Watch the presentation of this webinar here: https://bit.ly/3NDNIKe Automated, fit-for-purpose tools are essential in CAR-T processing to support sustainable manufacturing of clinical and market-approved cell therapy products. This webinar will discuss how the ekko™ Acoustic Cell Processing System uses acoustic technology as a touchless approach to manipulate cells, enabling a modular tool across the CAR-T manufacturing workflow. Typical performance of templated ekko™ System processes for DMSO washout of leukapheresis material, low volume and high cell concentrate for electroporation preparation, and harvest of expanded T cells will be reviewed. This webinar will also give an early glimpse at the ekko™ Select System for unmatched T cell selection. In this webinar, you will: • Uncover how the ekko™ System supports the broad industrialization of cell therapy, with particular focus on how to achieve low volume, high concentrate cell product for critical transduction and transfection steps • Discover how ekko™ System for wash and concentrate processes throughout the cell therapy workflow achieve high cell recovery, viability, and effective residual removal • Preview to ekko™ Select, our cell therapy selection platform, to achieve unmatched ease-of-use with direct processing from leukopaks reducing the need for preparation steps Presented by: Benjamin Ross-Johnsrud, Acoustic Technology Expert Robert Scott, Mechanical Engineer III

1. The life science business of Merck KGaA,
Darmstadt, Germany operates as
MilliporeSigma in the U.S. and Canada.
CAR-T Manufacturing
Innovations that Work -
Automating Low Volume Processes
and Other Key Steps
Robert Scott, R&D Engineer III
Benjamin Ross-Johnsrud, Technology Expert
June 9th, 2022

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

3. Agenda
1
2
3
Manufacturing Requirements for Cell Therapy Industrialization;
Utilizing Acoustic Technology in Automation
Case study: Overview of ekko™ System and its
use throughout the CAR-T manufacturing process
Introduction to ekko™ Select System for
T cell Selection
4 Closing

4. 1,308+ Regenerative Medicine Companies Worldwide
Cell-based immuno-oncology makes up a growing portion of pipeline
2,261
Clinical Trials Underway WW
RM/AT
Designation
1,026
Regenerative Medicine Companies, WW
Source: Alliance for Regenerative State of the Industry briefing Q1 2022
CAR-T Manufacturing Innovations that Work - Automating Low Volume Processes and Other Key Steps| 6/9/2022
4
Source Alliance for Regenerative Medicine
Annual Report 2021
1,308 Cell, Gene & Tissue Therapeutic
Developers WW
19% Year-over-Year Increase
Cell-based immuno-oncology (IO) and
cell therapy constitute majority of trials
47
132
43
Gene Therapy: 222
225
209
31
Cell Based IO: 465
130
232
62
Cell Therapy: 424
1
11
6
Tissue Engineering: 18
24
57
3
Gene Therapy: 84
187
187
3
Cell Based IO: 377
156
433
67
Cell Therapy: 656
6
8
1
Tissue Engineering: 15
1,129 Industry Sponsored Clinical Trials
1,132 Government and Academic Sponsored Clinical Trials

5. The late-stage pipeline is robust, 216 Phase 3 clinical trials and
regulatory decisions expected on multiple product candidates
IO and Cell-Based Product Approvals in 2021 Anticipated Near-Term Decisions
Source: Alliance for Regenerative State of the Industry briefing Q1 2022
CAR-T Manufacturing Innovations that Work - Automating Low Volume Processes and Other Key Steps| 6/9/2022
5
83% of 2021 ATMP
approvals in cell therapies
6 Cell Therapies anticipated;
1 BLA & MAA already
submitted (Janssen/Legend)
Brezyani* (US)
CAR-T Therapy
DLBCL
BMS
Abecma (US & EU)
CAR-T Therapy
Multiple Myeloma
Bluebird bio & BMS
Stratagraft* (US)
CAR-T Therapy
Severe Burns
Mallinckrodt
Carteyva (China)
CAR-T Therapy
LBCL
JW Therapeutics
Rethymic* (US)
Tissue Therapy
Congenital Athymia
Enzyvant
Janssen/Legend (US & EU)
Multiple Myloma
Atara Bio (US & EU)
EBV + PTLD
Iovance (US)
Melanoma
Gamida Cell (US)
Blood Cancer + HSCT
CARsgen
Multiple Myloma
Adaptimmune
Synoval Sarcoma
2022
2023

6. Successful clinical trials must be translated to marketable treatments
Key Concerns in Cell Therapy Processing
Process tools should be optimized to address these concerns
CAR-T Manufacturing Innovations that Work - Automating Low Volume Processes and Other Key Steps| 6/9/2022
6
Safety Speed Process Economics Flexibility
• Reduced operator
error
• Reduced risk of
contamination and
environment exposure
• Accelerated time to
clinic or market
• Faster implementation
• Ease of use
• Reduced processing
times
• Adaptable to changing
process needs
• Suitable for multi-
product
manufacturing
• Scalable
• Reduction in operating
costs
• Reduction in costs due
to performance issues
• Workflow efficiencies

7. Addressing the CAR-T Workflow through Closed and Automated Systems
Acoustic Processing Frees Users of Traditional Hurdles
Acoustic force acting on many
discrete particles results in
capturing and clustering
Acoustic pressure acting on
interface between clarified
fluid and fluid mixture of fluid
and particles
ekko™ Select System ekko™ System
• CD4 and CD8 cell selection
• Process directly from leukopak
• Fluidized selection column
supports increased recovery,
purity and reduced process
times
• Wash, concentrate and
media exchange
• Ability to process variety of
cell types with wide range of
incoming volumes and total
cells
Workflow
Product
Technical
Approach
Acoustic Edge Effect Direct Acoustic Capture
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7

8. Acoustic Cell
Processing with the
ekko™ System

9. Acoustic Cell Processing Technology Employed for ekko™ System
Acoustophoresis Process Steps
1. Flow chamber is primed with cell material
2. The transducer generates forward-propagating acoustic waves. These
waves are reflected, diffracted, and scattered via a reflector creating an
acoustic field
3. Acoustic forces trap cells in regions of low pressure throughout the
acoustic field. As additional cells are trapped, loose cell clusters are
formed
4. Buffer is processed through the acoustic chamber to wash captured cells
5. Through gravity, cell clusters gently settle out of the acoustic channel
into the collection reservoir
6. Chamber is purged with air and concentrated cells are collected in a low
output volume
CAR-T Manufacturing Innovations that Work - Automating Low Volume Processes and Other Key Steps| 6/9/2022
9
1 2 3 4 5 6

10. Automated tool to simply process development
and manufacturing
 Intuitive, easy set-up utilizing cartridge format single use
assembly with integrated tubing
 Connection to variety of transfer bags and equipment via
tube weldable PVC and TPE lines
Flexible for a variety of wash and concentration
applications
• Harvest wash
• Electroporation preparation
• Cryoprotectant addition and removal
• Single cell removal
• Buffer exchange
Freedom to Optimize with ekko™ Acoustic Cell Processing System
CAR-T Manufacturing Innovations that Work - Automating Low Volume Processes and Other Key Steps| 6/9/2022
10

11. ekko™ Software, the same intuitive navigation with
increased compliance to support regulatory filings
Our ekko™ System Software is designed to support compliance to 21 CFR Part 11 and developed according to GAMP5
guidelines. Our software has been independently audited by a third party to affirm the ekko™ System meets the requirements
set forth by the FDA in the 21 CFR Part 11 requirements and the ISPE GAMP5 Guidelines.
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11

12. ekko™ Architect
Process Development Freedom at your Fingertips
Set Protocol Steps
1 Adjust Parameters
2 Finalize Procedure
3
Fine-tune process steps based on
application across a variety of cell types:
• Buffer exchange
• Formulation
• Wash and concentration
• Volume reduction down to 5 mL
• Single cell depletion
Parameters for optimization include,
but are not limited to:
• Acoustic Power (W)
• Flow Rate (mL/min)
• Pump ramp (sec)
• Transition Condition – e.g. duration
Execute Run
• In Development Mode, the protocol can
remain editable
• In Manufacturing Mode, the procedure is
locked for execution in compliance with
21 CFR Part 11
CAR-T Manufacturing Innovations that Work - Automating Low Volume Processes and Other Key Steps| 6/9/2022
12

13. Acoustic Cell Processing Applications
DMSO Removal using the ekkoTM System to Achieve High
Recovery and DMSO Removal Across a Range of Input Conditions
• The ekkoTM System was used to wash out
DMSO from cryopreserved leukopaks.
Prior to processing, cryopreserved
leukopaks were diluted after thawing.
• Sample size and composition:
• N=3 for each leukopak size
• Analytics:
• Cell concentrations were determined via
CBC and cell viability was determined
using an NC-202. DMSO removal
determined via HPLC.
• A PBS buffer including 1% HSA, and 3mM
EDTA was used for the upfront dilution
and cell washing.
• The ekkoTM System achieved robust
performance, processing a wide range of
input material with an average recovery
of 88.5% and an average DMSO
removal of 97% while maintaining cell
viability.
CAR-T Manufacturing Innovations that Work - Automating Low Volume Processes and Other Key Steps| 6/9/2022
13
-5%
10%
25%
40%
55%
70%
85%
100%
1 2,5 5 10
Recovery
/
Viability
Change
/
DMSO
Removal
Input WBCs (e9 Total Cells)
Performance of ekkoTM System: DMSO washout of
frozen leukopaks: >95% DMSO removal with 85-
91% WBC recovery
Recovery Viability Change DMSO Removal

14. Acoustic Cell Processing Applications
The ekkoTM System Achieves High Recovery in Low Volume
Processing as Preparation for Electroporation or Transduction
• The ekkoTM System was used to concentrate T
cells for transduction preparation.
• Sample size and composition:
• T cells were concentrated then washed with PBS
supplemented with 1% HSA followed by commercial
electroporation buffer in a protocol aiming to
minimize output volume in order to pre-process for
electroporation.
• Final Product Volume: 5.0 mL ± 0.7 mL
• Analytics:
• Cell concentrations and cell viability were
determined by an NC-202.
• The ekkoTM System achieved robust performance,
concentrating 0.5e9 T cells from 0.5 L down to a final
volume of 5.0 mL with an average recovery of 95%
while maintaining cell viability.
CAR-T Manufacturing Innovations that Work - Automating Low Volume Processes and Other Key Steps| 6/9/2022
14
Condition 0.5e9, 0.5 L
(n=4)
Recovery (%) 95 ± 5
Output Volume (mL) 5.0 ± 0.7
Output Concentration (e6 cells/mL) 153 ± 45
Product Viability(%) 96.2 ± 0.5

15. Acoustic Cell Processing Applications
Robust ekko™ System Processing of Expanded T cells
Achieving Repeatable Recoveries Across a Range of Inputs
• The ekko™ System was used to concentrate and
wash expanded T cells for final harvest.
• Across 15 different experiments, a single ekkoTM
System protocol was able to achieve an average
viable cell recovery of 86.0% ± 3% while
maintaining cell viability.
• Analytics:
• Cell concentrations and cell viability were
determined by an NC-202. IL-2 Removal
determined by ELISA.
• The expanded T cells were washed with a PBS
buffer including 1% HSA.
• Across the 3 different experiments with 1e9 cells,
the IL-2 residuals were measured. The ekkoTM
System was determined to achieve an IL-2
washout >96%.
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15
-5%
10%
25%
40%
55%
70%
85%
100%
1e9, 1 L 1.5e9, 0.5 L 7.5e8, 1 L 3.0e9, 1 L 1.5e9, 2 L
Viable
Cell
Recovery
Input Cell Number, Input Volume
Performance of ekkoTM System Concentration
and wash of expanded T cells: robust cell
recoveries from 0.5-2L input
Recovery Viability Change
Test IL-2 Removal
Test 1 97.3%
Test 2 96.2%
Test 3 96.9%

16. Acoustic Cell
Selection with the
ekko™ Select
System

17. Acoustic Cell Selection via the ekkoTM Select System
How acoustics are used to select cells in automated manufacturing
CAR-T Manufacturing Innovations that Work - Automating Low Volume Processes and Other Key Steps| 6/9/2022
17
The acoustic properties of the
ekko™ Select Droplet, coupled
with their high biotin binding
capacity, affords
discriminate selection of
target cells from a mixed cell
population.
Upward flow of the selection
column suspends ekko™
Select Droplets, creating a
gently-mixed fluidized bed
that facilitates binding
between the affinity reagent
and biotin-labeled target cells.
ekkoTM Select Cell Selection
Platform. Closed, automated
prototype system
 Intuitive cartridge design enables
easy setup and operation
 Common ekkoTM user interface and
protocol development freedom with
ekkoTM architect
How it works: The ekko™ Select Column

18. Introducing the ekkoTM Select System
Cell selection with ekkoTM Select
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18
1. Target Cell Capture Step
• Unprocessed leukopak is labeled
with ekko™ Select Antibodies.
• Column is primed with buffer and
ekkoTM Select Droplets.
• Target cells bind to ekkoTM Select
Droplets.
• The acoustic field retains the
Droplet – target cell complexes.
2. Non-target Cell Removal Step
• Residual non-targets are removed
with ekko™ Select Wash Buffer.
3. Target Cell Harvest Step
• ekko™ Select Elution Buffer
detaches target cells.
• Target cells are captured in the
product bag.
1
2 3

19. Introducing the ekkoTM Select System
ekkoTM Select Prototype Performance Overview
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19
T cell Selection from Unprocessed Apheresis Products
Input: unprocessed apheresis
product
Platelets RBCs
T cells Non-T cell WBCs
WBC chromatogram: only T cells are retained in the column
Output: purified T cells
Platelets RBCs
T cells Non-T cell WBCs
• Platelets and RBCs are
>90% (in number) of the
initial Apheresis product
• T cells are <3%
• Platelets and RBCs are
<5% of the final T cell
product

20. Introducing the ekkoTM Select System
Delivering T cells with High Cell Recovery and Purity Across Wide
Range of Input Cell Numbers
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20
• The ekkoTM Select System was used
to isolate CD4 and CD8 T cells from
fresh, unprocessed apheresis
material from healthy donors.
• Sample size and composition:
• N=23, each run from unique
donor sourced from three
different vendors
• Analytics:
• Cell concentrations, viability and
phenotype were determined via
flow cytometry.
• The ekkoTM Select System
successfully isolated CD4 and CD8 T
cells across a wide range of input
conditions. It achieved an average
recovery of 78.3% and an average
purity of 96.7% while maintaining
cell viability.
-5%
10%
25%
40%
55%
70%
85%
100%
2-3 3-4 4-5 5-6
Recovery
/
Purity(CD3
in
CD45)
/
Viability
Change Input Target Cell Number Range (e9)
ekkoTM Select CD4 and CD8 T cell Selection: >70%
recovery and >95% final purity of T cells
Recovery Purity Viability Change

21. Conclusion

22. 22
CAR-T Manufacturing Innovations that Work – Automating Low Volume
Processes and Other Key Steps with ekko™ System and ekko™ Select
Safety Speed Process Economics Flexibility
Our automated and
closed ekko™ System
supports reduced
operator error and risk
of contamination and
environmental exposure.
The ekko™ System
supports fast
implementation into
process workflows due to
it’s ease of use and fully
automated protocols.
The ekko™ System is
adaptable and can be
easily optimized with the
ekko™ Architect protocol
development tool to
address your challenges.
The ekko™ System
improves workflow
efficiencies for its ability
to address and combine
multiple unit operations
– including low volume
processing.
ekko™
Select
ekko™
System
CAR-T Manufacturing Innovations that Work - Automating Low Volume Processes and Other Key Steps| 6/9/2022

23. Robert.scott@milliporesigma.com
linkedin.com/in/robert-scott-
a53542116/
Robert Scott
Benjamin.ross-johnsrud@milliporesigma.com
linkedin.com/in/benjamin-ross-johnsrud-
5b4b03186/
Benjamin Ross-Johnsrud
The vibrant M, Millipore, and ekko 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.
© 2022 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.