Watch the presentation of this webinar here: https://bit.ly/3b3Jc77 Gene therapies hold the promise to change lives. As your path to patients accelerates, how can you assure the robust process design, intensification and scalability that meets your evolving manufacturing needs? What benefits can a templated process bring to your commercial success? As gene therapy progresses toward broader clinical and commercial success, the industry is shifting from treating rare conditions to those of larger populations. This requires scalable solutions for process intensification. In this webinar, we’ll discuss scale-up development for a common viral vector in gene therapy, lentivirus, using the VirusExpress™ Lentiviral Production Platform in Mobius® single-use bioreactors. We will highlight critical considerations when moving from bench-scale to clinical scale process design with manufacturability in mind to ensure commercial readiness. Finally, we’ll review the significant benefits of implementing a templated manufacturing process. In this webinar you will learn: • Scale-up development of a suspension-based lentivirus production process • Designing a process that is manufacturing-friendly and supports commercialization • The benefits of having a templated manufacturing process

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

4. Scale-up
Development

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

14. Designing for
Manufacturability &
Commercialization

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

19. Benefits of
Template
Production
Processes

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.