Introduction to Tangential Flow Filtration (TFF)

This presentation provides an introduction to tangential flow filtration and reviews the following:
- TFF process basics and terminology
- TFF membrane technology
- TFF hardware, devices and systems
- Growing applications and the future

To learn more about this topic or collaborate with our technical experts, schedule an in-person or remote visit at our M Lab™ Collaboration Centers: www.emdmillipore.com/mlab

1. Merck KGaA
Darmstadt, Germany
Oscar Bernal Zuniga, PhD –Process Development Scientist
Introduction to
Tangential Flow
Filtration (TFF)

2. 01
02
03
04
Agenda
2
TFF Process Basics and Terminology
TFF Membrane Technology
TFF Hardware, Devices and Systems
Growing Applications and the Future

3. Normal Flow Filtration (NFF)
Feed Flow
(in)
Membrane surface
Filtrate
(out)
Volume filtered
Filtrate flux rate
What is Tangential Flow Filtration?

4. Pressure
Tangential Flow Filtration (TFF)
Volume filtered
Filtrate flux rate
Filtrate (Out)
Flux
Membrane surface
Feed Flow (In and Out)
What is Tangential Flow Filtration?

5. •Purification (separates components by size, removes impurities)
•Concentration (reduces volume, de-waters)
•Buffer exchange or diafiltration (formulation)
What Does TFF Do?
500 L
1g/L
50 L
10 g/L
Buffer A Buffer B
Purification Concentration Buffer Exchange

6. Important Basic Parameters
The volume of a solution that can be filtered with a given area of filter
Capacity (L/m
2
or g/m
2
)
Retention
The flowrate of liquid through a membrane per area
The ability of a filter to remove or retain a given component
Flux (L/min/m
2
or LMH)

7. A Basic TFF Set-up
7
Holder
Feed Pump
Retentate Valve
Pressure gauges
Flow meters
Tubing / piping
-Minimum working volume must be
determined (maximum concentration)
-System design & product recovery must be
considered (yield %)
-Cleaning and sanitization: pressure gauge
location, dead legs (cleaning, bioburden,
endotoxin)
Critical Considerations

8. Flows and Forces in a TFF Channel
C
b= protein concentration in bulk solution [g/L]
C
w= protein concentration at membrane [g/L]
k = mass transfer coefficient [L/m
2
*h]
Q
F
TMP
Q
p
Q
RC
b
C
w
Membrane
Membrane
k
Q
F= feed flow rate [L/h]
Q
p= permeate flow rate [L/h]
Q
R = retentate flow rate [L/h]
P
F= feed pressure [bar]
P
R= retentate pressure [bar]
P
p= permeate pressure [bar]
P
F P
R
P
p

9. DP [bar or psi] = P
F–P
R
Difference in pressure along membrane feed channel
•Resistance to feed flow in the channel
•Function of viscosity, feed flow rate, channel geometry
Pressure Drop (dP)
Membrane
Membrane
Inlet Pressure (P
F)
Retentate Pressure (P
R)
Permeate Pressure (P
p)
30 psi 20 psi
DP = 30 –20 psi = 10 psi
dP
1.38 bar
2.07 bar
0.69 bar

10. TMP [bar or psi] = [(P
F+ P
R) / 2 -P
p]
•Average driving force across the membrane (creates flux J)
•Created by applying retentate pressure & increased feed flow
Transmembrane Pressure (TMP)
Membrane
Membrane
Inlet Pressure (P
F)
Retentate Pressure (P
R)
Permeate Pressure (P
p)
30 PSI
20 PSI
2 PSI
TMP = (30 + 20)/2 -2 = 23 psi
TMP
2.07 bar
1.38 bar
0.14 bar
1.58 bar

11. •By adjusting the pump speed (Feed flow rate Q
F)
•By opening / restricting the retentate valve
(Retentate pressure P
Ror P
out)
•As TMP increases, filtrate flux increases
(pressure controlled) low polarization
How to Adjust TMP, and its Effect on Flux0
10
20
30
40
50
60
70
80
0 10 20 30 40 50 60
Flux [LMH]
TMP [psid]
Linear
Polarized

12. Polarization
Accumulation of retained solutes in a concentrated layer close to the membrane surface
Affected by cross-flow, TMP, temperature and concentration
Results in decline in filtrate flux over time during process
Reversible by better balance of cross flow / TMP conditions
All TFF operations have a degree of polarization
Unbalanced Systems Produce Polarization
C
b Bulk Concentration
C
w Wall Concentration
Polarization Layer
Membrane
Membrane

13. Controlled
Membrane
Polarization
No
Membrane
Polarization
Uncontrolled
Membrane
Polarization
Membrane Polarization Needs to be Controlled

14. •Refers to the decline in permeate flux over time resulting from interaction (adsorption & deposition)
between the solute or particles & membrane.
•It is 'irreversible’ during process-original flux cannot be restored by reversing process operating
conditions.
•Reversed only by cleaning
Fouling

15. Tangential Flow Filtration dynamics depends on the membrane used in the holder
Types of TFF
1
Microfiltration
(typically
0.1 –5um)
2
Ultrafiltration
(typically 1kD –
1,000kD MW)
3
Nanofiltration
(typically 200 –
1,000D MW)
4
Reverse Osmosis
(typically
40 –200D MW)
Pressure-driven membrane-
based separation process
in which particles and
dissolved macromolecules
larger than 0.1um
are rejected
Pressure-driven membrane-
based separation process
in which particles and
dissolved macromolecules
smaller than 0.1um and
larger than about 2nm
are rejected
Pressure-driven membrane-
based separation process
in which particles and
dissolved molecules
smaller than about 2nm
are rejected
Liquid-phase pressure-
driven separation process
in which applied
transmembrane pressure
causes selective
movement of solvent
against its osmotic
pressure difference
Clarification Concentration/ Diafiltration

16. Filtration Type vs. Typical Particle Size
Ref: http://www.cellsalive.com/howbig_js.htm

17. Upstream
•Harvest
•Clarification
•Perfusion
Where Does TFF go in a Process?
Volume
reduction
Cell culture
Harvest
Clarification
Chromatography
Virus
removal
Tangential flow
filtration
Sterile
filtration
Formulation
and Final Fill
Large Molecule
Downstream
•Purification
•Formulation
•Volume reduction

18. Goals of TFF
Concentration/diafiltration (buffer exchange)
Product retained by the membrane and concentrated
Buffer passes through the membrane
New buffer may be added to product (diafiltration)
Contaminants and/or impurity removal
Product retained by the membrane
Contaminants and/or impurities pass through the
membrane

19. What About Microfiltration?
Permeateflow(flux)controltoafixed,robustsetpointthroughoutaprocessis
achievedthroughtheuseofapermeatepump–thefluxcontrolledTFF
processesarealsoreferredtoas2-pumpTFF

20. Flux Effects on Performance
As flux increases:
Observed sieving (S
o) of a partiallyretained species increases
However, polarization (C
wall/C
bulk) of a fullyretained species also increases
−Beyond a certain flux (polarization level), the observed sieving (S
o) of the
partiallyretained species may tend to decrease.
−High MW species will preferentially polarize and block the target
component
−Transport resistance through polarized layer, fouling etc.
TMP
Polarized zoneFouling zone
Flux (J)
Sieving, S
o
or Mass Flux
Sieving
Mass Flux
TMP
Critical Flux
“determines
the Optimum
Operating Point”
Domain of Optimum Operation
MassFlux=JC
Permeate=JC
FeedS
o

21. 01
02
03
04
Agenda
21
TFF Process Basics and Terminology
TFF Membrane Technology
TFF Hardware, Devices and Systems
Growing Applications and the Future

22. Let’s Think About Membranes
What is a UF membrane ?
Thin barrier with ability to separate
particles and/or molecules by size
Typical Membrane Considerations:
What are we removing/retaining?
−NMWL Selection guidelines
Types/Material of Construction
−Chemical Compatibility
Interactions with feed stream solutes
−Protein Binding
Screen Type
−Flux benefits, pressure limitations
Biomax® membrane 300 kD top view
magnified 80000x

23. How Does a Membrane Work?
Retention (rejection)
−Large particles/solutes are rejected or retained by the membrane structure
Sieving (passage)
−Small particles/solutes pass or sieve through the membrane
Biomax® membrane 300 kD top
view magnified 80000x

24. •Micron pore size
•0.10 -0.65 µm
•Separate cells and cell debris from proteins
•PVDF, Cellulose, and PES most commonly used
Microfiltration
Durapore
®
membrane GVVP 0.2um magnified 650x

25. •Sized in molecular weight
•1 kD -1000 kD
•Separate proteins from small molecular weight
contaminants
•Regenerated cellulose and polyethersulfone (PES) are
commonly used
Ultrafiltration
Biomax
®
PES membrane 30 kD
magnified 1000x
Ultracel
®
composite regenerated cellulose
membrane 10 kD magnified 500x

26. What is NMWL/MWCO ?
•A convenient reference label for UF membranes
•No industry standard (or specification) between UF membrane manufacturers
How do manufacturers choose NWML ?
•Typically based on MW of 80-95% of a retained MW solute
•Can depend on solute marker, test apparatus and operating conditions
Nominal Molecular Weight Limit (NMWL) and Molecular Weight
Cut-off (MWCO)

27. Molecular Weight Cut -off
4 particles with similar
molecular weights and
subunits may look quite
different

28. Molecular Weight Cut -off
On a given filter they may BEHAVE very differently as well…
Total Retention Incomplete Retention

29. Molecular Weight Cut -off
29
Always match the pore size to the application!
For retention of product:
-Use a NMWCO of 1/3 –1/5X that of product
For product passage
-Use a NMWCO of 3 -5X that of the product

30. Feed screens create:
•Gentle turbulence at the membrane surface to control the effects of concentration polarization and
maintain higher flux
•Hydraulic resistance resulting in higher pressure drop
Screen Function in TFF
Higher polarization
Lower flux
Lowerpressure drop
Lower polarization
Higher flux
Higher pressure drop
Effect increases at
higher viscosities

31. Screens & Their Tradeoffs
31

32. 01
02
03
04
Agenda
32
TFF Process Basics and Terminology
TFF Membrane Technology
TFF Hardware, Devices and Systems
Growing Applications and the Future

33. TFF Device Formats
SpiralWound HollowFiberFlat Sheet

34. Flow Path in a Flat Sheet Cassette
Feed
Retentate
Permeate

35. Linear Scalability
•Identical retentate and
permeate channel
•Multiple membrane areas
15.5
cm
Pellicon
®
3 cassette 0.11 m
2
Pellicon
®
3 cassette 0.57 m
2
Pellicon
®
3 cassette 1.14 m
2
Stacked to 80 m
2
per holder
Linear Scalability is defined as the ability to use lab process parameters
developed in PD at larger scale with identical performance
Pellicon
®
3 cassette 88 cm
2

36. Ultracel
®
PLC
5, 10, 30, 300,
1,000kD
Durapore
®
0.1, 0.22, 0.45, 0.65µm
Biomax
®
5, 8, 10, 30, 50, 100,
300, 500, 1,000kD
Ultracel
®
PLC
3, 5, 10,
30kD
Biomax
®
10, 30,
50kD
Ultracel
®
PLC
5, 10, 30, 100,
300, 1,000kD
PL 1, 3, 10kD
Biomax
®
5, 8, 10, 30, 50, 100,
300, 500, 1,000kD
Durapore
®
0.1, 0.22, 0.45, 0.65µm
Cassette Areas Change to Process -Specific Batch Volumes
Screening/feasibility Pilot/production
Pellicon
®
3 88cm
2
Pellicon
®
3 0.11m
2
Pellicon
®
3 0.57m
2
Pellicon
®
3 1.14m
2
50mL –1L 2L and up
80mL –10L 10L and up
Pellicon
®
2 Mini 0.1m
2
Pellicon
®
2 0.5m
2 Pellicon
®
2 Maxi 2/2.5m
2
Pellicon®
3 cassette
Pellicon®
2 cassette
Pellicon® XL 50
Pellicon®
XL50
cassette

37. TFF Hardware

38. What is Expected of a TFF System?
That it can adapt especially regarding scale to the different development
phases of a product to support the fast market introduction of new
molecules
38
Single-Use System –Mobius
®
FlexReadySolution for TFF
Smart Flexware™
clamshell: reduced hold up
& dead volumes

39. TFF System Family
Custom TFF System
0.5 to 10 m
2
0.5 to 40 m
2
0.1 to 1.14 m
2
Cogent®
Process Scale
Single-
use Multi-use
Multi-use
Cogent® M1Cogent®
µScale
Labscale TFF
System
Mobius®
FlexReady
Solution for
TFF

40. Small Scale –Cogent
®
μScale TFF System
1 L polypropylene
tank with a removable
vacuum seal lid
Integrated magnetic
stir mechanism for
efficient, but gentle
mixing
Dual channel peristaltic pump
head with STA-PURE® (platinum-
cured silicone expanded PTFE)
tubing enables low pulsation (<
+/-3 psi)
Pellicon
®
filter
holder designed to
hold up to three
Pellicon
®
3 88 cm
2
cassette (264 cm
2
)
The weight scale option also
enables semi-automatic
operation process when a
target filtrate weight has
been achieved
Multilingual Touch
Screen: P&ID screen for
simultaneous real time
monitoring of all active
process parameters

41. Large Scale -Cogent
®
Process Scale
Full process automation :
•Enables to easily and consistently produce
clinical and preclinical scale quantities of
high-value drug products to cGMP standard
•Optimized design.
•NovAseptic
®
valves.
•Pellicon
®
TFF device holders
•Optional mini-loop on retentate.
➔Low minimum working volume
-2.5L for 2.5-5.0 m²
-5.5L for 5-15m²
➔Maximum product recovery

42. What are the Challenges in the Biomanufacturing Industry?
Trends in
biopharmaceutical
manufacturing:
•Reduced costs
•High feed/titers
•Reduced validation
•Increased efficiency and
system flexibility
Pre-
clinical
Commercial Manufacturing
Phase
III
Phase I
Phase II
Therapeutics
Running processes single -use may increase consumable costs but
can reduce overall cost -> faster implementation, lower buffer use,
eliminate cleaning validation

43. Smart Flexware
®
Assemblies
What is a Smart Flexware
®
Assembly?
•A single-use flow path with no dead legs
•An assembly composed of PureFlex™ film,
fittings, tubing, and connectors
•Designed with the unique process flow
path welded inside the assembly
Advantages:
•Increased automation of manufacture to
decrease variability between assemblies
•Allow a streamlined flow path to maximize
process efficiency
•“Single-use” approach eliminates the
need for cleaning validation

44. 01
02
03
04
Agenda
44
TFF Process Basics and Terminology
TFF Membrane Technology
TFF Hardware, Devices and Systems
Growing Applications and the Future

45. Common and Growing Applications in Process
Concentration/ Buffer
exchange/High
viscosity formulations
Single-use TFF
Single-pass TFF Perfusion / Cell
Retention / Process
Intensification

46. High-Viscosity TFF and Impact of Screens
More open screen (D) weave is
optimized for the high viscosity range:
•Pressure drop within operating
specifications
•Higher flux than more open channels:
reduces process time
•Higher concentration target achievable
High viscosity formulations present challenges
Developed novel feed channel for processing high
concentration therapeutic antibodies
−Concentrations >150 g/L (e.g.final formulation)

47. 47
Performance for High Viscosity TFF with Ultracel
®
30kD membrane

48. 48
Performance for High Viscosity TFF with Biomax
®
30kD membrane

49. Speed
Reduce new facility build
times by 70%. Compress
production lead time by
80%.
Quality
10Xrobustness.
90%reduction in cost of
poor quality.
Flexibility
Reduce product change-over
time by 90%.
Cost
90%reduction in cost to
manufacture and CAPEX.
Business
Drivers
Market
Growth
Uncertainty
New Product
Classes
Cost
Pressure
Market
Trends
Market Trends, Business Drivers and Key Enablers*
to Drive Next Generation BioProcessing:
Process
Intensification
Process
Analytics
Software &
Automation
Key
Enablers
Single Use
BioContinuum™ Platform Digital BioProcessing (incl Sensors) Single Use

50. Why Would You Operate Single -Use?
Speed
Safety
Process
Economics
Flexibility
Faster product changeover
Accelerated time to clinic
or market
Faster implementation
Adaptability to changing
process needs
Multi-product mfg facilities
Scalable and movable
platforms
Lower maintenance: reduced
water/caustics, utilities, equipment
downtime, and unproductive labor
Smaller footprint
Lower investment
Reduced bioburden risks
Reduced cross-
contamination risks
Reduced operator and
environment exposure

51. Bioreactor
today
Standard
mAb
Template
@ 6 g/L
Example: Process improvement allows significant productivity gains
while eliminating capacity constraint and reducing costs
Hold Tanks
Based upon an actual case study and implementation data
Clarification Affinity
Chromatography
Virus
Inactivation
Purification
Chromatography
Polishing
Chromatography
Viral
Clearance
Concentration &
Diafiltration
Affinity
Chromatography
Virus
Inactivation
Bioreactor Clarification Concentration &
Diafiltration
Purification
Chromatography
Polishing
Chromatography
Viral
ClearanceSingle Pass
TFF
Improved
mAb Process
Impact
Hold Tank Bottlenecks due to titer increase
Intensified
Seed-train Inoculation
Intensified
Seed-train Inoculation
High cell density
seeding & Media
Optimization led to
increase titer in
production reactor
from 1.5g/L to 6.0g/L
Pellicon
®
Single
Pass TFF
Eliminate downstream
shortfall in hold tank
capacity
•Plant capacityrequiredfor
moleculeproductiondecreased
from90% to26%
•75% annual
COGS reduction

52. What is single-pass TFF?
Product is not concentrated after a
single pass
−Requires retentate return and multiple
passes through filter
Batch operation
−Requires skid with tank and pump
Batch
SPTFF
Product is concentrated after a
single pass through the filter
assembly
−No retentate tank needed
Continuous operation
−Run in-line with no tank or pump

53. Next Generation Bioprocessing
An alternative application to batch TFF…
Product sufficiently concentrated after one pass through filter
modules
No retentate recirculation required
Membrane arranged in series to increase path length and
improve performance
Run with Pellicon
®
2/3 cassettes & Pellicon
®
Capsules
Applications:
•Volume reduction In -between unit operations
Reduce/control intermediate pool volumes
•Final high concentration formulation
Post UF/DF, smaller secondary system (lower hold-up volume)
•Connected or Continuous processing
In-line operation coupling TFF to existing process steps
mAbs · Vaccines · Plasma · VGT

54. Applications: Versatility in Bioprocessing
Integrate anywhere where volume reduction is needed, including
−Before or after column chromatography or filtration steps
−At final concentration and formulation
Bioreactor Clarification Protein A
Capture
Virus
Inactivation
CEX Polishing AEX Polishing Viral
Clearance
Final
Filtration
Concentration
& Diafiltration
Depth
Filtration

55. Pellicon
®
Capsules for Single-use TFF
1
Plug and Play Design
•Holderless and no torque required
•Self-contained
•Easy to install, easy to remove
2
Ready to Use in Minutes
•Gamma sterilized
•No preservatives
•Significantly reduce pre-process steps
3
Pellicon
®
TFF Proven Performance
•Same membrane and screen as Pellicon
®
cassettes
•Comparable performance and scalable to our cassettes
•Efficient DF and chemical compatibility
Single-use TFF Capsules

56. Single-use Applications in mAb and ADC manufacturing
mAb drivers: changing molecules + biosimilars
= speed to clinic/market
ADC drivers: operator safety + product
contamination risks = containment
✓Less complexity:
-easy installation
-no sanitization
✓Faster batch turnaround/ speed to market
✓Post-use containment
✓Easy to remove and dispose
✓Efficient DF
✓DMSO/DMAC solvent compatibility
✓Linear scalability
0.0
20.0
40.0
60.0
80.0
100.0
120.0
140.0
0.05.010.015.020.025.030.035.040.045.0
Flux (LMH)
TMP (psi)
10 g/L BgG
Cassette 0.11 m², P3C030C01 (+10%)
Cassette 0.11 m², P3C030C01 (-10%)
Capsule 0.1 m², PCC030C01
Capsule 0.5 m², PCC030C05

57. Process intensification helps :
✓Save process time
✓Increase yield
✓Boost manufacturing capabilities
✓Reduce footprint
Cellicon™ Perfusion Solution
Using Perfusion for Process Intensification
1. We provide
perfusion during
seed train so that
the production
bioreactor can be
spiked with the
highest cell density.
3. Ultimately, this
provides increased
yield and
manufacturing
capabilities. It also
helps reduce the
resources needed
and space required.
2. We provide a cell
retention offering
that recirculates
cells out of the
bioreactor, removes
spent media, and
circulate cells back
into the bioreactor.
How do we implement perfusion?
Cell retention filter
• Part of a single use gamma-irradiated
assembly
• Flat-sheet tangential flow design
• 5um Durapore
®
membrane
• Suitable for 1-2.5 L bioreactor working
volume range
Controller
• Bench-top
• Allows for real-time process data
• Recirculation flow control
• Intuitive UI
• Remote capability
Cellicon™N-1 Perfusion
Solution
0
20
40
60
80
100
120
-1 1 3 5 7
VCD (E6 cells/mL) and Viability
(%)
Duration (days)
Cell Growth Data for N-1 Perfusion
Viable Cell…
Viability
0
2
4
6
8
10
0 500 1000 1500 2000
TMP (psi)
Throughput (L/m
2
)
Device Throughput Data for N-1 Perfusion
Achieves
high cell
densities!
The filter
is
properly
sized!

58. •TFF (UF/DF) Operations
Video
•UFDF Learning center
•Optimization UFDF Tech
Note found in learning
center
•Webpage
•Includes links to
webinars & product
literature
•LinkedIn: TFF Experts
Community
•Learn@M: register for a
TFF course
Process
Development
TFF
Web & Social
Links
TFF Resources

59. Schedule an in-person or remote
visit today.
EMDMillipore.com/mlab

60. The vibrant M, M Lab, BioContinuum, Biomax, Durapore, Mobius, Pellicon, PureFlex, Smart Flexware, Ultracel and Cellicon 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.
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