110
CORN I NG ASCENT FBR PD SYSTEM
8.5.2 Lysis
All fluid handing can be done outside the biosafety cabinet since sterility is not required. Prepare the lysis buffer in a 1L Erlenmeyer flask.
An example of a lysis buffer is as follows:
• 10 mM Tris-HCl pH 8.0, 2 mM MgCl
2
, 150 mM NaCl, 1% Tween® 20
• 25 U/mL of Benzonase
Add lysis buffer volume to the MCV using the suggested volumes listed below for the following setup:
• Lynx® connector to the bioreactor: 1 m
2
(650 mL); 2.5 m
2
(800 mL); 5 m
2
(950 mL)
• AseptiQuik® G connector to the bioreactor: 1 m
2
(500 mL); 2.5 m
2
(600 mL); 5 m
2
(700 mL)
1. Ensure proper connections.
• There are several options to add lysis buffer to the MCV.
• Option 1: Connect to the luer fitting dip-tube to the luer port on the MCV head plate. Apply positive pressure to the dip-tube to allow the
solution to flow into MCV.
• Option 2: Alternatively user can use AseptiQuik® connectors or tube weld on to the feed addition line of the consumable to connect lysis
buffer container.
• Option 3: Alternatively user can use an unused media addition line and connect via an AseptiQuik® connector.
TIP:
Ensure that the flexible tubing is in the pump head, not the silicone portion. An adaptor may need to be attached earlier in the run to
have appropriate connections.
2. Add lysis buffer to the MCV then perfuse through the bioreactor.
• For Option 2: On the HMI, go to Bolus Addition, press
to add lysis buffer.
• For Option 3: On the HMI, go to Media Addition, and set parameters to add lysis buffer to the MCV.
• In Operator mode, set the recirculation pump at 50 mL/min. counterclockwise (forward flow). Slowly fill the bioreactor for 15 min.
3.
Adjust the mass flow controller (MFC) setpoints. Place each MFC in Operator mode.
• Air 10 SCCM
• O
2
0 SCCM
• CO
2
0 SCCM
Set temperature to 37°C if desired. Set pH to 8.0.
NOTE:
The recirculation pump must be running to change the temperature, otherwise the interlock prevents any changes.
4.
Set the recirculation pump at 500 mL/min. counterclockwise (forward flow).
• Set pH to 8.0 to allow for the addition of sodium bicarbonate to maintain pH.
• Perfuse the bioreactor for 4 hours or until lysis is complete.
5. Drain lysis buffer from the bioreactor to the MCV.
• Open the air valve on media return line.
• Set the recirculation pump clockwise at 500 mL/min. (reverse flow) to drain the lysis buffer from the bioreactor, Lynx® connector, and
tubing into the MCV.
6. Sampling option.
Pull a sample from the sample collection port during the recirculation.
7. Collection.
• Connect the 3L Erlenmeyer collection bottle to the media waste AseptiQuik® connection.
• Go to Media Removal Phase, and press
to pump cell lysate from the MCV to a Media Removal bottle.
8.5.3 PBS Wash
Add DPBS wash to the MCV in the volume listed below for the following setup:
• Lynx® connector to the bioreactor: 1 m
2
(650 mL); 2.5 m
2
(800 mL); 5 m
2
(950 mL)
• AseptiQuik® G connector to the bioreactor: 1 m
2
(500 mL); 2.5 m
2
(600 mL); 5 m
2
(700 mL)
1. Ensure proper connections.
• There are several options to add lysis buffer to the MCV.
• Connect to the luer fitting dip-tube to the luer port on the MCV head plate. Apply positive pressure to the dip-tube to allow the solution
to flow into MCV.
• Use AseptiQuik® connectors or tube weld on to the feed addition line of the consumable to connect the lysis buffer container.
TIP:
Ensure the heat sealable flexible tubing is in the pump head, and not the silicone portion. An adaptor may need to be attached earlier
in the run to have appropriate connections.
• Use an unused media addition line and connect via AseptiQuik® connector.
2. Add DPBS to the MCV, then perfuse through the bioreactor.
• On the HMI, go to Bolus Addition, press
to add DPBS.
• Or on the HMI, go to Media Addition, and set parameters to add DPBS to the MCV.
• In Operator mode, set the recirculation pump at 500 mL/min. counterclockwise (forward flow) for 30 min.
