3. Place pressure sensors under the sterile workbench and rinse
the flow channel of the sensors with 1 M NaOH (e.g.,
3 Â 10 mL with a serological pipette). Rinse the flow channel
with 20 mM Tris buffer (pH 7.4) afterward.
4. Set up equipment under a sterile workbench.
(a) Connect pump and balance to the power outlet of the
bench.
(b) Place pump tubing into the pump head.
(c) Clamp the depth filter capsule to a tripod rod with flow
direction from top to bottom.
5. Clamp one pressure sensor in front of the entrance and one
behind the outlet of the capsule.
6. Connect balance and pressure sensors to the ports of the peristaltic pump. Additionally, connect the pump to a PC with the
appropriate software (WinWedge and Microsoft Excel).
7. Place the permeate flask on the balance and set the pressure
sensors equal to zero.
8. Equilibrate the capsule with at least 1 L of 20 mM TRIS buffer
(pH 7.4) and fill the buffer into the feed vessel.
9. Start the pump (filtration rate of 150 mL/min), monitor the
pressure, and record the permeate weight.
10. Discard the permeated buffer.
11. Connect a flask (e.g., 1 L) sterilely to the sampling tube of the
bioreactor and transfer the content of the bioreactor to the
flask, by closing the exhaust air connection with a clamp and
the activation of the aeration of the bioreactor (pneumatic
conveyance). Important: The pressure equalization must be
guaranteed in the flask (see Note 12).
12. Disconnect the flask (sterilely) from the bioreactor and connect
it to the filtration system inside the clean bench.
13. Switch on the pump with a flow rate of 150 mL/min to pump
the cell culture supernatant through the capsule.
14. Keep the filtrate in sterile bottles/tubes and store it intermediately at À80
C, or proceed directly with the chromatographic
purification (see Note 13).
3.2.2 Preparation
of Buffers and Membranes
1. Prepare ca. 500 mL PBS solution with 8% (w/v) PEG 8000 to
be used as the running buffer A. Dissolve the PEG using the
magnetic stirring thermomix device at 30–40
C and gently
mix (250 U/min) (see Notes 14 and 15).
2. Prepare ca. 50 mL PBS solution with 16% (w/v) PEG 8000 to
be used as the sample dilution buffer.
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Keven Lothert et al.
the flow channel of the sensors with 1 M NaOH (e.g.,
3 Â 10 mL with a serological pipette). Rinse the flow channel
with 20 mM Tris buffer (pH 7.4) afterward.
4. Set up equipment under a sterile workbench.
(a) Connect pump and balance to the power outlet of the
bench.
(b) Place pump tubing into the pump head.
(c) Clamp the depth filter capsule to a tripod rod with flow
direction from top to bottom.
5. Clamp one pressure sensor in front of the entrance and one
behind the outlet of the capsule.
6. Connect balance and pressure sensors to the ports of the peristaltic pump. Additionally, connect the pump to a PC with the
appropriate software (WinWedge and Microsoft Excel).
7. Place the permeate flask on the balance and set the pressure
sensors equal to zero.
8. Equilibrate the capsule with at least 1 L of 20 mM TRIS buffer
(pH 7.4) and fill the buffer into the feed vessel.
9. Start the pump (filtration rate of 150 mL/min), monitor the
pressure, and record the permeate weight.
10. Discard the permeated buffer.
11. Connect a flask (e.g., 1 L) sterilely to the sampling tube of the
bioreactor and transfer the content of the bioreactor to the
flask, by closing the exhaust air connection with a clamp and
the activation of the aeration of the bioreactor (pneumatic
conveyance). Important: The pressure equalization must be
guaranteed in the flask (see Note 12).
12. Disconnect the flask (sterilely) from the bioreactor and connect
it to the filtration system inside the clean bench.
13. Switch on the pump with a flow rate of 150 mL/min to pump
the cell culture supernatant through the capsule.
14. Keep the filtrate in sterile bottles/tubes and store it intermediately at À80
C, or proceed directly with the chromatographic
purification (see Note 13).
3.2.2 Preparation
of Buffers and Membranes
1. Prepare ca. 500 mL PBS solution with 8% (w/v) PEG 8000 to
be used as the running buffer A. Dissolve the PEG using the
magnetic stirring thermomix device at 30–40
C and gently
mix (250 U/min) (see Notes 14 and 15).
2. Prepare ca. 50 mL PBS solution with 16% (w/v) PEG 8000 to
be used as the sample dilution buffer.
234
Keven Lothert et al.