Watch the presentation of this webinar here: https://bit.ly/3b7vD60 Closed processing involves use of physical barriers to separate processing fluid from the external environment. This approach reduces capital expenditures and clean room classification while accelerating time to market. This webinar will present a TFF process run in a closed mode. Closed processing with single-use technologies is a critical enabler for efficient and robust manufacturing for novel modalities as well as continuous biomanufacturing processing. It can also reduce the dependence on classified clean rooms for traditional modalities. This approach helps to mitigate the risk of contamination by adventitious agents while enhancing operator safety.  In this presentation, we discuss the implementation of closed processing for downstream applications and present the design and performance testing of a single use manufacturing-scale tangential flow filtration system to be able to operate in both functionally and fully closed mode. In this webinar, you will learn: • The context of closed processing • Differences between closed and functionally closed processing • The drivers for adoption • Its practical implementation to a TFF step

1. The life science business of Merck KGaA,
Darmstadt, Germany operates as
MilliporeSigma in the U.S. and Canada.
Single-Use
Tangential Flow
Filtration for
Closed Processing
May 6, 2021

2. The life science business of Merck KGaA,
Darmstadt, Germany operates as
MilliporeSigma in the U.S. and Canada.
Sarah Le Merdy
Strategy Deployment,
Single-Use and Integrated Systems
Dana Kinzlmaier
Applications Engineer
SPEAKERS:

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

4. Agenda
1
2
3
Definitions and drivers
TFF processing in a closed
mode
System characterization
and performance testing
4 Conclusion and
perspectives

5. Definitions
and drivers

6. 6
What is closed processing about?
Isolating a
product from
the environment
and vice versa
“A process that ensures that biological material does not
come into contact with the external environment. Closed
processing enables grey space or CNC (Controlled Not
Classified) processing and is sometimes referred to as being
conducted in a “ballroom”.
According to ISPE:
“A process condition when the product, materials, critical
components or container/closure surfaces are contained and
separated from the immediate process environment within
closed/sealed process equipment. A process step (or system) in
which the product and product contact surfaces are not exposed
to the immediate room environment.”
BPOG: “The key factor for industry acceptance was the Pharmaceutical
Inspection Cooperation Scheme (PIC/S) determination that clean-room
requirements for bioP can be relaxed/eliminated if your process is closed”.
ISPE Baseline Guide: “The strategy of using uncontrolled or unclassified
spaces for closed piping systems has always been accepted by the
regulatory agencies as an acceptable risk”.

7. 7
FUNCTIONALLY
CLOSED
FULLY CLOSED
BRIEFLY OPEN
SYSTEM
• Never exposed to the environment
• Materials may be introduced to a closed system, in a way to avoid
exposure of the product to the room environment
• Exposes the product to the room environment
• The room environment is controlled to minimize the risk of
product contamination
• For bioburden-controlled processing, open operations are
expected to be performed in a classified environment
• May be routinely opened but returned to a closed state through
a sanitization or sterilization step prior to process use
• Defining and validating the required sanitization or sterilization is
the user's responsibility
Source : Challenging the Cleanroom Paradigm for Biopharmaceutical Manufacturing of Bulk Drug Substances
Aug 01, 2011, http://www.biopharminternational.com/challenging-cleanroom-paradigm-biopharmaceutical-manufacturing-bulk-drug-substances?id=&sk=&date=&pageID=2
… and many
different users'
interpretations!
How closed?
Different ways of achieving a closed state

8. Drivers and benefits
‘Closed’ single-use is an accelerator of single-use benefits
1
Protect from contamination – Ultimately
reducing cleanroom requirements by minimizing
contamination independently from the production
environment
2
Flexibility to market - Means to reduce time to
market using multimodal solutions and/or reducing
turnover time between campaigns, in grey space
3
Reduction of interferences – Limiting the
number of human manipulations and high-risk
open state throughout the process to limit risk of
failure
8

9. Early and late steps are typically closed by definition
Cell culture, upstream and final fill:
- Closed by design
- Closed by operation
Downstream culture of reuse
- Reusable expendables (TFF devices, chromatography media)
with optimized lifecycle
- Steps still to be converted to single-use operation (clarification)
or with large footprint
Drivers and benefits
Closing what is currently open: Downstream steps
9
Moving downstream steps to single-use and ultimately closed
technologies is a natural next step of the evolution of biomanufacturing

10. Drivers and benefits
Different drivers lead to closed processing
10
• Manufacturing at ‘point of
use’
• Small, fast set-up capabilities
• Safety of product and patient
“The main objective is the
assurance of an uncompromised
product using a processing
mode which doesn't have
potentially harmful steps.”
• Reduce human interferences
• Reduce environment control
requirements
“The flowpaths used in closed
mode help increasing process
safety and participate to easier,
faster, and lighter
environmental control.”
• Filtration not possible
• Biosafety: no egress
• Animal vaccine never sterile
filtered
Future state:
• Multimodal solutions
• Ballroom concept
Viral vector
& live viruses
Recombinant
proteins
Cell therapy

11. TFF Processing in
closed mode

12. Open versus closed processing step
Tighter control and management of inputs and outputs in closed
Open
Multi-use sensors can
be used
Ideally fully automated
‘All’ expendable Similar
to MU processing
‘Open’ lines
authorized,
connect/disconnect
containers
Similar to multi-use
TFF
Similar to multi-
use TFF
Typically class C
SYSTEM &
FLOWPATH
EXPENDABLE SET-UP PROCESSING SAMPLING ENVIRONMENT
Closed
(Fct)
SU sensing
technologies
Aseptic Connectors
Ideally fully auto
Can be sanitized or
sterilized in place
Collecting bags for all
fluids from sanitization
step
Post-sanitization all
fluids in/out are
contained
Closed Potentially lower to
class D
Open allowed post-
product recovery
Closed
(Fully)
Drip-less
disconnections
Are pre-sterilized and
equipped with aseptic
connectors or built in
flowpath
Bags for all fluids from
process start (water,
buffers, waste/drain,
product…)
From set-up to
dismounting all fluids
are contained
Closed, ideally part
of the flowpath
assemblies
Potentially lower to
class D
Potentially ‘never’
open, closed and
dripless
disconnections/
dismounting/
discarding
12

13. Open:
Connections and
disconnections in a non-sterile
environment during the
different steps in the TFF
operation (flushing, cleaning,
draining, etc)
Flowpath and system design
Closed operation flowpath requires additional fixed lines
WFI Air NaOH Buffer Product
13
Filtrate

14. Flowpath and system design
Closed operation flowpath requires additional fixed lines
Closed:
Assemblies used for cleaning,
flushing and draining incorporated
into one single assembly (multiple
sub-assemblies mated with aseptic
connectors)
Control of fluid flow during flushing
and draining done with pneumatic
pinch clamps
14
Filtrate
WFI Air NaOH Buffer Product

15. Functionally closed
- Select any device format
which can be sanitized in place
- Take into account sanitization
agent and flushing volumes
Expendable selection
15
Fully closed
- Select pre-sterilized device
format
- Take into account
connectivity/adaptability to
flowpath
Closed-enabled formats
It is not necessarily about
selecting a ‘new’ or ‘specific’
filtration device type…

16. System
Characterization and
Performance Testing
Flexware® Assemblies for Closed TFF Processing

17. Introduction to Equipment used in Experimental Set-up
Mobius® FlexReady System for TFF and Pellicon® Capsule
Mobius® FlexReady
System for TFF
Pellicon® Capsule
& Manifold
Available Recycle Tank Volume [L] 50, 100, 200 -
Membrane Area [m2] 0.5 – 5
0.5 m² - 1.5 m²
Manifold: 1 m²
Multiple of 1.5m²
Flowrate [L/min]
2 – 20
Reco. flowrates for TFF
4-8 L/min/m2
-
Details
Pre-sterilized flowpath
sub-assemblies
Pre-sterilized
ready-to-connect
17

18. Mobius® FlexReady System for TFF
Fully Closed Flowpath
1 Recycle Vessel Assembly 100 L
2 Transfer Pump Assembly
3 Feed Pump Assembly
4 Transfer Pump Manifold Assembly
5 Smart Flexware® Assembly
6 Pellicon® Capsule Assembly
7 Retentate Sampling Port Assembly
8 Flush/Recovery Assembly
9
Permeate Assembly (with SU sensor
flow cell)
10 Permeate Sampling Port Assembly
1
4
2
3
7
5
8
9
10
6
18

19. 19
Application Testing
System Characterization and Process Testing
1 Minimum working volume
As a function of flow rate and viscosity
2
Flowpath pressure drop
As a function of viscosity
Total pressure drop
Retentate line pressure drop
3 Process run
Concentration and Diafiltration of Human Plasma IgG
4
Product recovery
Low viscosity applications
Elevated viscosity applications

20. System Characterization
Minimum Working Volume
20
Is the minimum volume at which the system can be operated at given process flowrate without causing air
entrainment in the process fluid. It accounts for volume in the tubing, valves, instruments, membranes, and
minimal volume in the recycle tank.
Why is it important?
To ensure air is not entrained into the system during processing.
Minimum Working Volume
Minimum working volume of flowpath with
1 m2 Pellicon® Capsule assembly at a typical
feed flow rate of 4 - 8 L/min is <1.3 L for
solutions with viscosity between 1 – 50 cP
• Capsule assembly not installed during testing
• Measurements performed with
• Purified Water: 1 cP viscosity
• Glycerin/Purified Water solution:
• 10, 25 and 50 cP viscosity

21. System Characterization
Total Pressure Drop of the System
21
Total pressure drop of a system is the line drop from the feed pump discharge through the feed lines,
devices and retentate line back to the recycle vessel.
Why is it important?
High Total System Pressure Drop could limit the flow rate that can be driven through devices to increase
mass transfer and drive high flux. It is especially true for high protein concentrations and high viscosity
solutions. • Capsule assembly not installed
during testing
• Measurements performed with
purified water and Mixture of
Glycerin/ Purified Water
Total Pressure Drop of the system in the flow rate
range of 4-8 L/min is less than 6 psi in 1- 50 cP
viscosity range
No significant pressure drop increase due to
added sterile connectors

22. System Characterization
Retentate Pressure Drop
22
The retentate pressure drop is the line drop from the outlet of the membrane holder through the retentate
line back to the recycle vessel.
Why is it important?
High retentate line pressure drop could limit the minimum transmembrane pressure that can be achieved
even with the retentate control valve fully open.
• Capsule assembly not installed
during testing
• Measurements performed with
Purified Water and Mixture of
Glycerin/ Purified Water
Retentate Pressure Drop in the flow rate
range of 4-8 L/min is less than 5 psi in
1- 50 cP viscosity range
No significant pressure drop increase due
to added sterile connectors

23. Equilibration Concentration Diafiltration Final concentration Recovery
Closed Processing with Single Use Flexware® Assemblies
and Devices
23
• Equilibration buffer
10 mM PBS, pH 7.2
• 20 L/m2
 18.5 L
 20 g/L to 60 g/L IgG
 Viscosity: 1.5 – 1.8 cP
 Diafiltration at 6 L
 25 mM Sodium
Acetate, pH 5.5
 8 Diavolumes
 60 g/L to 200 g/L IgG
 Viscosity: 1.8 – 13 cP
 Elevated Viscosity
Applications
 Low Viscosity
Applications
 Manual & Automated
All streams have to be collected into bags
Buffer
Product
Waste
Permeate
Product
In
Waste
Retentate
Out

24. Product Recovery
XV001
XV009
XV003
XV002
XV404
XV102
PCV101
Recycle
Vessel
XV441 XV402 XV431 XV421
XV401
XV411
XV403
XV405
XV007
XV006
XV406
WFI F401 NaOH Buffer Source
Drain
Recovery
Drain
To Flush
Bag
P002
P001
XV004
XV005
PCV001
XV001
XV009
XV003
XV002
XV404
XV102
PCV101
Recycle
Vessel
XV441 XV402 XV431 XV421
XV401
XV411
XV403
XV405
XV007
XV006
XV406
WFI F401 NaOH Buffer Source
Drain
Recovery
Drain
To Flush
Bag
P002
P001
XV004
XV005
PCV001
XV001
XV009
XV003
XV002
XV404
XV102
PCV101
Recycle
Vessel
XV441 XV402 XV431 XV421
XV401
XV411
XV403
XV405
XV007
XV006
XV406
WFI F401 NaOH Buffer Source
Drain
Recovery
Drain
To Flush
Bag
P002
P001
XV004
XV005
PCV001
XV001
XV009
XV003
XV002
XV404
XV102
PCV101
Recycle
Vessel
XV441 XV402 XV431 XV421
XV401
XV411
XV403
XV405
XV007
XV006
XV406
WFI F401 NaOH Buffer Source
Drain
Recovery
Drain
To Flush
Bag
P002
P001
XV004
XV005
PCV001
24
Step 1
Pump the Product out of
the recycle vessel through
the recovery line
Step 2
Gravity Drain the
Retentate Line between
the bottom of the recycle
vessel and transfer line
Step 3
Buffer Flush through the
transfer and retentate lines,
devices to the recovery line
Step 4
Flush the Retentate Line
between the transfer line
and bottom of the recycle
vessel with a small amount
of buffer, then pump it out
through the recovery line

25. Low Viscosity Applications, Manual Mode of Operation
25
Product Recovery
Protein Recovery Summary
Conc, Pre-Recovery [g/L] 53
Viscosity [cP] 1.6
Yield [%] 102.9
Final Conc [g/L] 47
Step 3 Step 4
Step
1&
2

26. Elevated Viscosity Applications, Manual Mode of Operation
26
Product Recovery
Protein Recovery Summary
Conc, Pre-Recovery [g/L] 186
Viscosity [cP] 10
Yield [%] 99.0
Final Conc [g/L] 165
Step 3 Step 4
Step
1&
2

27. 27
Product Recovery
Automation: A Key to Process Consistency
Common Control Platform® (CCP®) Software
• Written application recipes can be edited and stored
for different processes
• Report generator quickly creates batch reports
Advantages of Automation
• Run to run consistency
• Lower risk of operator error
• Easier troubleshooting
Product Recovery, Automated Mode
 Product recovery recipes written, tested and
optimized to achieve maximum product recovery
with minimal dilution.

28. Low Viscosity Applications, Automated Mode of Operation
28
Product Recovery
Protein Recovery Summary
Conc, Pre-Recovery [g/L] 51
Viscosity [cP] 1.5
Yield [%] 97.6
Final Conc [g/L] 44
Step 3 Step 4
Step 1& 2

29. Elevated Viscosity Applications, Automated Mode of Operation
29
Product Recovery
Protein Recovery Summary
Conc, Pre-Recovery [g/L] 202
Viscosity [cP] 14.5
Yield [%] 94.9
Final Conc [g/L] 170
Step 3 Step 4
Step 1& 2

30. 30
Summary
Closed TFF System and Process Characterization
1
Minimum working volume
• Minimum working volume using 1 m² Pellicon® Capsule assembly
is < 1.3 L for solutions with viscosity between 1 – 50 cP
2
Flowpath pressure drop
• No significant pressure drop increase due to added sterile connectors
• For typical range of flow rates for a TFF process and viscosity 1-50 cp
• Total system: < 6 psi
• Retentate line: < 5 psi
3
Process run
• Protein solutions can be concentrated to high final concentrations
• System can handle processing elevated viscosity solutions with ease
4
Product recovery
• Proposed recovery methods result in ≥ 95% protein recoveries with
minimal protein dilution
• Process can be adjusted to comply with customer specifications and needs

31. 31
A closed state can be achieved in different
ways and would present benefits for
downstream steps.
While drivers vary between applications, the
same specific mindset needs applying to make
it successful with no compromise on
performance.
Ultimately, closed processing steps will be an
integral part of connected, continuous
processing.
Conclusion and
perspectives

32. sarah.lemerdy@emdgroup.com
dana.kinzlmaier@milliporesigma.com
Sarah Le Merdy
Dana kinzlmaier
The vibrant M, Millipore, Flexware, Mobius and Pellicon 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.
© 2021 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.
Acknowledgments
Special Thanks to North America MSAT Team, Patrick McGee, Elizabeth Goodrich,
Akshat Gupta, Emily Peterson, Gayathri Raja and Product Marketing Team,
Sylvie Riou and Christophe Dufossé
Thank you