Addressing the Challenge of Scalability in Viral Vectors
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The document discusses the significance of scaling in viral vector manufacturing for gene therapies, emphasizing the transition from traditional adherent cultures to suspension platforms for improved scalability. It highlights various production parameters, including titers and recovery rates, for different viral vector types and outlines the ongoing push for regulatory approvals and clinical trials in the gene therapy market. Additionally, it presents an overview of the manufacturing processes at Merck KGaA, focusing on the importance of robust technology and facility design to support large-scale production.
Addressing the Challenge of Scalability in Viral Vectors
- 1. Merck KGaA Darmstadt, Germany October 1, 2020 Henry George and Lisa Freeman-Cook The Importance of Scale in Viral Vector Manufacturing
- 2. 2 The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada.
- 3. 3 Agenda Introduction to Viral Vector Manufacturing: Why Scale Matters Downstream: viral vector purification and testing 1 2 3 Upstream: suspension cell line and chemically defined media The Importance of Scale in Viral Vector Manufacturing| 01.10.2020
- 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
- 6. Increasing clinical activity in larger indications Gene Therapy Market 6 • Viral vectors will remain the modality of choice for the next 10+ years • Increasing clinical activity for viral gene therapies targeting larger indications Source: Alliance for Regenerative Medicine 10-20 FDA approvals per year by 2025 Anticipated based on current pipeline of cell and gene therapy products 804 Clinical trials Ongoing for Gene Therapy, including Musculoskeletal, hematology, oncology* *Includes Gene-Modified & Cell-Based IO The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 7. Gene Therapy Regulatory Timelines Compressed 7 Orphan and other designations have sped up the clinical trial process The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 8. Adherent Culture Cultured in T-flask or cell factory/stack Easy to set-up, but hard to scale-up Manual operation, labor intensive and has process safety concerns 8 Traditional Adherent Culture Suitable for bench scale process establishment and small scale production The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 9. 9 Adherent Systems can be challenging to scale The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 10. Suspension (VirusExpress™ Lentiviral Production Platform) Crude harvest titer = 5e10 TU/L DSP recovery = 20% Scale per therapy type: − HSC vector: 1 dose per 1 L upstream − Autologous T cell vector: 20 doses per 1 L upstream Scalable to large volumes (≥200 L batches) 10 Adherent vs. Suspension Processes Scale of LV Vector Therapies Adherent LV Production Crude harvest titer = 2e10 TU/L DSP recovery = 20% Scale per therapy type: − HSC vector: 0.4 doses per 1 L upstream − Autologous T cell vector: 8 doses per 1 L upstream Practical scale limited to 50 L batches The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 11. Patient population (examples) Estimated 100,000 patients have SCD in the United States Estimated 300,000 born annually with SCD in sub-Saharan Africa Scale required to treat patient population VirusExpress™ Lentiviral Production Platform’s suspension process generates approximately 1 dose per 1 L upstream at scale − 100,000 L of LV vector upstream just for US SCD patients 11 Example: Sickle cell disease (SCD) Lentiviral (LV) Vector Scale for Large Indication The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 12. AAV Production by Transient Transfection Crude harvest titer = 5e13 vg/L DSP recovery = 4–20% Scale and titer need depends on target organ − Ophthalmologic therapy (e.g., retinal dystrophies): − 2–33 patients per L upstream depending on productivity and dose (1011–1012) − Adherent process may be sustainable for these indications − Muscular therapy (e.g., muscular dystrophies): − 0.0004–0.1 doses per L upstream depending on productivity and dose (1014–1015) − Dose and patient population make suspension production a requirement 12 Suspension vs. Adherent Processes Scale of AAV Vector Therapies The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 13. 13 Example: Duchenne Muscular Dystrophy AAV Vector Scale for More Common Indications Patient population 1 in 3,500 male births per year 1,000 births annually in the US alone Scale required to treat patient population 0.01 patient doses per 1 L upstream (Assuming 1e13 vg/L USP productivity and 1e15 vg dose) − 100,000 L per year AAV vector upstream just for US patients The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 15. 15 Advantages Easy scale-up and process optimization Reduce contamination risks Reduce footprint Simplified operation and 57% cost savings when using suspension culture in Mobius® Single-use Bioreactor Benefits for Suspension Cell Culture in Mobius® Single-use Bioreactor The Importance of Scale in Viral Vector Manufacturing | 01.10.2020 $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 ®
- 16. 16 VirusExpressTM 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 transfection, and transient production. Validated with CD medium in bioreactors up to 40 L scale. Lentiviral Production Process Demonstrated scalable process from flasks to 50L. The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 18. ▪ Single cell cloning and screening for improved growth and production characteristics • cGMP cell banking and testing by BioReliance® Testing Services Human Embryonic Kidney Cells Cell Lines Selected for Improved Viral Production HEK293 Virus Express™ Production Platform 293T Improved cell growth and virus production capability HEK293 T improved transfection and r-protein production capability HEK293 Reliable growth and transfection https://www.atcc.org/~/media/Attachments/Micrographs/Cell/CRL-1573.ashx ADVEC (1970’s) Stanford U (1980’s) (2019) 18 The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 19. Lentivirus production in selected HEK 293T clones Parental cell performance vs. clonal variants 48 Hour Viral Harvest 72 Hour Viral Harvest 5x106 TU/mL 4 x107 TU/mL Parental HEK293T Virus Express ™ HEK293T Clone 2 Clone 3 r GOI transfection The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 21. 21 CD Media development EX-CELL® CD HEK293 Viral Vector Medium Media design specifications ▪ Support fast cell growth (<24 hr doubling times) ▪ High peak cell density (>15 x 10e6 cells/ml) ▪ No other supplementation (beyond L glutamine) required ▪ Minimal clumping in suspension culture ▪ Bioreactor “friendly” ▪ High transfection efficiency and compatibility with PEI ▪ High virus productivity capability ▪ Animal origin free/chemically defined ▪ Supply at large scale to support clinical/manufacturing ▪ Qualified raw materials with supporting regulatory documentation The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 22. 22 • Purple lines are version 1 media and Blue lines are EX-CELL® CD HEK293 Viral Vector Medium • Bioreactors in HEK293 Viral Vector media reached peak VCD of 17-19E6 vc/mL on Day 5 • Shake flasks controls reached similar peak VCD for both conditions EX-CELL® CD HEK293 Viral Vector Medium Media Improvements lead to over 2X peak viable cell density over previous version The Importance of Scale in Viral Vector Manufacturing | 01.10.2020 ®
- 23. VirusExpress™ Lentiviral Production Platform cells grown in CD medium show minimal clumping: shakers vs bioreactors 23 250mL Shake Flask 50L Mobius® Single-use Bioreactor The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 25. Transient transfection based upstream process Inoculate production vessel with cells Day 0 Four plasmid system using PEI Day 1 Harvest with 1E7 – 2E7 TU/mLDay 3 Growth Lentivirus productionTransfection Total elapsed time for transient LV production process is 3 days Shaker flask process consistently yielded > 1E+07 TU/mL GOI: tGFP 25 The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 26. Process Optimization- Testing the Robustness of the Platform Time Course Studies ~1.8 fold increase in LV titer from 24 to 48 hr PT with no further increase from 48 to 72 hr pT 48 hr PT transfection used as harvest time/downstream process development activities Differences across Sites • Incubators • People • Transduction Assay (TU) Protocol • Read-out of TU Assay Batch mode 26 The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 27. Effect of Total DNA/1E6 Cells on Lentivirus Production LV functional titer was highest with 0.44µg DNA/1E6 cells (@ 48hr PT) 27 Process Optimization The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 28. Merck KGaA Darmstadt, Germany Scale into Bioreactors
- 29. Cell Growth in Bench Scale Bioreactor – Mobius® 3L Single-use Bioreactor Defining Bioreactor Process Parameters for VirusExpress™ Production Platform Cell Growth Higher agitation rate yields similar growth characteristics as small scale shake flasks pH has an effect on growth of cells and setpoint needs to be defined for optimal growth VirusExpress™ Production Platform cells grow similarly with open pipe or micro-sparger gassing configuration either with low or high DO concentration Agitation pH DO & Sparger Type 29 Cell Culture Duration (Days)Cell Culture Duration (Days) Cell Culture Duration (Days) 1. Focus on cell growth The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 30. Lentivirus Upstream Process Defining Bioreactor Process Parameters for Lentivirus Production 2. Focus on virus production Inoculate cells Day 0 Day 1 Day 2 Day 3 Transfect with plasmids Virus Harvest The range of agitation rates tested did not have an affect on LV production Similar LV titers were obtained in bioreactors and shaker flasks Highest lentivirus titer (48 h) was obtained in bioreactor controlled at pH D 30 The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 31. Lentivirus Upstream Process Scale-up of 3L Bench Scale Process to 50L Bioreactor Where Po = Ungassed power input V = Liquid working volume Np = Impeller power number ρ = Fluid density N = Impeller agitation rate D = Impeller diameter Mobius® 3L Single- use Bioreactor Mobius® 50L Single-use Bioreactor Process scale up from Mobius® 3L to 50L single-use bioreactor based on power input per volume 31 3. Focus on scale-up The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 32. Development of Upstream Lentivirus Production System LV Production in Mobius® 50L Bioreactor Product + LVfunctionaltiter(TU/mL) HEK 293T-3F11 + CDM Mobius® 50L Single-use Bioreactor Process-scale Performance ▪ Target titer of ≥ 1e+07 TU/mL achieved for 3 production runs The Importance of Scale in Viral Vector Manufacturing | 01.10.202032
- 33. LVfunctionaltiter(TU/mL) Increased Titer seen at 3L, 10L, 50L ▪ Target titer of 2.4 to 5.2e+07 TU/mL achieved The Importance of Scale in Viral Vector Manufacturing | 01.10.2020 Higher Titers seen with Improved Media Formulation 4,16E+07 5,20E+07 2,38E+07 1,00E+06 1,10E+07 2,10E+07 3,10E+07 4,10E+07 5,10E+07 6,10E+07 3L 10L 50L Work in Progress 33
- 35. Templated Lentivirus Production Process for ex vivo Applications VirusExpress™ Production Platform + Downstream Processing IV - AEX Chrom VI - Sterile filtration III - Benzonase® endonuclease + 2-8°C hold V - TFFII - ClarificationI - Virus Production in 50L Mobius® Single-use Bioreactor The Importance of Scale in Viral Vector Manufacturing | 01.10.202035
- 36. Templated Lentivirus Production Process for ex vivo Applications VirusExpress™ + Downstream Processing IV - AEX Chrom VI - Sterile filtration III - Benzonase® endonuclease + 2-8°C hold V - TFFII - ClarificationI - Virus Production in 50L Mobius® Single- use Bioreactor The Importance of Scale in Viral Vector Manufacturing | 01.10.2020 Scalable and robust technologies Pods/capsules for clarification eliminate need for centrifugation Modular designs and range of sizes for scalability from bench to large- scale manufacturing TFF and chromatography skids with automated control Single-use consumables Reduce contamination risk Eliminate need for assembling/cleaning stainless steel housings Eliminate need for packing/sterilizing columns 36
- 37. VirusExpress™ Production Platform + Downstream Processing Process Recoveries for 50L Runs 37 Consistent upstream and downstream processing yields consistent process recovery 15% 21% 18% 0% 5% 10% 15% 20% 25% 1 2 3 FunctionalParticleRecovery(%) Production Run Process Recovery The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 39. Analytics: In Process and Release Testing 39 AEX Chrom Sterile filtration Benzonase® Endonuclease + 2-8°C hold TFFClarificationVirus Production in bioreactor p24, TU or vg/mL titer HEK293 Host Cell Protein / Host Cell DNA / Plasmids Residual Benzonase / DNA Size Distribution Bioburden, Endotoxin Mycoplasma, RCL, Adventitious Virus p24, TU or vg/mL titer Sterility, Identity, RCL The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 40. 40 Carlsbad Manufacturing Facilities The Importance of Scale in Viral Vector Manufacturing | 01.10.2020 Custom Built Viral Manufacturing Facilities 100% Single Pass HEPA Filtered Air Rooms Exhausted Separately to Prevent Cross- Contamination Operated Under Biosafety Level 2 Enhanced (BSL2+) Procedures Unidirectional Personnel, Materials, Waste, and Product Flows Inspected and approved for commercial manufacturing by FDA and EMA
- 41. 140,000 sqft Facility Cell Expansion Suites Bioreactor Suites Downstream Suites Fill Suite w/ Isolator Inspection/Labeling Carlsbad Manufacturing Expansion The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 42. 42 Summary: Considerations for Scale in Viral Vector Manufacturing 1 2 3 Improved virus manufacturing Platform Downstream Focus on robust, scalable technologies and single-use consumables. Upstream Moving from traditional adherent culture to a suspension platform enables scalability. Testing and Facility Analytical testing and facility design considerations to support development to commercial scale manufacturing. The Importance of Scale in Viral Vector Manufacturing | 01.10.2020
- 43. The vibrant M, Mobius, EX-CELL, BioReliance and Benzonase are registered trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. VirusExpress is a trademark 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.