, U. of PennPhil Zoltick, U. of PennRick Morgan, NIH, NCIRick Morgan, NIH, NCIJeremy Luban, ColumbiaJeremy Luban, Columbia
IU- VPFIU- VPFKen CornettaKen CornettaScott CrossScott CrossLisa DuffyLisa DuffyLaksmi Sastry, PhDLaksmi Sastry, PhDDaniela Bischof, PhDDaniela Bischof, PhD
Clara HazelgroveClara HazelgroveSue KoopSue KoopLina SegoLina SegoJing YaoJing YaoSamantha GriffinSamantha GriffinAparna JastiAparna JastiLorraine MathesonLorraine MathesonErol CetinokErol Cetinok
Lab AlumniLab AlumniGuiandre JosephGuiandre JosephChristoph KahlChristoph KahlShangming Zhang, M.D.Shangming Zhang, M.D.
Support byNHLBI: HHSN26820074820 and P01 HL53586 (Dinauer)NCI: N02-RC-67002NCRR: U42 RR11148Lilly Endowment: Indiana Genomics Initiative (INGEN)
Department ofMedical and
Molecular Genetics
Understanding defective particles versus Understanding defective particles versus defective infection.defective infection.
gag/polgag/pol
envenv
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