3. Prepare ca. 200 mL of a PBS solution supplemented with
0.5 M NaCl for use as the running buffer B.
4. Prepare ca. 500 mL of deionized water.
5. Prepare 500 mL of 0.5 M NaOH and 1 M NaCl in water for
use as the cleaning buffer.
6. Filter all buffers using the 0.22 μm bottletop filter connected to
a vacuum pump into fresh glass bottles.
7. Degas all buffers by placing the bottles in an ultrasonic bath for
at least 10 min with the cap loosely placed on top (see Note
16).
8. Prepare membranes of 13 mm diameter to be used as stationary
phase as follows:
(a) Use 13 mm puncher and rubber hammer to punch discs
of appropriate size from the 50 mm cellulose sheets.
(b) Prepare about 12 discs per chromatographic run.
(c) Place the punched discs into a petri dish filled with PBS
until use (see Notes 17 and 18).
3.2.3 Preparation of the
Chromatographic Feed
Depending on the volume to be purified, slightly different parameters have to be applied. In the following, the method is
described for an initial method testing. Further information on a
potential scale-up are found in the respective notes. Always prepare
the sample directly before performing the SXC. Longer storage
times of the sample–PEG mixture might change the sample composition due to an enhanced virus aggregation.
1. Add 6 mL of the clarified virus harvest to a fresh tube.
2. Let 6 mL of the 16% PEG solution slowly run in PBS while
gently shaking the tube (see Note 19).
3. Soak up 10 mL of the sample directly into a syringe using a
blunt cannula (see Note 20).
4. Remove and discard the cannula and close the syringe using a
Luer-lock syringe cap (see Notes 21 and 22).
3.2.4 Steric Exclusion
Chromatography
1. Assemble the filter device according to the respective manufacturer’s instructions. Usually, the set-up is as follows from bottom to top: lower part of the filter holder, sealing ring, support
screen, sealing ring, upper part of the filter holder (see Notes
23 and 24).
2. Place ten layers of the cellulose membranes on top of the
support screen and top them with the sealing ring (see Note
25).
3. Close the filter holder device and insert it into the flow path at
the usual column position (see Note 26).
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0.5 M NaCl for use as the running buffer B.
4. Prepare ca. 500 mL of deionized water.
5. Prepare 500 mL of 0.5 M NaOH and 1 M NaCl in water for
use as the cleaning buffer.
6. Filter all buffers using the 0.22 μm bottletop filter connected to
a vacuum pump into fresh glass bottles.
7. Degas all buffers by placing the bottles in an ultrasonic bath for
at least 10 min with the cap loosely placed on top (see Note
16).
8. Prepare membranes of 13 mm diameter to be used as stationary
phase as follows:
(a) Use 13 mm puncher and rubber hammer to punch discs
of appropriate size from the 50 mm cellulose sheets.
(b) Prepare about 12 discs per chromatographic run.
(c) Place the punched discs into a petri dish filled with PBS
until use (see Notes 17 and 18).
3.2.3 Preparation of the
Chromatographic Feed
Depending on the volume to be purified, slightly different parameters have to be applied. In the following, the method is
described for an initial method testing. Further information on a
potential scale-up are found in the respective notes. Always prepare
the sample directly before performing the SXC. Longer storage
times of the sample–PEG mixture might change the sample composition due to an enhanced virus aggregation.
1. Add 6 mL of the clarified virus harvest to a fresh tube.
2. Let 6 mL of the 16% PEG solution slowly run in PBS while
gently shaking the tube (see Note 19).
3. Soak up 10 mL of the sample directly into a syringe using a
blunt cannula (see Note 20).
4. Remove and discard the cannula and close the syringe using a
Luer-lock syringe cap (see Notes 21 and 22).
3.2.4 Steric Exclusion
Chromatography
1. Assemble the filter device according to the respective manufacturer’s instructions. Usually, the set-up is as follows from bottom to top: lower part of the filter holder, sealing ring, support
screen, sealing ring, upper part of the filter holder (see Notes
23 and 24).
2. Place ten layers of the cellulose membranes on top of the
support screen and top them with the sealing ring (see Note
25).
3. Close the filter holder device and insert it into the flow path at
the usual column position (see Note 26).
Viral Nanoplex Vaccine Production
235