Access the interactive recording here: https://bit.ly/37nl3Ex Webinar summary: An efficient production platform is essential for successful commercial implementation of gene therapy programs. AAV and Lentivirus manufacturing process are often developed with compressed timelines, reduced process optimization and low product yields which can have significant effect on costs. In this webinar, you will learn: * How manufacturing costs are examined for adeno-associated virus and lentivirus production with several different for each vector * That key process characteristics like production titer, production of empty viral particles, downstream product recovery, and the batching strategy can effect the overall manufacturing cost * How holistic evaluation is an important tool during process development to help prioritize different approaches to improve viral vector production processes Abstract: An efficient production platform is essential for successful commercial implementation of gene therapy programs. Viral vector manufacturing processes are often developed under timelines which are considerably shorter than those for more mature biopharmaceuticals. Consequently, the level of process optimization is reduced and challenges related to low product yields are common. These factors, as well as the small batch sizes common for these processes, can have significant effect on manufacturing costs.

1. Merck KGaA
Darmstadt, Germany
David Bohonak, PhD
November 21, 2019
Process development
guidance for AAV
and lentivirus
manufacturing based
on cost modeling

2. 2
The life science business of
Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma
in the U.S. and Canada.

3. 3
Viral vectors for gene therapies are costly
$0 $500 000 $1 000 000 $1 500 000 $2 000 000
Marketed
gene therapies
Vaccines
Price per dose

4. 4
Small patient populations are likely to see
high prices and manufacturing costs
2012, Glybera (rare pancreatitis)
2017,Luxturna (rare ocular disease)
2017,Kymriah (Rare lymphoma)
2017, Yescarta (Rare lymphoma)
2019,Zolgensma (SMA)
2020,SRP9001 (D. Muscular dystrophy)
2021, Valectocogene (Hemophilia A)
2021, Lentiglobin (Thalassemia)
10
100
1 000
10 000
100 000
0 1 2 3 4 5 6 7 8 9 10
#ofpatients
Year of launch or expected launch
Launched therapeutics Late stage and near launch
Bubble size is proportional to treatment cost

5. High costs for gene therapies with high viral dose requirements
# of Doses and Cost of Goods (COGs)
Based on process modelling
0.01L batch for 1 dose
$6,000/dose manufacturing cost of goods
0.1L batch for 1 dose
$9,000/dose manufacturing cost of goods
10L batch for 1 dose
$30,000/dose manufacturing cost of goods
100L batch for 1 dose
$120,000/dose manufacturing cost of goods
In vivo AAV treatment
Manufacturing scales and manufacturing costs based on our
process cost modeling for AAV production
5

6. 6
Little opportunity for optimization during process development
• Shortened timelines
• No established template

7. 7
Prioritizing process development activities
Costs Impact
Function of:
• Type, amount, and/or cost of
available resources
• Pipeline and other opportunities
• Time required
Function of:
• Likelihood and magnitude of
success
• Fit with existing facilities,
platforms, etc.
• Reduction of cost of goods
manufactured

8. 8
Prioritizing process development activities
Costs Impact
Function of:
• Type, amount, and/or cost of
available resources
• Pipeline and other opportunities
• Time required
Function of:
• Likelihood and magnitude of
success
• Fit with existing facilities,
platforms, etc.
• Reduction of cost of goods
manufactured

9. 9
Overview
Quantify how improved process capabilities can reduce manufacturing costs
 Develop process cost models for typical production processes for viral vectors for gene therapies:
– adeno-associated virus (AAV)
– lentivirus
 Evaluate changes to process parameters, manufacturing strategy, and cost of goods for 3 case
studies:
Increase AAV titer in
bioreactor
Decrease production of
empty AAV capsids
Increase lentivirus
recovery during filtration
1 2 3

10. 10
Increase upstream titer
• Evaluate changes to the titer
of AAV at harvest
• Assume negligible changes to
downstream capacities
- clarification, endonuclease, and
UF/DF base on volume
- chromatography based on
number of viral particles
1
42% DSP recovery
Suspension cell
culture
Transient
transfection
570 doses/year
3 x 1014 vg/dose
Keyassumptions

11. 0
100
200
300
400
500
600
€ -
€ 10 000
€ 20 000
€ 30 000
€ 40 000
€ 50 000
€ 60 000
0,E+00 2,E+11 4,E+11 6,E+11 8,E+11
Bioreactorvolume(L)
Manufacturingcostperdose
Bioreactor titer (vg/mL)
11
Increase upstream titer
Reduce bioreactor size
Bioreactor volume
1

12. 12
Increase upstream titer
Reduce bioreactor size
0
100
200
300
400
500
600
€ -
€ 10 000
€ 20 000
€ 30 000
€ 40 000
€ 50 000
€ 60 000
0,E+00 2,E+11 4,E+11 6,E+11 8,E+11
Bioreactorvolume(L)
Manufacturingcostperdose
Bioreactor titer (vg/mL)
Bioreactor volume
Cost per dose
1
Target yield: 3 x 1011 vg/mL

13. 13
Increase upstream titer
Reduce bioreactor size
0
100
200
300
400
500
600
€ -
€ 10 000
€ 20 000
€ 30 000
€ 40 000
€ 50 000
€ 60 000
0,E+00 2,E+11 4,E+11 6,E+11 8,E+11
Manufacturingcostperdose
Bioreactor titer (vg/mL)
Upstream costs
Downstream costs
Diminishing
reduction in overall
costs at higher
titers due to fixed
downstream costs
1
Cost per dose

14. 14
Fewer batches Vs smaller batches
Assessing impact of higher titer
€ -
€ 10 000
€ 20 000
€ 30 000
€ 40 000
€ 50 000
€ 60 000
0,E+00 2,E+11 4,E+11 6,E+11 8,E+11
Manufacturingcostperdose
Bioreactor titer (vg/mL)
1 batch
per year
41 batches
per year
Constant number
of batches
Decreasing bioreactor size
Constant
bioreactor size
Greater cost savings
from decreasing
number of batches, but
downstream costs
increase when number
of batches is small
1

15. 15
Decrease empty
AAV capsid production
Evaluate effect of reducing
empty capsid titer:
• Harvest titer of full capsids
remains constant
• Binding capacity of chromatography
steps based on total viral particle
loading (full + empty)
2
Adeno-associated viruses
by G. Beards / CC-BY-SA-3.0
1 x 1011 vg / mL
1,100 doses/year
3 x 1014 vg/dose
3 x 1013 vp / mL
2 x 1013 vp / mL
200L suspension
cell culture
Keyassumptions

16. € -
€ 5 000
€ 10 000
€ 15 000
€ 20 000
€ 25 000
15% 40% 70% 95% 95% w/o AEX
Manufacturingcostperdose
Percent full capsids in harvest
16
Decrease empty AAV capsid production
Reduce chromatography scale and resin usage2
Minimal CoGs
improvement if
percent full is
already ≥ 40%
Target production : 40% full capsids

17. 17
€ -
€ 5 000
€ 10 000
€ 15 000
€ 20 000
€ 25 000
15% 40% 70% 95% 95% w/o AEX
Manufacturingcostperdose
Percent full capsids in harvest
Decrease empty AAV capsid production
Reduce chromatography scale and resin usage2
Minimal CoGs
improvement if
percent full is
already ≥ 40%
(unless AEX
step can be
eliminated)
Or target therapeutically
appropriate level

18. 18
Downstream recovery of lentivirus
Evaluate CoGs reduction as recovery of final
sterile filtration step is increased:
• Constant bioreactor volume /
variable number of batches
• 43% recovery for overall DSP excluding
filtration step
3
50L suspension
cell culture
540 doses/year
1 x 109 vg/dose
1 x 107 vg / mL
Keyassumptions

19. 19
Downstream recovery of lentivirus
Reduce number of batches
0
10
20
30
40
50
60
€ -
€ 5 000
€ 10 000
€ 15 000
€ 20 000
€ 25 000
0% 20% 40% 60% 80% 100%
Batchesperyear
Manufacturingcostperdose
Lentivirus recovery during final sterile filtration
3
Decreasing number of batches
Lentivirus production
increases proportionally
with recovery (fewer
batches)
– But costs do not
decrease much when
recovery is
> ~30%

20. 20
Downstream recovery of lentivirus
Reduce number of batches
0
10
20
30
40
50
60
€ -
€ 5 000
€ 10 000
€ 15 000
€ 20 000
€ 25 000
0% 20% 40% 60% 80% 100%
Batchesperyear
Manufacturingcostperdose
Lentivirus recovery during final sterile filtration
3
Lentivirus production
increases proportionally
with recovery (fewer
batches)
– But costs do not
decrease much when
recovery is
> ~30%
Cost per dose
Decreasing number of batches
Target yield: 30%

21. 21
Downstream recovery of lentivirus
65% of lentivirus CoGs are fixed
52%
12%
8%
14%
13%
Capital
Consumables
Materials
Other (fixed)
Labor
3
21%
32%
23%
18%
6%
Materials
Capital
Other (fixed)
Labor
Consumables
Lentivirus case study AAV case study

22. 22
Increasing both the titer and recovery of AAV
Cost reductions beyond limits of individual improvements
60%
40%
30%
20%
10%
50%
2.0E10 5.0E10 2.0E111.0E11 1.5E11
 Increasing harvest titer and
downstream recovery concurrently
to intermediate levels reduces
costs more than increasing in only
one parameter dramatically
 Decreasing production of empty
capsids had much smaller effect
on reducing manufacturing costs <€20,000
€20,000 − €40,000
Cost per dose
€40,000 − €80,000
>€80,000
10x cost
reduction
6x cost
reduction

23. • Fixed costs often dominate overall CoGs during
production of viral vectors
• Particularly for smaller scale processes
(e.g. Lentivirus)
• Limited small-scale options in DSP
• For production of full vs. empty AAV capsids, CoGs
reductions may be minimal unless producing
therapeutically appropriate levels in bioreactor
• When prioritizing areas for process
improvement, key considerations include:
• Likely effect on CoGs
• Manufacturing strategy
Takeaways
23

24. Thank you for your time!
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david.bohonak@milliporesigma.com
David Bohonak