Tangential Flow Filtration
- TFF Processes
For Hanvet
By Tavis Tan
This presentation is the work
product of Pall Corporation
and no portion of this
presentation may be copied,
published, performed, or
redistributed without the
express written authority of
a Pall corporate officer.
© 2017 Pall Corporation.
1
Do You Recall?
General TFF System Configuration
2
2
Do You Recall?
TFF Process Flowchart
3
3
TFF Process Overview
Pre-Use Conditioning
1)
INSTALL cassette with torque wrench

MEASURE hold-up, minimum working volumes
2)
FLUSH to remove storage agent
3)
SANITIZE filter and system with NaOH solution
4)
FLUSH to remove NaOH solution
5)
MEASURE Normalized Water Permeability (NWP)

To determine membrane cleaning effectiveness
6)
INTEGRITY TEST

To determine integrity of filter and system
7)
CONDITION with buffer

To condition filter for pH, ionic strength, temp
4
4
Pre Use Conditioning
1) Cassette Installation
Torque Sequence
5
5
Pre Use Conditioning
1a) Hold-up & Minimum Working Volumes
 System Hold-up Volume
– The total volume contained within the feed / retentate flow path
 Most of this volume is recoverable
 Minimum Working Volume
– The system hold-up volume plus the minimum volume of liquid that must
remain at the bottom of the feed vessel to prevent air from getting drawn
into the system
 Limits maximum concentration achievable
 Increases with increase in cross flow rate
 Tank design affects minimum working volume
6
6
Pre Use Conditioning
2) Initial Flushing
Objective :
 To remove bulk of storage agent
(glycerin / sodium azide or NaOH)
 To remove additional foulants dissolved during storage
(i.e. reused)
7
7
Pre Use Conditioning
3) Sanitization
 Typically accomplished by chemical mechanism in cassettes
Objective :
 To lower the microbial population in the system
 To remove metabolites (proteins and sugars) which will
promote microbial growth
8
8
3) Sanitization
Sanitization Agents
 Chlorine
– Bleach (sodium hypochlorite NaOCl)
 pH 6 – 8, 10 – 50 ppm
 Kills bacteria, spores, moulds, yeast and certain viruses
 Peracetic Acid
– CH3C03H (Acetic acid + hydrogen peroxide)
 100 - 300 ppm
 High biocidal effectiveness, good alternative to chlorine
 Sodium Hydroxide
– NaOH
 pH > 12
 Not as effective as chlorine but adequate in most cases
9
9
3) Sanitization
Sterilization
 Complete destruction of microorganisms in the system
using heat
– Steam-in-place (SIP)
– Wet autoclave at 121°C for 30 minutes
 Does not remove pyrogens and endotoxin
 NOTE: Most cassettes cannot be steamed!
10
10
Pre Use Conditioning
4) Post-Sanitization Flushing
Objective :
To remove all fluid extractables
 Removes all traces of sanitization agent
 Assures membrane is completely wetted before determining
NWP
 Removal of extractables is time dependent – not volume
dependent
11
11
Pre Use Conditioning
5) Measurement of NWP
NWP t°C = Normalized Water Permeability
Measure of the water filtrate flux rate as a function of transmembrane
pressure at a given set temperature
NWP 20°C = [ J (LMH) / TMP (psig) ] x tc
where
J = Filtrate flux rate
TMP = Transmembrane Pressure
tc = temperature correction factor
12
12
5) NWP
Normalized Water Permeability
Water Flux Rate (LMH)
 WP must be determined for "new"
*
membranes to establish the value
against which recovery is measured.
60
*
40
20
 WP is related to the pore diameter
and number of pores / unit area of
the membrane.
*
 WP is temperature dependent.
5
10
15
TMP(psig)
13
13
5) NWP
Water Permeability
Example:
Permeate Flux (LMH)
Determining Membrane NWP
Water Permeability = 110 LMH @ 10 psi
= 11 LMH/psi
NWP20°C = 11/10 X TCF
Temperature Correction Factor
= 1.109
(Correcting the water temperature from 16°C to
20°C)
Temp = 16°C
X
150
X
100
50
X
0
5
10
Normalised Water Permeability (NWP20°C)
= 11 X 1.109
= 12.2 LMH
15
Transmembrane Pressure (psi)
14
14
5) NWP
Temperature Correction Factor
T oC
11
12
13
14
15
16
17
18
19
20
TCF20oC
1.271
1.235
1.202
1.169
1.139
1.109
1.081
1.053
1.027
1.000
T oC
21
22
23
24
25
26
27
28
29
30
TCF20oC
0.978
0.955
0.933
0.911
0.890
0.871
0.851
0.833
0.815
0.798
T oC
31
32
33
34
35
36
37
38
39
40
15
TCF20oC
0.781
0.765
0.749
0.734
0.719
0.705
0.692
0.678
0.665
0.653
T oC
41
42
43
44
45
46
47
48
49
50
15
TCF20oC
0.641
0.629
0.618
0.607
0.596
0.586
0.576
0.566
0.556
0.547
5) NWP
When to Determine NWP
 Immediately after initial pre-conditioning?
 One or two days after initial pre-conditioning
 After a full cleaning regime?
 After the first process run?
16
16
5) NWP
Cross Flow vs DP
 CF is directly related to DP with
filtrate closed and constant
viscosity
 Changes in DP for same CF may
indicate
– cassette compression
– or that there may be a
blockage in the feed ports to
the cassette
– or there has been a change in
fluid viscosity
17
17
Pre Use Conditioning
6) Integrity Test
 Purpose: To assure proper
assembly and membrane /
membrane element integrity
 Techniques used:
– Pressure Hold (Qualitative) – For
system integrity
– Air Diffusion (Quantitative) – For
membrane integrity
 Use mass flow meters for fast,
18
accurate determination
18
Pre Use Conditioning
6) Integrity Test
System Configuration
M ass
Flow meter
Cassette
Hardware
Air
Source
Integrity
Inlet
Valv e
Feed
Test Profile
Retentate
Filtrate
Pump
Integrity
Outlet
Valv e
Waste
19
19
Pre Use Conditioning
7) Buffer / Temperature Conditioning
 Ensure buffer is in suitable conditions:
– Ionic strength
– pH
– Temperature
 Prevent possible precipitation when product is
added to TFF system
 Remove air that can block pores and reduce flux
rate
 Prevent micro bubbles (shear)
 Prevent contraction or expansion caused by
changes in temperature
20
20
TFF Process Overview
Processing
8)
OPTIMIZE processing
 To determine operating parameters
9)
CONCENTRATE
 To reduce sample volume
10)
DIAFILTER (or exchange buffer)
 To reduce salt concentration
11)
RECOVER product
 To maximize recovery of product
from the system
21
21
Processing
TFF Process Optimization
1) Planning
2) Pre and Post Use Conditioning
3) Optimization
4) Concentration
5) Diafiltration
6) Product Recovery
22
22
TFF Process Optimization
1) Planning
 Clearly establish process objectives
 Trial considerations
– Establish volume to be used for trial
– Membrane area to volume ratio
 Approximate to full scale process
– Concentration factor required
 Hold-up volume and dilution
– Diafiltration requirements?
– Product stability (e.g. temperature)
23
23
TFF Process Optimization
1) Planning
 Identify and obtain required materials, TFF equipment
and cassettes
– Select correct platform
– Select membrane rating/MWCO
– Determine area to be used for trial
– Acquire appropriate volume of sample
 Sample should be truly representative of product
 Measure actual volume used
 Assay sample for mass balance
– Have additional cassettes available in case?
– Consider large scale process and process requirements
(e.g. SIP)
24
24
TFF Process Optimization
1) Planning
 Equipment requirements
– Pump
– Hold-up/Min working volumes
 Know it and minimize it
– Correct range of instruments
– Calibrated instrumentation
 Pressure gauges
 Thermometer
– Temperature control?
– Flowmeter?
– Use spreadsheet if possible
25
25
TFF Process Optimization
Flux vs Transmembrane Pressure
GEL LAYER
MEMBRANE
CONTROLLED REGION CONTROLLED REGION
Filtrate Flux Rate
WATER
PROCESS FLUID
CONSTANT CF
(D P)
WHERE CF =
CROSS FLOW VELOCITY
OPTIMAL
Transmembrane Pressure
26
26
TFF Process Optimization
Optimum conditions for selected Crossflow for concentration step...
Permeate Flux Rate
(LMH)
Flux Vs TMP
50.00
1.25
40.00
1.00
30.00
0.75
20.00
0.50
10.00
0.25
0.00
0.00
0.50
1.00
1.50
0.00
2.00
Transmembrane Pressure
Process Flux
Differential Pressure
27
27
TFF Process Optimization
Process Sample
 Concentrate sample at "optimized" conditions
 Measure Flux vs. Concentration Factor
 Sample both retentate and permeate
 Perform diafiltration as required
 Recover Product
 Measure product recovery
28
28
TFF Process Optimization
TFF System Configuration -- Concentration
Recirculation
29
29
TFF Process Optimization
TFF System Configuration -- Diafiltration
Recirculation
30
30
TFF Process Optimization
Optimal Concentration (CD) to Perform Diafiltration
CD =
CG
=
1.0
e
CG
2.7183
= 0.368 CG
Filtrate Flux Rate (LMH)
100
90
80
70
K
60
50
40
CG
30
20
10
0
1
2
3
4
5
6
7
8
9 10
20
Concentration Factor
31
31
TFF Process Optimization
Diafiltration Volume, VD
 Each VD is a volume of buffer equal to the volume of process solution
present in the process reservoir and circulation loop at the start of the
diafiltration.
 To predict the number of diafiltration volumes required to “wash out” a
target percentage of a permeable molecule, (refer to chart).
 Note: Concentration can affect performance of the diafiltration in 2 ways
– 1. Process flux
– 2. Transmission of permeable molecules
 Increase in conc = increase in resistance of flow through membrane
 Increase in resistance = decrease in permeate flow rate (process flux, J)
 In some apps, conc of retained molecules at the membrane surface can
result in difficulty for smaller permeable species to pass through membrane.
 Passage or Transmission, T = Cpermeate/Cfeed
32
; T(%)
= T x 100
32
TFF Process Optimization
Diafiltration Volume, VD
2x concentrate
33
33
TFF Process Optimization
Determining the Number of Diafiltration Volumes
J = Cp/Cf = T%
Constant Volume Diafiltration
0
% Permeable solids recovered in filtrate
Passage/Transmission %
of molecule
40
60
10
80
90
20
94
96
98
30
% Passage
100
99
0
1
2
3
4
60
80
5
6
7
40
50
8
9
10
11
12
Diafiltration Volumes
E.g.
T% = 10%, 1X VD = 10% permeable; 2X VD = 15% permeable
T% = 50%, 1X VD = 45% permeable; 2X VD = 68% permeable
T% = 100%, 1X VD = 70% permeable; 2x VD = 88% permeable, etc.
34
34
13
14
A Comparison of Different Concentration And Diafiltration
Combinations Used For The Processing Of Albumin
System Size
10m 2
Batch Vol.
(L)
Protein
Conc. %
Filt. Rate
(L/Hr)
Avg. Rate
(L/Hr)
Perm. Vol.
(L)
Filt. Time
(Hr)
CASE A
1) Initial
2) Conc. 5X
3) Diafilt. 5X
TOTALS
1000
200
200
5
25
25
940
140
160
460
150
800
1000
1800
1.7
6.7
8.4
CASE B
1) Initial
2) Diafilt. 5X
3) Conc. 5X
TOTALS
1000
1000
200
5
5
25
940
1060
160
1000
520
5000
800
5800
5.0
1.5
6.5
CASE C
1) Initial
2) Conc. 2X
3) Diafilt. 5X
4) Conc. 2.5X
TOTALS
1000
500
500
200
5
10
10
25
940
600
680
160
740
640
370
500
2500
300
3300
0.7
3.9
0.8
5.4
Source: Scale-up Considerations for Membrane Processes
R.S. Tutunjian, Bio/Technology Inc. 3, July 85
35
35
TFF Process Optimization
6) Product Recovery
 Close filtrate
 Recirculate concentrate for ~15 min.
 Displace / Collect concentrate
 Minimum volume buffer recirculation
 Displace / Collect remaining sample
Actual recovery procedure has to be optimized
for each process
36
36
Gel Layer
 Represents a high concentration of
PRODUCT at the membrane surface
 Represents a significant percentage of total
PRODUCT
 Accounts for a significant loss of PRODUCT if
not properly recovered.
37
37
Summary of UF Optimization protocol
1) Measure sample activity and volume
2) Measure NWP and DP vs. CFF for water
3) Determine air integrity and system hold-up volume
Buffer condition before adding sample
4) Generate Flux vs. TMP curve at selected CFF
– Measure passage and recovery
– Evaluate Flux vs. Passage
– Repeat Flux vs. TMP at different CFF if results are
not optimal
38
38
Summary of UF Optimization protocol
5) Concentrate Sample at optimal TMP
– Measure Flux vs Concentration Factor
– Measure product passage
– Concentrate beyond required factor
6) Add required diafiltration volumes
– Measure Flux vs Diafiltration Volumes
7) Recover product
8) Determine optimal cleaning protocol
39
39
TFF Process Overview
Post-Use Conditioning
12) FLUSH with buffer (optional)
 To remove easily removed foulants prior
to introducing chemicals
13) CLEAN in place (CIP) with NaOH solution
14) FLUSH to remove NaOH solution
15) MEASURE NWP
 To determine membrane cleaning
effectiveness
16) STORE cassette in NaOH solution
 To ensure filter remains wet
 To prevent microbial growth
40
40
Post Use Conditioning
Reasons for Cleaning
 Allow for reuse of cassettes (economics)
 Maintain efficient productivity by removing fouling
materials from the membrane
 Maintain a clean and sanitary system by removing
microorganisms and their metabolites
 Prevent contamination of subsequent production
batches!
41
41
Post Use Conditioning
Selection of Cleaning Agent
 Cleaning agents selected:
– Based on combination of effectiveness and compatibility
– With the membrane, membrane device and the process
 Need to determine and identify:
– Type of fouling agents
 Likely contaminants in product or solutions
– Membrane type and device / system
 Compatibility of cleaning agent with system
– Application requirements
 Compatibility of cleaning agent with process
42
42
Post Use Conditioning
Types of Cleaning Agents - Acids
Type
Foulant
Conditions
Nitric Acid
HNO3
Phosphoric
H3PO4
Citric Acid
Mineral scale
0.1N, 35-45oC,
pH 1
0.1N, 35-45oC,
pH 1
1%, 35-45oC,
pH1
Inorganics
Nucleic Acids
Iron
43
43
Post Use Conditioning
Types of Cleaning Agents
– Alkalis, Oxidizers
Type
Foulant
Conditions
Sodium Hydroxide
Proteins, enzymes,
vaccines,
bacterial cells / lysates,
polysaccharides,
organic colloids,
pyrogens, lipids
0.1-0.5 N NaOH,
35-45oC, pH > 13
Sodium Hydroxide /
Sodium Hypochloride
Inorganics
Nucleic Acids
0.3-0.5 N NaOH,
200-400 ppm NaOCl,
35-45oC, pH>12
44
44
Post Use Conditioning
Types of Cleaning Agents - Surfactants
Type
Foulant
Conditions
Sodium Dodecyl Sulfate
(SDS)
Triton X 100
Tween 80
Precipitated proteins
bacterial cells / lysates,
polysaccharides,
lipids, oils, antifoams
0.1%
35-45oC,
pH 4-9
45
45
Post Use Conditioning
Critical Cleaning Process Parameters
The goal is to determine
– the minimum time
– the minimum concentration of cleaning agent
required to assure effective, reliable, repeatable,
efficient and economic cleaning
46
46
Post Use Conditioning
Critical Cleaning Process Parameters
 Time
 Cleaning Agent Concentration
 Temperature
 Flow Rate – Fluid Velocity
 Water Quality
47
47
Post Use Conditioning:
Critical Cleaning Process Parameter
Time
– For a given cleaning agent, concentration and solution
temperature, the minimum cleaning solution contact time
must be determined.
– Typical time frame: 30 - 120 minutes
– Longer is not better
Temperature
Increasing temperature will:
 Decrease strength of foulant /
surface bonds
 Increase solubility of foulant
 Increase hydrolysis and oxidation rates
Typical range: 25- 45°C
48
48
Post Use Conditioning:
Critical Cleaning Process Parameters
Crossflow Requirements
– To reduce boundary layer effects, it is essential to maintain
adequate shear at liquid/surface interface.
– For cleaning, use 1 ½ - 2X CFF rate used for processing
– Minimum velocity (pipe) of 3 m/s will generate flow in the
turbulent range
49
49
Post Use Conditioning:
Critical Cleaning Process Parameters
Water Quality
 Pharmaceutical grade
 Water For Injection (WFI)
Water used for cleaning and making up cleaning
solutions should be of equivalent quality to the
water used in the process.
50
50
Post Use Conditioning:
Membrane Recovery
 Ability to show recovery of “new” membrane NWP
after cleaning is an accepted measure of membrane
recovery
 Cassettes can be dissected and membrane
surfaces analyzed for remaining contaminants
(destructive)
51
51
Post Use Conditioning:
Determine Cleaning Effectiveness
 The target for effective cleaning is to recover the
initial water permeability of the membrane.
NWP (after cleaning)
Membrane Recovery = --------------------------------- x 100%
NWP (initial)
 Typical recovery should exceed 80%.
 NWP = Water Permeability at 20°C
52
52
Post Use Conditioning:
Storage of Membrane Cassettes
 Prevent polymeric membrane from drying out
 Prevent microbial growth during storage
 Cassettes should be stored in a suitable agent, in a
closed, sealed container at temperatures between
4 –20°C
53
53
Post Use Conditioning:
Storage of Membrane Cassettes
Agent
Condition
Water
Time Frame
1 day max
Sodium Hydroxide
0.05 – 0.1 N
< 6 months*
Sodium Azide
Glycerol
0.05 – 0.1%
15-20%
< 1 year
* It is recommended that storage agents be flushed out and replaced at least every 6 months.
54
54
Post Use Conditioning:
Cleaning Validation
 Provide documented evidence that the cleaning
procedure assures the equipment is suitably clean
 In order to protect the product’s identify, safety,
purity, and potency.
55
55
Post Use Conditioning:
Validation of Membrane Recovery
 Need to demonstrate the ability to effectively and
consistently clean the membrane of all residual
matter, after each process run, back to its initial “unused” condition.
 Single-use eliminates the need to clean and go
through the process of cleaning validation
56
56
For an Effective Cleaning Protocol
– need to establish
 Cleaning agent(s)
– Mode of Cleaning: hydrolysis, oxidation, solubilization,
emulsification, enzymatic digestion,etc.
 Conditions
– Contact time, temp, volume, conc., etc.
 When does cleaning protocol start?
 Post cleaning procedure
– Flushing to remove all traces of cleaning agents
– Storage
57
57
Post Use Conditioning:
Objective Targets for Cleaning
The target for effective cleaning is to recover the
initial water permeability (NWP) of the
membrane.
The target for consistent cleaning is to reproduce
the effective cleaning over repeated cycles.
58
58
Post Use Conditioning:
Cleaning Study – Case Study
 Materials
– Omega 10KD membrane
– Centrasette Screen Channel Cassette
 Foulant: 1% BSA /PBS buffer
 Process Conditions
– Steady state 2-hour process cycles at 25 psig TMP
– 2-hour cleaning cycle at given conditions
 Procedure
– SOP direct from Pall Cassette Care & Use Manual
59
59
Post Use Conditioning:
Cleaning Study – Case Study
A: None
Cleaning
Agents Legend
B: 0.1N
NaOH
A= None - 1% BSA Recirc.
B= 0.1N
NaOH @ CFF=0.6
B2: 0.1N
NaOH
B2= 0.1N@ CFF=0.6
C= 0.5N
NaOH
C: 0.5N
NaOH
D= 0.1N + 200ppm OCl
D: 0.1N NaOH + 200ppm OCl
Membrane Cleaning Recovery
Centrasette 10KD Cassette
OS010C06 Lot# 38075001
Figure 1A: Measured NWP results before and after cleaning for 4 consecutive recirculation cycles with 1% BSA
12
11
NWP (LMH/psig)
10
B
B
9.48
9.25
D
9.55
9
B
B
B
8.65
8.8
8.75
9.1
B
8
B
6.9
C
B
7.15
6.8
7
B2
6.95
6.55
6
5
4
3
2
4.6
4.65
A
A
4.85
A
3.3
A
1
2
3
Cycle Run
60
4
60
JJR0599C13
Post Use Conditioning:
Cleaning Study – Case Study
Membrane Cleaning Recovery
Centrasette 10KD Cassette
OS010C06 Lot# 38075001
A: None
B: 0.1N NaOH
B2: 0.1N NaOH @ CFF=0.6
C: 0.5N NaOH
D: 0.1N NaOH + 200ppm OCl
Cleaning Agents Legend
A= None - 1% BSA Recirc.
B= 0.1N NaOH
B2= 0.1N@ CFF=0.6
C= 0.5N NaOH
D= 0.1N + 200ppm OCl
Figure 1B: % Recovery before and after cleaning for 4 consecutive recirculation cycles with 1% BSA
98
100
% Membrane Recovery
90
B
101
91
B
B
96
93
D
B
80
73
70
B
75
72
C
B
B
60
50
49
40
A
73
69
B
51
49
A
A
35
30
A
20
10
0
1
2
Cycle Run
61
3
4
61
JJR0699C01
Post Use Conditioning:
Cleaning Study – Case Study
A: None
Cleaning Agents Legend
B: 0.1N
NaOH
A= None
- 1% BSA Recirc.
B= 0.1N NaOH
B2: 0.1N
NaOH @ CFF=0.6
B2= 0.1N@ CFF=0.6
C= 0.5N NaOH
C: 0.5N
NaOH
D= 0.1N + 200ppm OCl
D: 0.1N NaOH + 200ppm OCl
Membrane Cleaning Recovery
Centrasette 10KD Cassette
OS010C06 Lot# 38075001
12
Figure 2A: Measured NWP results before and after cleaning for 3 consecutive recirculation cycles with 1%
D
11
D
D
9.65
10
D
9.75
9.45
9.1
NWP (LMH/psig)
9
8
B
6.8
7
B2
C
6.95
6.55
6
5
4
3.3
3.55
3
A
A
A
2
A
2.35
2
1
4
5
Cycle Runs
62
6
7
JJR0599C13
62
Post Use Conditioning:
Cleaning Study – Case Study
Membrane Cleaning Recovery
Centrasette 10KD Cassette
OS010C06 Lot# 38075001
A: None
Cleaning
Agents
Legend
B: 0.1N
NaOH
A= None - 1% BSA Recirc.
B2:
0.1N
NaOH @ CFF=0.6
B= 0.1N
NaOH
B2= 0.1N@ CFF=0.6
C:C= 0.5N
0.5N
NaOH
NaOH
D= 0.1N + 200ppm OCl
D: 0.1N NaOH + 200ppm OCl
Figure 2B: % Recovery before and after cleaning for 3 consecutive recirculation cycles with 1% BSA
102
100
96
90
% Membrane Recovery
103
100
D
D
D
D
80
70
60
50
40
30
37
35
A
A
25
21
20
A
A
10
0
4
5
Cycle Run
63
6
7
JJR0699C01
63
Post Use Conditioning:
Cleaning Summary
 Cleaning is a critical part of the production process
 It is necessary to demonstrate that a cleaning
protocol is effective and consistent
 A cleaning study should document the effect of
changes in the cleaning process variables:
– Cleaning agent concentration
– Time
– Temperature
– Fluid Velocity
64
64
Thank You
Tavis Tan