Scientific & Technical Report
PN33339
The Partnership of the Minimate™ TFF
Capsule with Liquid Chromatography Systems
Facilitates Lab-scale Purifications and Process
Development Through In-Line Monitoring
Introduction
Tangential Flow Filtration
Tangential flow filtration (TFF) is an effective
method for both diafiltration and concentration
operations in purification strategies for biomolecules.1,2 The TFF process recirculates
sample flow over the surface of a membrane,
controlling gel layer formation and effectively
reducing fouling. The operator develops an
effective and reproducible process by
optimizing flow rates and controlling transmembrane pressure. This results in improved
flux rates and productivity.3 A TFF process
developed on lab-scale devices can be
readily scaled to larger process volumes.
Traditionally, TFF separations have used
peristaltic pumps and flexible tubing to
provide the needed pressure and flow.
Although standard lab pump systems are
sufficient for most TFF applications, the use
of high-performance liquid chromatography
(LC) systems can offer some additional
advantages with respect to process
development and control. This paper
describes the novel use of an LC system
as the control platform for performing TFF
using a Minimate TFF capsule.
Liquid Chromatography and TFF
Protein purification is rarely a simple process
and almost never a single step. Instead, it
requires an ordered sequence of purification
steps assembled into a process compatible
with the biochemistry of the target protein
to achieve the required purity. Purification
protocols include a variety of separation
techniques that may include general filtration/
clarification, size exclusion chromatography,
affinity chromatography, ion exchange
chromatography, ultrafiltration, and possibly
a sterilization step. TFF is frequently used in
a process before chromatographic steps to
desalt and buffer exchange (diafiltration) the
sample into a suitable state for binding or to
adjust the sample concentration. It may also
be applied as a bridge between chromatographic steps when the elution buffer at the
end of one process is incompatible with the
binding conditions of a subsequent step.
At the end of a process, it may be used to
adjust the final concentration and ionic
strength of the product as required.
Few protein chemists are without access
to an LC pump system that has pumping
capacities approaching 100 mL/min. These
systems can serve as robust alternatives to
the use of a peristaltic pump for TFF protocols
with significant additional benefits. Here we
report the use of an ÄKTA Explorer◆ liquid
chromatography system (GE Healthcare)
to operate the Minimate TFF capsule.
Specific advantages of using an LC pump
system to drive TFF are:
• Use of chromatography equipment already
in place saves equipment cost, benchspace, and training time.
• Precise control of pressure and flow
rate assures uniform development of
the gel layer to improve reproducibility
between runs.
• Ability to monitor and record process parameters in
real time (pH, conductivity, absorbance, temperature, feed flow rate, valve positions, system pressure)
gives a researcher an auditable trail for process
validation.
• Reduction in system working volume with narrow
bore PEEK◆ and Tefzel◆ tubing allows greater
concentration factors.
• Operation of system valves to multiplex purification
procedures using the same pumping system
(sequential operations on multiple cartridges and
columns). TFF devices can be sanitized in place
while other column operations are underway.
steps. An optimized fluid path minimizes hold-up and
working volumes and allows easy scale up to larger
TFF systems like Centramate™ and Centrasette™
systems. The Minimate TFF capsule and larger devices
utilize consistent materials of construction to assist in
process validation. Finally, each pharmaceutical grade
Minimate TFF capsule is 100% integrity tested during
manufacture to ensure reliable performance.
Technical Data
Effective Filtration Area
Recommended Crossflow Rate
Operating Temperature Range
Maximum Operating Pressure
pH Limits
Product Hold-up Volume (feed/retentate)
50 cm2 (0.05 ft2)
30-80 mL/min
(0.6-1.6 L/min/ft2)
5-50 °C (41-122 °F)
4 bar (400 kPa, 60 psi)
at 20 ºC (68 ºF)
1-14
~1.6 mL
Ultrareservoir™ Container, 500 mL
The Ultrareservoir container is designed to hold up to
500 mL of sample and give efficient mixing of product.
The sloped bottom and optimally located feed and
return ports reduce the system hold-up volume, which
allows high concentration factors to be achieved. The
reservoir lid seals tightly, allowing additional sample
volume or diafiltration solution to be drawn into the
reservoir by vacuum that is created as filtrate is generated through the TFF device. This allows continuous
diafiltration to be performed without the need for a
transfer pump.
Minimate TFF Capsule with LC Pump System
Materials and Methods
Minimate TFF Capsule
Minimate TFF capsules contain Omega™ polyethersulfone membrane with an effective filtration area of
50 cm2. The Omega ultrafiltration (UF) membrane,
available in a wide range of molecular weight cut-offs,
offers low adsorption characteristics resulting in high
product recoveries. Sample batch sizes of up to 1 liter
can be desalted and subsequently concentrated to
volumes as low as 5 mL with little user intervention.
The reusable/disposable capsule can perform single
or sequential concentration/diafiltration steps using the
same device in a closed loop connection, saving time
and avoiding product loss associated with transfer
2
Equipment and Reagents
1. ÄKTA Explorer Chromatography Workstation with
Unicorn◆ 4.0 Software (GE Healthcare)
2. Digital Pressure Gauges (Cole Parmer, Model 1200)
3. 1XPBS (phosphate buffered saline) Solution
(Invitrogen, Carlsbad, CA)
4. 1M NaCl in PBS
5. Bovine Serum Albumin (BSA) solution: 1 mg/mL
dissolved in PBS with 1M NaCl
6. 0.5M sodium hydroxide in water
7. 0.1M sodium hydroxide in water
Step-by-Step Procedures
Table 1
Parts List for Connecting the Minimate TFF Capsule to
ÄKTA Explorer FPLC System
Description
Qty
Tefzel tubing, 1/8" (use the minimal amount of tubing)
PEEK tubing (green), 0.030 x 1/16" (use the minimal
amount of tubing)
Tefzel tubing, 1/16" (use the minimal amount of tubing)
Adapter assembly, 1/8" NPT to 5/16, 24 flat
bottom female
Tubing connector, 1/8" male
Ferrule for 1/8" tubing (use the minimal amount
of tubing)
Union, 5/16" female 1/16" male
Fingertight connector, 1/16"
Pressure gauge TEE
Digital pressure gauge, 0-300 psi range
10-32 female to male luer assembly
Micro-metering valve, 1/16"
10-32 female to female luer assembly
3 ft
3 ft
3 ft
3 each
3 each
3 each
1 each
12 each
3 each
3 each
2 each
2 each
2 each
Set-up
1. Connect an outlet valve line from the ÄKTA
(e.g., F8) to the “From Device” outlet of the
Ultrareservoir container.
2. Connect a buffer valve line from the ÄKTA
(e.g., A18) to the “To Pump” outlet of the
Ultrareservoir container.
3. Choose a column valve position from ÄKTA (e.g.,
position 4) for the Minimate capsule. Connect
bottom column valve to a digital pressure gauge
(P1) and then connect P1 to one of the “Feed/
Retentate” ports of the Minimate TFF capsule.
Connect top column valve to a digital pressure
gauge (P2) and then connect P2 to the other
“Feed/Retentate” port of the capsule. Orient the
capsule in a vertical position with the end connected to the top valve facing up. Connect the
upper “Permeate/Drain” port of the capsule to a
digital gauge (P3). Connect a length of tubing to
the outlet from the pressure gauge and put the
other end in a waste container. Connect the
other “Permeate/Drain” port to a three-way valve
connector. Use the valve to close off the port.
4. Put separate buffer valve lines from the ÄKTA into
the following solutions:
A13: PBS
A14: 0.5M sodium hydroxide
A12: 0.1M sodium hydroxide
A11: BSA solution
Membrane Equilibration and Line Flushing
1. Using manual instruction, set the flow path to
the following:
Column Position: 4
Outlet Valve: Waste F1
Buffer Valve: A13
Flow Direction: Up Flow
2. Start the pump by setting a flow rate of 20 mL/min.
Increase flow rate until the feed pressure reaches
2.7 bar (40 psi).
3. Inspect the line connection to make sure there
is no leak.
4. Run for at least 5 minutes or until the pH, conductivity, and absorbance signals flatten out.
5. Stop pump.
Diagram 1
Partnership of TFF with Column Chromatography
A Minimate TFF capsule is installed
between the top and bottom
column valves. Pump flow is
directed into the TFF feed port, and
the retentate flow goes through the
analysis sensors and back to the
Ultrareservoir container. Pressure
sensors or gauges upstream and
downstream of the capsule monitor
pressure, and a third gauge is
placed along the filtrate path to
monitor backpressure. In diafiltration
mode, the Ultrareservoir container is
connected to the diafiltration buffer
vessel. In concentration mode, the
Ultrareservoir container is left open
to the atmosphere.
www.pall.com/lab
3
Buffer Exchange
A buffer exchange protocol was developed using 1M
NaCl in PBS as the starting sample. This solution was
diafiltered with PBS to remove salt and then back to
high salt with a solution of 1M NaCl in PBS. Once the
process conditions were developed, the process was
repeated using a starting solution of 1 mg/mL BSA,
1M NaCl in PBS.
1. Equilibrate the Minimate TFF capsule according to
the procedure described in the preceding section.
2. AutoZero the UV absorbance monitor of the ÄKTA
and set the column position to “Bypass.”
3. Set the buffer valve line to A11 and pump flow rate
to 50 mL/min. Fill the Ultrareservoir container with
200 mL BSA solution (1 mg/mL dissolved in PBS
with 1M NaCl).
4. Put the diafiltration buffer feed line (connected
to the top part of the Ultrareservoir) into a flask
containing PBS.
5. Using manual instruction, set the flow path to
the following:
Column Position: 4
Outlet Valve: F8
Buffer Valve: A18
Flow Direction: Up Flow
6. Start the pump at 20 mL/min. Adjust the flow rate
so that the feed pressure (P1) is just under 2.7 bar
(40 psi).
Note: Flow will start from the permeate tube. Diafiltration
solution will start to be drawn into the Ultrareservoir container in a few minutes. If this does not occur, check
that the reservoir lid is completely inserted and sealed.
7. Stop the pump once the conductivity signal reaches
a plateau.
8. Put the diafiltration buffer feed line into a flask
containing 1M NaCl in PBS solution.
9. Repeat steps 5-7.
Sample Concentration
After the buffer exchange is complete, a sample can
be concentrated by opening the diafiltration buffer
feed line to air. This will break the vacuum generated
under the diafiltration mode.
4
1. Leave the diafiltration buffer feed line open to air.
2. Using manual instruction, set the flow path to the
following:
Column Position: 4
Outlet Valve: F8
Buffer Valve: A18
Flow Direction: Up Flow
3. Start the pump at 20 mL/min. Adjust the flow rate
so that the feed pressure (P1) is just under 2.7 bar
(40 psi).
4. Collect the permeate in a graduated cylinder. (This
will allow the permeate volume to be measured and
concentration factor to be determined.)
5. Stop the pump once the desired concentration
is reached.
Washing, Regenerating, and Storing
the Minimate Capsule
1. Using manual instruction, set the flow path to
the following:
Column Position: 4
Outlet Valve: Waste F1
Buffer Valve: A13 (PBS)
Flow Direction: Up Flow
2. Start the pump by setting a flow rate of 20 mL/min.
Increase flow rate until the feed pressure reaches
2.7 bar (40 psi).
3. Wash the system until the UV, pH, and conductivity
signals stabilize.
4. Switch the buffer valve line to A14 (0.5M sodium
hydroxide).
5. Wash the system until the UV, pH, and conductivity
signals stabilize.
6. Stop the pump. Let the Minimate capsule soak
in 0.5M sodium hydroxide for 1 hour.
7. Switch the buffer valve line to A12 (0.1M sodium
hydroxide).
8. Wash the system until the UV, pH, and conductivity
signals flatten out.
9. Stop the pump. The Minimate capsule can be
stored in 0.1M sodium hydroxide at 4-20 °C.
B
60
250
50
40
150
30
20
50
0
A280
20
40
60
80
0
C
Increasing Salt
100
450
90
400
Conductivity
350
70
60
250
50
200
40
150
30
100
20
50
10
A280
0
0
10
20
30
40
50
200
40
100
30
20
A280
50
60
70
80
Time (min)
D
70
80
0
-10
Increasing Salt
100
90
80
350
70
60
250
50
40
150
30
20
50
10
0
-100
60
Conductivity
Absorbance (mAU)
60
40
50
450
Conductivity (mS/cm)
70
Conductivity
30
80
300
80
20
10
Time (min)
90
10
120 140
-50
D
100
0
100
Conductivity (mS/cm)
Decreasing Salt
300
Conductivity (mS/cm)
70
Conductivity
Time (min)
400
Absorbance (mAU)
350
-50
500
0
90
80
Figure 1
Development of Buffer Exchange Parameters
with In-line Monitoring
A
100
Conductivity (mS/cm)
A sequential buffer exchange back to the high salt
buffer was possible without
disconnecting the device
D
from the system by replacing the low salt (PBS)
diafiltration buffer with a high salt buffer (PBS,
1M NaCl). Again, successful buffer exchange was
detected in-line with the exchange of four buffer
volumes (Figures 1C, 1D). Absorbance at 280 nm
was monitored to act as a sample process control
to determine if the absorbance readings are deflected
by the changing salt concentrations. The ability to
monitor and document the process “live” allows the
researcher to save time and buffer, as well as provides
a basis for future scale up decisions.
Decreasing Salt
450
Absorbance (mAU)
Continuous Diafiltration for Process Development
To develop a process protocol, a mock bufferexchange protocol was created where a buffer-only
sample (1N NaCL in PBS) was first diafiltered with
PBS to reduce the salt concentration (Figures 1A,
1B). In-line monitoring allowed the documentation
of salt concentration changes in real-time using the
conductivity sensor and data acquisition program
built into the ÄKTA Explorer. Consistent with previous
observations,1 the passage of four volumes of
diafiltration buffer was adequate to achieve > 98%
buffer exchange.
Absorbance (mAU)
Results and Discussion
0
10
20
30
A280
40
50
-50
60
10
0
Time (min)
Diafiltration (buffer exchange) processing was documented from
high salt to low salt then back to high salt. Repeated runs using a
10K Minimate capsule were processed at 20 (graphs A and C) and
25 (graphs B and D) mL/min recirculation rates. Salt and protein
(A280) concentrations were monitored using in-line sensors.
www.pall.com/lab
5
C
60
Conductivity
40
30
0
20
-20
20
0
100
120
90
80
A280
70
80
60
60
50
C
Conductivity
40
40
30
20
20
A600
0
0
10
20
30
40
50
60
-20
Time (min)
6
70
80
90 100 10
0
Conductivity (mS/cm)
Absorbance (mAU)
D
Decreasing
140
100
40
30
50
0
10
20
20
A600
40 50
30
Conductivity (mS/cm)
50
A280
100
60
70
-50
80
90 100 10
0
Time (min)
I
D
Increasing Salt
C
100
90
300
80
70
I
250
60
200
50
150
C
A280
40
100
30
50
20
A600
10
60 80 100 120 140 160 180
0
Time (min)
0
-50
0
20
40
Conducitivity (mS/cm)
Conductivity
Diafiltration (buffer exchange) processing was documented from
high salt to low salt then back to high salt with a protein solution
(1 mg/mL BSA). Repeated runs using a 10K Minimate capsule
were processed at 25 (graphs A and C) and 20 (graphs B and D)
mL/min recirculation rates. Salt, protein (A280), and turbidity (A600)
were monitored using in-line sensors.
Time (min)
B
60
150
0
Absorbance (mAU)
80
Conductivity (mS/cm)
Absorbance (mAU)
100
A600
40 60 80 100 120 140 160 180 200
70
350
180
80
80
Conductivity
200
400
120
A280
90
450
Decreasing
130
100
250
Figure 2
Validation of Diafiltration Parameters in the
Presence of Protein
A
Increasing Salt
300
Absorbance (mAU)
Buffer Exchange at Constant
Protein Concentration
Once a simple buffer-exchange protocol was developed, a second protocol was used to demonstrate
diafiltration of a protein solution (1 mg/mL BSA) going
first from high salt to low salt buffer (Figures 2A, 2B)
and then back to a high salt buffer (Figures 2C, 2D). In
addition to monitoring protein and salt concentrations,
in-line sensors documented the A600 values to ensure
that the protein solution had not precipitated during
processing. A precipitated protein solution would
produce turbidity and cause a reading at A600.
The use of in-line monitors allows the researcher
to immediately trace the incident back to the salt
concentration where protein precipitation first occurred,
thus saving valuable time in troubleshooting.
Precise Control of Sample Concentration
In concentration mode, the Ultrareservoir container
is left open to atmosphere. The concentration of the
retained solute increases as permeate is removed,
decreasing the process volume. The in-line monitors
allow the researcher to track the progress of the
concentration of the protein sample. While the salt
concentration (which is 100% permeable to the
membrane) remains constant, the protein solution
concentration increases. The use of sensors allows
the researcher to stop the process once the desired
concentration is achieved. Documenting turbidity
25
200
80
70
700
60
Conductivity
50
500
A280
300
-100
40
30
80
20
A600
10
100 120 140 160 180 200 220 240 260
0
Time (min)
20
Conductivity
150
15
A280
100
10
50
A600
5
10
15
20
25
Conductivity (mS/cm)
Absorbance (mAU)
90
900
100
250
0
100
1100
5
30
35
40
0
-50
Time (min)
The concentration of a 1 mg/mL BSA solution was accomplished
by leaving the diafiltration feed line open to air. The run using a
Minimate 10K capsule was processed at 25 mL/min recirculation
rate. Salt, protein (A280), and turbidity (A600) were monitored using
in-line sensors.
Sequential Buffer Exchange and Concentration
An advantage of processing using in-line sensors was
demonstrated by performing sequential processing
operations. Diafiltration of the protein sample
(1 mg/mL BSA) reduced the salt concentration by
> 98% with about four volumes of buffer. As soon as
the conductivity leveled off, indicating that desalting
was complete, sample concentration was started
by simply disconnecting the buffer line to the Ultrareservoir container. Concentration of retained solute
increased as permeate was removed, decreasing the
process volume. This process is facilitated using the
features of an LC system, such as the multiport valves
and in-line sensors. The ability to monitor “live” allows
greater control over the process during the run and
provides documentation that can be used to develop
and validate the scale-up protocol.
Sequential diafiltration followed by concentration was documented
for a single run using a 1 mg/mL BSA solution in 1X PBS, 1M NaCl
starting solution. The run using a Minimate 10K capsule was
processed at 25 mL/min recirculation rate with the buffer exchange
from high salt to low salt. The diafiltration buffer feed line was then
opened to air, allowing the concentration of the sample. Salt, protein
(A280), and turbidity (A600) were monitored using in-line sensors.
Scalability
TFF process development can be performed on a
Minimate TFF capsule with a simple lab-scale system
incorporating a peristaltic pump or with an LC pump
and in-line monitoring system as described above.
Both allow development of procedures that are
ultimately scalable. The advantage of the LC system
is greater control and documentation. The automation
and documentation aspects simplify development of
validated protocols and provide an audit trail for labs
under Good Lab Practices (GLP). This is extremely
useful in the scale-up process.
While the Minimate TFF capsule is a valuable tool for
small process volumes (typically under 1 liter), larger
volumes can be processed by plumbing several
Minimate TFF capsules in parallel. The Minimate TFF
capsule was designed with the same flow path length
as larger TFF devices (Centramate and Centrasette
systems) allowing predictable performance and saving
valuable optimization time when scaling beyond lab
scale to volumes used in pilot and production plants.
www.pall.com/lab
7
Conductivity (mS/cm)
Figure 3
Precise Control of Protein Concentration
Figure 4
Sequential Operations for Diafiltration and Concentration
Absorbance (mAU)
(A600) provides a good diagnostic to determine
experimentally the sample concentration limit prior to
protein precipitation. The 1 mg/mL BSA solution could
be concentrated over four-fold within 40 minutes without the formation of turbidity (Figure 3). While these
conditions were chosen for this demonstration, in
reality, much higher concentration factors for BSA can
be achieved. Other protein solutions may precipitate
more easily depending on solubility.
References
Literature Resources
1. L. Schwartz, 2003. Diafiltration: A Fast, Efficient Method
for Desalting or Buffer Exchange of Biological Samples.
Pall Scientific & Technical Report.
• Product Data, Minimate Tangential Flow Filtration System
and Minimate TFF Capsule, PN 33366
2. L. Schwartz, 2003. Desalting and Buffer Exchange by
Dialysis, Gel Filtration or Diafiltration. Pall Scientific &
Technical Report.
3. L. Schwartz and K. Seeley, 2002. Introduction
to Tangential Flow Filtration for Laboratory and
Process Development Applications. Pall Scientific
& Technical Report, PN 33213.
• Technical Article, Desalting and Buffer Exchange by
Dialysis, Gel Filtration, or Diafiltration, available online
at www.pall.com/lab
• Technical Article, Diafiltration: A Fast, Efficient Method
for Desalting or Buffer Exchange of Biological Samples,
available online at www.pall.com/lab
• Technical Article, Increased Productivity Using Minimate
Capsules to Replace Stirred Cell Systems, PN 33342
• Technical Article, Introduction to Tangential Flow Filtration
for Laboratory and Process Development Applications,
PN 33213
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PN33339