Maxcyte, Inc. · NASDAQ:MXCT
Maxcyte — Technical Presentation (2007)
26 slides from Maxcyte's technical presentation, 2007
Slide 1: Large Scale Production of Lentiviral Vectors Using Adherent and Suspension-Adapted Cells
Large Scale Production of Lentiviral Vectors Using Adherent and Suspension- Adapted Cells James P. Brady, Ph.D, Matthew Malehorn, Joseph C. Fratantoni, M.D., Linda N. Liu, Ph.D. and Madhusudan V. Peshwa, Ph.D. MaxCyte, Inc.
Slide 2: Agenda
Agenda • Background • Lentivector Production in Adherent Cells • Lentivector Production in Suspension Cells • Summary & Conclusions
Slide 3: Lentiviral Vectors
Lentiviral Vectors Advantages ¾ Integrate into genomes of dividing and non-dividing cells ¾ Easily pseudotyped ¾ Large transgene capacity Challenges ¾ Biosafety concerns – viral components must be encoded by multiple plasmids ¾ Stable producer cell lines are difficult to generate due to toxicity of
Slide 4: Electroporation: Efficient Delivery of Various Molecules into Primary Cells
Electroporation: Efficient Delivery of Various Molecules into Primary Cells Electroporation mRNA, siRNA DNA Protein Adapted from Glover et al. Nature Genetics (2005)
Slide 5: MaxCyte GT System
MaxCyte GT System Computer controlled, cGMP compliant, aseptic processing Processing Volume: 0.02 – 100 mL (5 sec – 5 min) Processing Capacity: 5 x 105 – 1 x 1010 cells OC-series CL 1 CL 2
Slide 6: Core Technology Platform
Core Technology Platform Flow Electroporation Scalability Is One of the Key Elements Moving a Product Concept from Bench to Bedside
Slide 7: Transient Transfection for Cell/Gene Therapeutics Production
Transient Transfection for Cell/Gene Therapeutics Production High yield, reproducible & scalable process 250x 100 90 80 Cell Viability (%) eGFP+ Cells (%) 70 Small scale (24x106 cells) Scale-up (6x109 cells) 60 50 40 30 20 10 0 4 8 12 16 20 24 28 Collected Fractions
Slide 8: Agenda
Agenda • Background • Lentivector Production in Adherent Cells • Lentivector Production in Suspension Cells • Summary & Conclusions
Slide 9: Four Plasmid HIV-Based Lentivector System
Four Plasmid HIV-Based Lentivector System “Vector” “Gag/pol” pCDNA-CS-CGW pMDLgpRRE CMV:eGFP CMV:gag/pol Packaging signal RRE 9.2 kb 8.9 kb pMDG “VSV-G” CMV:VSV-G “REV” pRSV-REV 6.1 kb RSV LTR:REV 4.2 kb Plasmids courtesy of Dr. Ken Cornetta, Indiana University School of Medicine
Slide 10: Optimization of Parameters for Small-Scale Lentivector Production in Adherent Cells
Optimization of Parameters for Small-Scale Lentivector Production in Adherent Cells Collect 293FT cells (IVGN) by Parameters Tested trypsinization (5-20 x 106 cells/EP) ¾ Relative plasmid molar ratios Resuspend cells in EP buffer + plasmid DNA ¾ Total DNA concentration ¾ Cell concentration during St
Slide 11: Lentivector Production in T25 flasks
Lentivector Production in T25 flasks Day (-1): Day 0: Days 1 & 2: Seeded cells into Collected cells, counted, EP’d Collected media T25 Flasks at 5 & returned to T25 with 4 mL different densities medium + Na Butyrate + [cell] = 1 x108/mL # Cells # Cells Plated Transfected [DNA] = 400 µg/mL Flask 1 1.
Slide 12: Transfected Cells, 24 Hrs Post EP
Transfected Cells, 24 Hrs Post EP Flask 1 Flask 2 Flask 3 Flask 4 Flask 5
Slide 13: Lentivector Titers in T25 Flasks
Lentivector Titers in T25 Flasks 1.0E+08 3.4 3.8 3.1 2.5 1.6 9.7 1.1 1.1 1.0E+07 0.8 TU/mL 24 hrs 0.2 1.0E+06 48 hrs 1.0E+05 Flask 1 Flask 2 Flask 3 Flask 4 Flask 5
Slide 14: Large-Scale Lentivector Production with Electroloaded 293FT Cells
Large-Scale Lentivector Production with Electroloaded 293FT Cells Day (-2) or (-3): Day 0: Days 1, 2 & 3: Seeded cells into a Harvested cells Collected media 10 tier cell factory Transfected by flow EP in CL-2 & static EP in OC-100 4 x 108 1-2.5 x 109 CL-2 1L [cell] = 1 x108/mL [DNA] = 200 µg/mL Rel
Slide 15: Lentivector Titers and Total Lentivector Production in 10-Tier Cell Factories
Lentivector Titers and Total Lentivector Production in 10-Tier Cell Factories # Cells # Cells EP DNA Plated Transfected Volume Used 4 x 108 Experiment 1 2.5 x 109 25 mL 5 mg (day -3) 4 x 108 Experiment 2 1.5 x 109 15 mL 3 mg (day -2) 1.0E+08 1.0E+11 2.1 x 1010 Transducing Units (T.U.) 1.2 x 1010 1.5
Slide 16: Large Scale Lentivector Production Efficiencies
Large Scale Lentivector Production Efficiencies 12 6.0E+06 10 5.0E+06 8.2 8.2 4.1 4.1 T.U./µg DNA 8 T.U./Cell 4.0E+06 6 3.0E+06 4 2.0E+06 2 1.0E+06 0 0.0E+00 Experiment Experiment Experiment Experiment 1 2 1 2
Slide 17: Agenda
Agenda • Background • Lentivector Production in Adherent Cells • Lentivector Production in Suspension Cells • Summary & Conclusions
Slide 18: Advantages of Suspension Cells
Advantages of Suspension Cells ¾ Ease in scale-up ¾ Reduced cost of labor & materials ¾ Serum-free media ¾ Homogeneous culture ¾ Streamlined downstream processing
Slide 19: Adapting 293FT Cells to Suspension Growth in Serum Free Medium
Adapting 293FT Cells to Suspension Growth in Serum Free Medium 3.0E+06 100% 90% 2.5E+06 80% % Viable 70% Cells/mL 2.0E+06 60% 1.5E+06 50% 40% 1.0E+06 30% 20% 5.0E+05 10% 0.0E+00 0% 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 8 Freestyle™ Day Day medium 500 µg/mL 0% ACA 0.1% ACA 1.0% ACA Geneticin® ACA = Anti-Cl
Slide 20: Optimizing Instrument Settings for Transfecting Suspension Cells: 24 hr FACS Analysis
Optimizing Instrument Settings for Transecting Suspension Cells: 24 hr FACS Analysis 9 x 105 cells + 6 µg pCMV:eGFP % eGFP Viability 100% 2500 90% 80% 2000 70% 60% 1500 MFI 50% 40% 1000 30% 20% 500 10% 0% 0 Condition Condition Condition Condition Condition Condition Condition Condition Condition Con
Slide 21: Small-Scale Lentivector Production in Suspension-Adapted FT Cells
Small-Scale Lentivector Production in Suspension-Adapted 293FT Cells T175 shake flask Freestyle™ medium 500 µg/mL Geneticin® 1 x107 cells [cell] = 5 x107/mL [DNA] = 400 µg/mL EP 3 Conditions Relative Molar Ratios Vector 2 Rev 1 T25 Shake Flask 4 mL Freestyle™ Gag/pol 1 No Geneticin® VSV-G 0.1 5 mM N
Slide 22: Lentivector Titers Generated by Small-Scale Electroloading of Suspension Cells
Lentivector Titers Generated by Small-Scale Electroloading of Suspension Cells 1.0E+08 1.8 1.9 1.6 0.9 1.0E+07 T.U./mL 0.7 0.5 24hrs 1.0E+06 48hrs 1.0E+05 Condition Condition Condition 2 3 4
Slide 23: Large-Scale Lentivector Production in Suspension-Adapted FT Cells
Large-Scale Lentivector Production in Suspension-Adapted 293FT Cells Day (-3): Day 0: Days 1, 2, 3 & 4: Seeded cells into 10 Harvested cells Collected media shake flasks Transfected by flow EP in CL-2 & static EP in OC-100 1.7 x 108 1 x 109 10 X 100 mL CL-2 4 X 100 mL 10 mL [cell] = 1 x108/mL [DNA]
Slide 24: Lentivector Titers Resulting from Large Scale Electroloading of Suspension Cells
Lentivector Titers Resulting from Large Scale Electroloading of Suspension Cells 1.0E+08 0.7 0.8 1.0E+07 24 hrs T.U./mL 0.4 0.3 48 hrs 0.2 72 hrs 1.0E+06 96 hrs 0.06 0.03 0.03 1.0E+05 10 mL 100 µl
Slide 25: Total Lentivector Production Resulting from Large-Scale Electroloading of Suspension Cells
Total Lentivector Production Resulting from Large-Scale Electroloading of Suspension Cells 1.0E+10 4.7 x 109 Transducing Units (T.U.) 1.0E+09 96 hrs 72 hrs 48 hrs 24 hrs 1.0E+08 1.0E+07 10 mL EP (1 x 109 cells)
Slide 26: Conclusions
Conclusions ¾ Adherent 293FT cells electroloaded with four lentivector component plasmids using the MaxCyte GT instrument yielded lentiviral titers >3x107 T.U./mL. ¾ Suspension-adapted 293FT cells could be transfected efficiently with plasmid DNA using the MaxCyte system. ¾ Lentivector titers >1x107