Faster Media, Faster Outcomes 11.17.2020

Faster Media,
Faster Outcomes
Faster Media, Faster Outcomes
12/4/2020 // confidential

2
Previous webinar: Better Media, Better
Outcomes (available to view on Nucleus
Biologics’ website)
Today: Faster Media, Faster Outcomes
Future webinar: Intelligent Media
Formulation Using Machine Learning
2

3
Who We Are
David Sheehan
Founder and CEO
Nucleus Biologics
Roddy O’Connor, Ph.D
Research Assistant
Professor
University of Pennsylvania
Alex Klarer
Head of CellTherapy
Development
BioCentriq
David Smith Ph.D
VPTechnical
Operations
Ori Biotech

4
September Webinar Recap
Media Can Be a Competitive Advantage in Your Therapy Development
Perceived Impediments to Custom
Cost?
Development time?
Expertise?

Custom Media vs. Proprietary Off-the-Shelf Media (POTS)
Custom
• Built based on ingredients specified by scientist.
• Can start with common basal media formulation or be completely custom.
• Will usually be optimized to the unique attributes of the therapy company’s
ecosystem.
POTS
• Cell-specific media developed by media manufacturer.
• Formula is secret both in composition and concentration.
• Formula can only be augmented by the user at the point of manufacturing.
Faster Media, Faster OutcomesFaster Media, Faster Outcomes

6
Media is a Critical Part of Your Therapy Ecosystem
The dilemma of
media choice in
cell and gene
therapy
POTS
Unknown
ingredients, not
your IP, sole
sourced
Fast regulatory,
supposedly in-
stock
Custom media
6 month+ lead
time, difficult to
iterate
Own the media,
know every
ingredient
What is the Development Cycle and Cost?

7
Research POTS
Suppliers
Order and Delivery4 weeks
Media
Testing/Development w/
and w/Out Supplemental
Reagents
Order Additional
Reagents to Test, and
Delivery
4 weeks
>> Ready to order, off the shelf
media w/ additional reagents added
at point of use
Media
Testing/Development
w/ and w/out
Supplemental
Reagents
2 weeks
6 weeks
6 weeks
Place Order – Delivery 2
Weeks
Configuration and
quoting
Media
Testing/Development
Configuration and
quoting
6 weeks
Manufacturing
Research Components4 weeks
20
weeks
6 weeks
Manufacturing20
weeks
6 weeks
Traditional Custom Media
development cycle is 1 year
Traditional
Custom
POTS

8
POTS Formulation - Trial and Error
The number of conditions tested goes up with POTS because the formulations are unknown
Companies
Thermo
Lonza
Miltenyi
Irvine Scientific
R&D Systems
STEMCELLTechnologies
Cellgenix
Reagents
IL-2
IL-7
IL-15
Serum
Physiologix
HEPES
Glutamine
Thousands of test conditions to look at without
knowing what is in the basal media you are using
Testing 7 reagents
creates 125 unique
combinations and that
doesn’t count
concentration changes.
Multiply that by the
number of basal media
tested

Media Supplier
Configuration and
quoting
Research Suppliers
Small Lot
Manufacturing
4 weeks
Nucleus, a
New Model for
Custom
POTS
Research Components 2 weeks
Media
Testing/Development
6 weeks
Configuration and
quoting
Small Lot
Manufacturing
4 weeks
Order and Delivery
From Multiple
Suppliers
4 weeks
Media
Testing/Development w/
and w/Out Supplemental
Reagents
Order Additional
Reagents to Test, and
Delivery
4 weeks
6
minutes
Media
Testing/Development
6 weeks
>> Ready to order, scale
production of custom media in
any packaging required
>> Ready to order, off the shelf
media w/ additional reagents added
at point of use
Media
Testing/Development
w/ and w/out
Supplemental
Reagents
It now takes the same amount of time to develop custom media as
it does to create a formulation based on proprietary media
2 weeks
6 weeks
6 weeks
22
weeks

OK, development time is equal
but what about cost?
12/4/2020 10

11
Brand Media $/L Protein Reagents Total
OpTmizer $166 $100 (serum at 5%) $20 (Il7, IL15) $286
XVivo
Therapeak
$182 $100 (serum at 5%) $20 (Il7, IL15) $302
Immunocult $320 $320
TexMACS $170 $100 (serum at 5%) $20 (IL7, IL15) $290
Average $299.50
Accounting for Media - $299.50/L for POTS

12
Base Medium
(X-Vivo, OpTmizer)
Reagents (IL-2,
IL-15)
Protein
(HSA, Human AB)
POT
S
NB-Lux
$
$
$ $
$ $ $$166
$20
$100
$299.50 + Labor +Waste / Liter $189 / Liter @ 200L
$470/ Litter @ 2L
$ $
$$
Cost to Order
Multiple known
basal medias +
reagents you
choose
Test multiple
formulations quickly.
Known basal media +
protein + cytokines
QC:
POTS - each component
Custom - final product

13
Development Costs
Small Lot Custom materials are more expensive, but your labor time drives development costs
0K 20K 40K 60K 80K 100K 120K
NB-Lux
POTS
Cost
Can you go faster if you know the ingredients?
Materials Labor Labor:
Assume 22 weeks
dev time at
$100/hr
Materials:
$26K for Custom
$18K for POTS

14
Lifecycle Costs – POTS 50% Higher Cost than
Custom As You Go Into Commercial Production
$M
$2M
$4M
$6M
$8M
$10M
$12M
$14M
Dev Phase 1 Phase 2 Phase 3 Commercial Low
(#1000)
Commercial High
(#5000)
CUMULAITVECOST
NB-Lux Custom Thermo POTS Lonza POTS
Most biotechs trade at
30X earnings. This is
$120M of company
value
$4M/year

15
Summary So Far…
• Development costs for media are dominated by labor hours not materials
• Development time is the same for NB-Lux as it is for POTS
• At scale production cost of NB-Lux custom media is 33% less than POTS
• If you own the formula, it’s your IP and you control who manufactures it,
the format, and second sources
• Cost of a failed lot - how do you troubleshoot a failed lot if you don’t know
what’s in your media?
At scale production, you’re looking at millions of dollars of cost
differential just for media.

12/4/2020 16
Case Study #1

17
Custom Media is Required to Optimize
your Cell Therapy
Problem Statement:
Inherent limitations with proprietary off-the-shelf media as the impact of
individual components on CART-cell efficacy can’t be studied
The Goal:
The goal was to design medium specifically lacking a single amino acid to test its
contribution to mitochondrial function in CART-cells
Methods:
Design medium lacking arginine using the NB-LUX platform. Supplement this
medium with varying levels of 13C6 arginine
Results:
The final media was easily designed, cost-efficient, and yielded considerable
insights into the role of arginine in CART-cell metabolic function

18
Cancer patient
T cells removal
from a patient
1
Genetic redirection ofT
cells
ex vivo expansion
2
3
Reintroduction of
modifiedT cells
4
Generating CAR-T Cells for Cell-Based Therapies

19
α-ketoglutarate
succinyl-CoA
succinate
fumarate
citrate
oxaloacetate
L-malate
acetyl-CoA
IDH
MD
FH
SD
acetyl-CoApyruvate
PD
Glucose
Citrate
Acetyl CoA
Palmitate
TCA Cycle
13C5 Gln
GlnCO2
13C5 Gln
Using a [U-13C5]-Glutamine Tracer to
Examine Glutamine Metabolism in T Cells

20
0
20
40
60
80
100
%labelinSuccinyl-
CoA
Untreated
13C5 gln Bulk
T
13C5 gln
CD4+
13C5 Glutamine
0
20
40
60
80
100
M+0 M+1 M+2
%labelinacetyl-CoA
pool
Untreated
Bulk T
CD4+
CD8+
13C5 Glutamine
α-ketoglutarate
succinyl-CoA
succinate
fumarate
citrate
oxaloacetate
L-malate
acetyl-CoA
IDH
MD
FH
SD
Citrate
Acetyl CoA
Palmitate
TCA Cycle
13C5 Gln
GlnCO2
How can we optimize the medium for these studies?
Representative data from
O’Connor et al., 2018
Glutamine Supports Mitochondrial
Function in Activated T Cells

21
SY5Y
(1m
M
)
T
cells
(1m
M
)
T
cells
(4m
M
)
0
20
40
60
80
100
%LabeledOrnithine
13C6 L-Arginine
SY5Y
(1m
M
)
T
cells
(1m
M
)
T
cells
(4m
M
)
0
20
40
60
80
100
%LabeledOrnithine
13C6 L-Arginine
Customized Medium Supports Our Efforts To Determine
The Role of Amino Acids in CAR T-cell Metabolism

22
Custom Media is Required to Optimize
Your Cell Therapy
Conclusion:
Identified the need to have media without certain components to test the
impact on the effectiveness of the CART cells
Solution:
Order small lot Arginine-free media with ease (5 minutes) from NB-Lux
and supplemented it with varying levels to test its role inT cells
Impact:
Performed experiments to show that higher levels play an important role
in metabolic function inCART cells. Expand study to look at other amino
acids. Long term expectation is an advanced formulation for enhanced
CART cell potency.

12/4/2020 23
Case Study #2
Media forVaccines- Formulation to ordering

24
Xeno Free Vaccine Media
Problem Statement:
A biotech generating a novel vaccine was struggling to eliminate animal origin
components from their media without affecting critical quality attributes.
The Goal:
The goal was to maintain proliferation and therapeutic potency with a simple
xeno free media that if possible, also lowered the cost of production.
Methods:
Nucleus Biologics FormulationTeam through a combination of high throughput
screening and experience-based component selection was able to create an
optimized formulation within 2 cycles of testing.
Results:
The final media was AOF, equivalent or slightly better in proliferation and
significantly lower cost. Formulation was transferred to NB-Lux for small lot
ordering.

2512/4/2020
Xeno Free Vaccine Media - Results
2 step process – get rid of animal origin components and then optimize media by
modifying amino acids and other component level changes

26
Developing Custom Media Enables Multifactor Analysis
0
0.5
1
1.5
2
2.5
3
Media A Media B Media C Media D
RELATIVEYIELD
POTS Simulated Results
0% HABS 2.5% HABS 5% HABS 10% HABS
0.0x Glutamine
0.5x Glutamine
1.0x Glutamine
1.5x Glutamine
0
0.5
1
1.5
2
2.5
0.0x
Arginine
0.5x
Arginine
1.0x
Arginine 1.5x
Arginine
RelativeYield
0-0.5 0.5-1 1-1.5 1.5-2 2-2.5
Custom Media Simulated
Results

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Comparison of Approaches
• Time to develop media was 3 months
• Trial and error testing with POTS
would have been same time
• Removed animal components and
optimized for critical quality attributes
of customer
• Formula now stored on NB-Lux and
can order any size lot in any package
configuration

28
NB-Lux as a Solution For Custom Media
Start small! Use us as validation lots before you order big orders
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29
If NB Lux development doesn’t take
longer, is less expensive, and is more
optimized, what’s stopping you from
ordering custom media?
Q&A Session
29
Next Webinar:
Intelligent self-guided
formulation services

12/4/2020 30
Thank you!
Connect with us
LinkedIn: www.linkedin.com/company/nucleus-biologics
Twitter: @nucleusbiologic

Faster Media, Faster Outcomes 11.17.2020

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