SOP21_ Millipore Pellicon XL Tangential Flow

Bucks County Community College
275 Swamp Road
Newtown, PA 18940
Document Number: 23
Revision Number: 0.0
Effective Date: 09 November 2015
Page 1 of 11
Title: Operation of Millipore™ Labscale -Pellicon XL Tangential Flow
Filtration System
Approvals
Prepared By: ________________________
Biotech Technician
Date
______________________
26 October 2015
Reviewed By: _________________________
Biotech Supervisor
Date
_______________________
02 November 2015
1. Purpose:
1.1. To describe the operation of the Millipore™ Labscale –Pellicon XLTangential Flow
Filtration System to concentrate or diafilter cell culture harvest during downstream
processing. Diafiltration is used for the removal of salt, for buffer exchange and to
concentrate cell culture harvest from bioreactors.
2. Scope:
2.1. Applies to concentration and diafiltration of cell culture harvest from the downstream processing
unit of the Biomanufacturing course at Bucks County Community College
3. Responsibilities:
3.1. It is the responsibility of the course instructor/lab assistant to ensure that this SOP is performed
as described and to update the procedure when necessary.
3.2. It is the responsibility of the students/technicians to follow the SOP as described and to inform
the instructor about any deviations or problems that may occur while performing the procedure.
4. References:
4.1. Millipore™ Labscale Tangential Flow Filtration System User Guide
4.2. “Diafiltration for Desalting or Buffer Exchange”, L. Schwartz, Bioprocess International
May 2003; 43-49
5. Definitions:
5.1. Diafiltration- ultrafiltration technique that uses permeable membranes to remove,
replace, or lower the concentration of salts or solvents from solutions containing
proteins, peptides, nucleic acids, and other biomolecules
5.2. Retentate/concentrate-solution retained by a membrane
5.3. Permeate-solution that passes through the membrane
6. Precautions:
6.1. Wear appropriate personal protection equipment ( lab coat, gloves, eye protection) when
operating the system
6.2. Use caution when opening and closing valves. Failure to open or close valves at the appropriate
times could result in trapped high pressure, sample loss, damaged cassettes, chemical spills, and
pump damage.
Bucks County Community College
275 Swamp Road
Newtown, PA 18940
Document Number: 23
Revision Number: 0.0
Effective Date: 09 November 2015
Page 2 of 11
Title: Operation of Millipore™ Labscale -Pellicon XL Tangential Flow
Filtration System
6.3. Prior to each use, carefully inspect all hoses and tubing for kinks or restrictions that could result
in pressure buildup and possible rupture of hoses or tubing.
6.4. Prior to each use, carefully check that tubing at pump outlet to feed in port (FEED IN) and
cleaning tubing are Sta-Pure (white) tubing. Failure in correctly installing Sta-Pure (white)
tubing can result in over pressurized tubing and burst.
6.5. Transfer pump tubing can rupture due to continuous contact with moving parts. Follow tubing
manufacturer’s recommended replacement interval.
6.6. Never move the system while there is liquid in the tank, while system processes are running or
the cassettes is in place. Labscale Tangential Flow Filtration System User Guide 7
6.7. Never operate the system without the tank cover in place. Never operate the system if any
moving parts are exposed. Never operate the system with the pump housing open.
7. Materials:
7.1. Millipore Labscale Tangential Flow Filtration System
7.2. Millipore XL 50 Pellicon Tangential Flow Filter (TFF)
7.3. Waste Collection vessels
7.4. 0.1N NaOH
7.5. Deionized Water
8. Procedure:
8.1. Preparation and Setup
8.1.1. Ensure that the Labscale Tangential Flow System ( including 500 mL reservoir,
stir base, and pump module ) is installed according the instructions in the user
manual ( pages 14-30) .
8.2. Install Pellicon XL Device on the System
8.2.1. Remove plug from the FEED, RET, PERM1, and PERM 2 ports
8.2.2. Align Pellicon XL device ports with the labscale 500 mL reservoir ports. Make
sure that the PERM and RET device ports of device and reservoir match. Press
firmly onto reservoir ports and turn the lock nuts until snug. NOTE: Be sure pump
tubing is installed before proceeding and Sta-Pure (white) tubing is installed
between pump outlet and feed in port (FEED IN). See Figure 29 for diagram of
system set-up with Pellicon XL device installed
Bucks County Community College
275 Swamp Road
Newtown, PA 18940
Document Number: 23
Revision Number: 0.0
Effective Date: 09 November 2015
Page 3 of 11
Title: Operation of Millipore™ Labscale -Pellicon XL Tangential Flow
Filtration System
8.3. Flushing
8.3.1. Disconnect retentate silicone (translucent) tubing from RET IN port and place end
of retentate tubing in waste collection vessel.
8.3.2. Place end of permeate silicone (translucent) tubing into waste collection vessel.
Open retentate valve—turn counterclockwise .
8.3.3. Remove reservoir cover and fill reservoir with 500 mL of deionized water.
Remove plug from VENT port and open tank outlet valve.
8.3.4. Turn pump on - increase pump speed until feed pressure gauge reads 1.38 Bar (20
psi).Note: Check system connections for leaks—tighten connections if required
8.3.5. Continue pumping to the waste collection vessel until level in the reservoir drops
to 350 mL. Turn pump off.
Bucks County Community College
275 Swamp Road
Newtown, PA 18940
Document Number: 23
Revision Number: 0.0
Effective Date: 09 November 2015
Page 4 of 11
Title: Operation of Millipore™ Labscale -Pellicon XL Tangential Flow
Filtration System
8.3.6. Reconnect the retentate silicone (translucent) tubing to the RET IN port and turn
pump on - increase pump speed until feed pressure gauge reads 1.38 Bar (20
psi).Note: Check system connections for leaks—tighten connections if required
8.3.7. Adjust retentate valve restriction by slowly turning retentate valve clockwise until
the retentate pressure gage reads 0.69 Bar (10 psi).
8.3.8. Adjust pump speed and retentate valve restriction to achieve 2.07 Bar (30 psi)
feed pressure and 0.69 Bar (10 psi) retentate pressure.
8.3.9. Continue flushing until the level in the reservoir drops to 50 mL. Turn pump off.
8.3.10. Drain System
8.3.10.1. Disconnect pump outlet Sta-pure (white) tubing from pump outlet port and
place in waste collection vessel.
Bucks County Community College
275 Swamp Road
Newtown, PA 18940
Document Number: 23
Revision Number: 0.0
Effective Date: 09 November 2015
Page 5 of 11
Title: Operation of Millipore™ Labscale -Pellicon XL Tangential Flow
Filtration System
8.3.10.2. Disconnect retentate silicone (translucent) tubing from RET IN port. Fluid
should now drain by gravity. If additional drainage is required, a syringe can
be placed on the end of the retentate tube and fluid can be blown down.
8.3.10.3. Replace retentate silicone (translucent) tubing in retentate port. Reconnect
pump outlet Sta-Pure (white) tubing.
8.3.10.4. Disconnect pump outlet Sta-Pure (white) tubing from FEED IN port and
place in collection vessel. Open tank outlet valve, turn pump speed up to drain
reservoir.
8.3.10.5. Reconnect the pump outlet Sta-Pure (white) tubing to the FEED IN port.
8.4. Integrity Testing with Pelllicon XL Device on System
8.4.1. Integrity Test Set Up
8.4.1.1.Close tank outlet valve and disconnect pump inlet tubing (silicone,
translucent) from the tank outlet valve. Close the retentate valve.
8.4.2. Integrity Test Procedure
8.4.2.1.Obtain air integrity test pressure and airflow specifications from the
Certificate of Quality supplied with the device.
8.4.2.2.2. Insert syringe in the female luer end of the pump inlet tubing. (silicone,
translucent). Inject air into the tubing until the feed pressure gauge reading
reaches the recommended air integrity test pressure.
8.4.2.3.Add 0.1 mL of water to a 1 mL pipette. Insert bottom of pipette into the end of
the permeate tubing (silicone, translucent).
8.4.2.4.4. Measure airflow by measuring the displacement of water in the pipette for
30 seconds. Multiply the displaced volume by 2 to calculate the airflow
(mL/min) and record.
8.4.2.5.5. Remove the pipette from the permeate tubing (silicone, translucent). Open
the retentate valve slowly to release the air pressure in the system.
8.4.2.6.6. Reconnect the pump inlet tubing (silicone, translucent) to the tank outlet
valve.
8.4.2.7.The Pellicon XL device is integral if the measured airflow is lessthan or equal
to the airflow specification on the Certificate of Quality.
Bucks County Community College
275 Swamp Road
Newtown, PA 18940
Document Number: 23
Revision Number: 0.0
Effective Date: 09 November 2015
Page 6 of 11
Title: Operation of Millipore™ Labscale -Pellicon XL Tangential Flow
Filtration System
8.5. Preconditioning
8.5.1. Place end of permeate tubing silicone (translucent) in waste collection vessel.
8.5.2. Remove reservoir cover and fill reservoir with 50 mL of an appropriate buffer.
Remove plug from VENT port.
8.5.3. Open tank outlet valve. Turn pump on - increase pump speed until feed pressure
gauge reads 1.38 Bar (20 psi).Check system connections for leaks—tighten
connections if required
8.5.4. Continue pumping to the waste collection vessel until the level in the reservoir
drops to the bottom of the reservoir stir bar well.(Stop pump before air is pumped
into the system.). Turn pump off.
8.5.5. Drain Permeate Tubing
8.5.5.1.Disconnect the permeate tubing (silicone, translucent) from the permeate
outlet (PERM 2) port. Drain permeate tubing into the waste collection vessel.
8.5.5.2.Reconnect the permeate tubing to the PERM 2 port.
Bucks County Community College
275 Swamp Road
Newtown, PA 18940
Document Number: 23
Revision Number: 0.0
Effective Date: 09 November 2015
Page 7 of 11
Title: Operation of Millipore™ Labscale -Pellicon XL Tangential Flow
Filtration System
8.6. Concentration of Sample
8.6.1. Remove reservoir cover and fill reservoir with sample to be concentrated.
8.6.2. Ensure vent port is open by removing plug from VENT port and leaving open or
install a Millex Filter. Open tank outlet valve.
8.6.3. Turn pump on—increase pump speed until feed pressure gauge reads 1.38 Bar (20
psi).Note: Check system connections for leaks—tighten connections if required
8.6.4. 4Adjust retentate valve restriction by slowly turning retentate valve clockwise
until the retentate pressure gauge reads 0.69 Bar (10 psi).
8.6.5. Adjust pump speed and retentate valve restriction to achieve desired feed retentate
pressures (recommend 2.07 Bar (30 psi) feed / 0.69 Bar (10 psi) retentate).Do not
exceed 4.14 Bar (60 psi) feed pressure.
Bucks County Community College
275 Swamp Road
Newtown, PA 18940
Document Number: 23
Revision Number: 0.0
Effective Date: 09 November 2015
Page 8 of 11
Title: Operation of Millipore™ Labscale -Pellicon XL Tangential Flow
Filtration System
8.6.6. Continue processing until the desired concentration or volume is reached. Turn
pump off.
8.7. Product Recovery
8.7.1. Disconnect pump outlet tubing (Sta-Pure, white) from pump outlet port and place
in product recovery collection vessel.
8.7.2. Disconnect retentate tubing (silicone, translucent) from retentate in port. Fluid
should now drain by gravity. If additional drainage is required, a syringe can be
placed on the end of the retentate tube and fluid can be blown down
8.7.3. Replace retentate tubing (silicone, translucent) in retentate port. Reconnect pump
outlet tubing (Sta-Pure, white).
8.7.4. 4. Disconnect FEED IN tubing and place in collection vessel. Open tank outlet
valve, turn pump speed up to drain reservoir.
Bucks County Community College
275 Swamp Road
Newtown, PA 18940
Document Number: 23
Revision Number: 0.0
Effective Date: 09 November 2015
Page 9 of 11
Title: Operation of Millipore™ Labscale -Pellicon XL Tangential Flow
Filtration System
8.7.5. 5. Reconnect the pump outlet tubing (Sta-Pure, white) to the FEED IN port.
8.8. Diafiltration-Vacuum
8.8.1. Cut silicone (translucent) tubing and insert a male luer x 1/8. barb port in one end
of the tubing as shown below. Place the other end of the diafiltration tubing in a
vessel of appropriate buffer.
Bucks County Community College
275 Swamp Road
Newtown, PA 18940
Document Number: 23
Revision Number: 0.0
Effective Date: 09 November 2015
Page 10 of 11
Title: Operation of Millipore™ Labscale -Pellicon XL Tangential Flow
Filtration System
8.8.2. Connect male luer end of diafiltration tubing to the diafiltration (DIA/RECIRC)
port. Plug VENT port.
8.8.3. Insure that the end of the tubing remains below the buffer level at all times. As
you remove permeate, a vacuum is created in the reservoir which will siphon
diafiltrate into the reservoir to maintain constant volume.
8.9. Cleaning
8.9.1. Disconnect retentate tubing (silicone, translucent) from RET IN port and place in
waste collection vessel. Place end of permeate tubing in waste collection vessel.
8.9.2. Open retentate valve turn counter clockwise .
8.9.3. Remove reservoir cover and fill reservoir with 500 mL of cleaning solution.
Ensure vent port is open by removing plug from VENT port and either leaving open
or installing a Millex Filter.
8.9.4. Open tank outlet valve.
8.9.5. Turn pump on - increase pump speed until feed pressure gauge reads 1.38 Bar (20
psi).Note: Check system connections for leaks—tighten connections if required
8.9.6. Continue pumping to the waste collection vessel until the level in the reservoir
drops to 250 mL. Turn pump off. Reconnect the retentate (silicone, translucent)
tubing to the RET IN port.
Bucks County Community College
275 Swamp Road
Newtown, PA 18940
Document Number: 23
Revision Number: 0.0
Effective Date: 09 November 2015
Page 11 of 11
Title: Operation of Millipore™ Labscale -Pellicon XL Tangential Flow
Filtration System
8.9.7. Connect the male luer end of the permeate tubing to the recirculation
(DIA/RECIRC) port. Turn pump on and increase pump speed until feed pressure
gauge reads 1.38 Bar (20 psi). Check system connections for leaks—tighten
connections if required
8.9.8. Adjust retentate valve restriction by slowly turning retentate valve clockwise until
the retentate pressure gage reads 0.69 Bar (10 psi). Adjust pump speed and retentate
valve restriction to achieve 2.07 Bar (30 psi) feed pressure and 0.69 Bar (10 psi)
retentate pressure.
8.9.9. Recirculate the cleaning solution for 30 - 60 minutes. Turn pump off.
8.9.10. Drain system as in Step 8.5.5
8.9.11. Flush device as in Step 8.3
8.9.12. See instructions enclosed with the Pellicon XL device for storage information
8.9.13. Clean the base
8.9.13.1. Disconnect power and wipe the exterior surface of the reservoir and the
base with mild soap and water.
9.
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