Developing a Scalable Upstream Bioreactor Process for Lentiviral Vector Production in Suspension
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This document describes the development of a scalable upstream bioreactor process for lentiviral vector production using suspension cell culture. The goals were to design a manufacturing-friendly process that supports commercialization. A 50L bioreactor process was developed using a chemically-defined medium and HEK293T cells optimized for suspension growth. The process was demonstrated to be scalable through geometric similarity across bioreactor sizes from 3L to 2000L. This template process provides benefits like speed to patients, reliability, robustness, and efficiency for lentiviral vector manufacturing.
Developing a Scalable Upstream Bioreactor Process for Lentiviral Vector Production in Suspension
- 1. Eva Fong February 25, 2021 Developing a scalable upstream bioreactor process for lentiviral vector production in suspension
- 2. 2 The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada. Developing a Scalable Upstream Bioreactor Process for Lentiviral Vector Production in Suspension
- 3. 3 Agenda Scale-up development of a suspension-based lentivirus production process The benefit of having a template manufacturing process 1 2 3 Designing a process that is manufacturing-friendly and supports commercialization The Importance of Scale in Viral Vector Manufacturing| 01.10.2020 Developing a Scalable Upstream Bioreactor Process for Lentiviral Vector Production in Suspension
- 5. in vivo and ex vivo Viral Gene Therapy 5 Gene Modified Cell Therapy Gene Delivery Therapy Oncolytic Virus Therapy Immunotherapy Viral Gene Therapy Gene Therapy (broad definition) Gene Therapy (narrow definition) e.g. AAV (in vivo) e.g. Lentivirus (ex vivo) HSC CAR-T, TCR-T Developing a Scalable Upstream Bioreactor Process for Lentiviral Vector Production in Suspension
- 6. VirusExpress™ Lentiviral Production Platform Key challenges in lentiviral production 6 Scale-out limitations Serum-containing medium Highly labor intensive Open process True scale-up Chemically defined medium Reduced labor More closed process Developing a Scalable Upstream Bioreactor Process for Lentiviral Vector Production in Suspension
- 7. VirusExpress Lentiviral Production Platform 1 2 3 EX-CELL® CD HEK293 Viral Vector Medium Chemically defined media for PEI transfection and vector production. VirusExpress 293T Lentiviral Production Cells 293T clone optimized for suspension growth and PEI based transient LV production. Growth and virus production demonstrated in bioreactors up to 40 L scale. Lentiviral Production Process Demonstrated scalable process from flasks to 50L. 7 Developing a Scalable Upstream Bioreactor Process for Lentiviral Vector Production in Suspension
- 8. Bench Scale Bioreactor Process Development Experimental Approach 8 Phase 1 – Develop parameters for cell growth Phase 2 – Develop parameters for virus production Design of Experiment Design Data Analysis Execute Defined process parameters - pH, DO & Sparger Type and Agitation Rate Understand process performance 3 L Mobius® 50L Mobius® Proceed to scale-up development Developing a Scalable Upstream Bioreactor Process for Lentiviral Vector Production in Suspension
- 9. Scaling – Geometric Similarity Mobius® Single-use Bioreactor Family 9 Developing a Scalable Upstream Bioreactor Process for Lentiviral Vector Production in Suspension Ratio Working Volume : Total Volume 0.8 0.8 0.8 0.8 0.8 Impeller Diameter : Vessel Diam. 0.6 0.3 0.3 0.3 0.3 Vessel Height : Vessel Diameter 1.8:1 2.0:1 2.0:1 2.0:1 2.0:1 Liquid Height : Vessel Diameter 1.4:1 1.7:1 1.6:1 1.6:1 1.6:1 Dual sparger (open pipe/ micro) YES YES YES YES YES Min – Max Working Volume (L) 1 – 2.4 10 – 50 40 – 200 200 - 1000 400 – 2000 2000 L 1000 L 200 L 50 L 3 L H D Dimp
- 10. VirusExpress Lentiviral Production Platform Bioreactor Scale-up Challenges Many factors influence optimal cell growth, viability and viral vector production • Mass transfer of gasses (kLa, vvm) Sufficient O2/Air delivery • Mixing efficiency (Re) A higher Reynold’s number is an indication of turbulent flow • Tip Speed (m/s) Influences mixing time and shear stress to cells • Power per unit volume (P/V)* Influences mixing; based on impeller design and fluid density * Common approach for bioreactor scale up includes determining an agitation rate that delivers equivalent energy dissipation rate or power per unit volume (W/m3). Agitation rates for both growth and lentivirus production studies were determined using the power per unit volume equation where (Po/V) = ρ*(N/60)^3*D^5*Np*Ni/V. The impeller design, fluid density, and agitation rate are considered in the equation. 10 Developing a Scalable Upstream Bioreactor Process for Lentiviral Vector Production in Suspension
- 11. Inoculate 50L bioreactor for N-1 Day 0 Volume-up to N-stage production volume Day 3 Transfection Day 4 Monitor & Sample Day 5 Harvest Day 6 Growth Lentivirus Production Transfection Our scale-up approach Maintain the same power per unit volume Linear scale-up of gasses Linear scaling of transfection parameters (transfection cocktail to culture volume) Utilize the same production vessel for cell expansion (N-1) and for production (N-stage) Mimic the two phase approach utilized at bench scale; Phase 1 – optimize for cell growth, Phase 2 – optimize for virus production VirusExpress Lentiviral Production Platform 50 L Mobius Bioreactor LV Production Process Flow 11 Developing a Scalable Upstream Bioreactor Process for Lentiviral Vector Production in Suspension
- 12. Inoculate 50L bioreactor for N-1 D0 Volume-up to N-stage production volume D3 Monitor D4-7 40 50 60 70 80 90 100 0 5 10 15 20 25 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 Viability (%) Viable Cell Density (E6 cells/mL) Culture Duration (Days) 50L BRX 500mL SF: N-1 stage Ctrl A 500mL SF: N-stage Ctrl A 500mL SF: N-1 stage Ctrl B 500mL SF: N-stage Ctrl B Volume Up • Cell growth at N-1 stage was able to achieve target VCD for transfection • Peak VCD achieved on D7 was ~ 17x10^6 vc/mL VirusExpress Lentiviral Production Platform Scale-up Phase 1: Growth Optimization 12 Developing a Scalable Upstream Bioreactor Process for Lentiviral Vector Production in Suspension
- 13. ▪ Consistent growth profiles and achieved target cell density at time of transfection ▪ Target titer of ≥ 1e+07 TU/mL achieved for our confirmation runs ▪ Cell viabilities remained relatively high at time of harvest, ≥ 85% VirusExpress Lentiviral Production Platform Scale-up Phase 2: Virus Production 13 Developing a Scalable Upstream Bioreactor Process for Lentiviral Vector Production in Suspension
- 15. Developing Processes for Manufacturability Manufacturing Considerations 15 Define Program Objectives Target product profile (TPP) Quality TPP Planning for clinical & commercial supply 1 Development of Product Design a process to meet quality requirements Scalable Robust Efficiency 2 Developing a Scalable Upstream Bioreactor Process for Lentiviral Vector Production in Suspension Continuous Improvement Implementing changes with more process knowledge 3
- 16. Scalability Developing Processes for Manufacturability 16 Developing a Scalable Upstream Bioreactor Process for Lentiviral Vector Production in Suspension Ratio Working Volume : Total Volume 0.8 0.8 0.8 0.8 0.8 Impeller Diameter : Vessel Diam. 0.6 0.3 0.3 0.3 0.3 Vessel Height : Vessel Diameter 1.8:1 2.0:1 2.0:1 2.0:1 2.0:1 Liquid Height : Vessel Diameter 1.4:1 1.7:1 1.6:1 1.6:1 1.6:1 Dual sparger (open pipe/ micro) YES YES YES YES YES Min – Max Working Volume (L) 1 – 2.4 10 – 50 40 – 200 200 - 1000 400 – 2000 2000 L 1000 L 200 L 50 L 3 L H D Dimp
- 17. Consistent process inputs and controls to ensure consistent product Inputs • VirusExpress HEK293T cells • Chemically Defined Medium • Transfection reagent • Plasmids • Consumables (Mobius Flexware) Equipment • Process parameter control (Mobius Bioreactor Control System) • Automation Outputs • Quality Target Product Profile • Consistent quantity Robustness Developing Processes for Manufacturability 17 Developing a Scalable Upstream Bioreactor Process for Lentiviral Vector Production in Suspension
- 18. $0 $20,000 $40,000 $60,000 $80,000 $100,000 $120,000 T flask Mobius Bioreactor 50 L Lentivirus run Labor Overhead Consumables + depreciation Simplified operation and a 57% cost savings when using suspension culture in a Mobius bioreactor Efficiency Developing Processes for Manufacturability 18 Developing a Scalable Upstream Bioreactor Process for Lentiviral Vector Production in Suspension
- 20. The Benefits of Scalable Template Processes Speed to Patient & Reliability 20 Developing a Scalable Upstream Bioreactor Process for Lentiviral Vector Production in Suspension
- 21. The vibrant M, EX-CELL, Mobius, and VirusExpress are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources. © 2020 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.