and allows a differentiation of the contaminating proteins and
virus particles. Furthermore, the system should contain a fraction collector, to allow a robust and reproducible collection of
the chromatographic fractions.
24. Depending on the batch size of the virus to be purified, larger
membrane holder modules can be used to scale up the membrane surface area (or volume) in relation to the viral load. For
an initial method development and optimization, a 13 mm
device will be sufficient.
25. Setting up the membrane stack within the membrane holder,
use forceps with rounded (not sharp!) edges and avoid air
bubbles between the individual layers.
26. If necessary, a layer of PTFE-band can be used to properly seal
the filter holder.
27. The chromatography system is commonly stored in 20% ethanol solution. Before flushing the system with buffer, the system
needs to be rinsed with water to remove the ethanol.
28. Loading the sample loop by a syringe can be challenging for
larger loop sample volumes. Thus, it might be easier to directly
prefill the loop under the bench for sample volumes between
50 and 150 mL. For sample volumes above 150 mL, an external sample pump should be used.
29. Prior to the loading of the virus particles, the system needs to
be equilibrated with at least ten column volumes, until all
relevant signals are constant.
30. Directly before the sample is loaded onto the column, the
UV 280 -detector and the light scattering detector must be set
to zero.
31. After sample loading, the subsequent washing step should be
completed after five to ten column volumes, or once the detector signals reach the baseline.
32. Priming the system tubing with the elution buffer, allows for a
faster elution and higher concentrated product fraction.
33. Elution is usually completed after ten column volumes. However, for small column volumes, in relation to the system
volume, a small fraction of the virus particles may still be in
tubing of the system. Here, a decision must be made between a
complete virus elution and a higher virus concentration.
34. The buffer system for the virus elution does not have to correspond with the sample buffer, allowing for a buffer exchange
within the chromatography run according to the requirements
of the subsequent unit operation.
Viral Nanoplex Vaccine Production
241
virus particles. Furthermore, the system should contain a fraction collector, to allow a robust and reproducible collection of
the chromatographic fractions.
24. Depending on the batch size of the virus to be purified, larger
membrane holder modules can be used to scale up the membrane surface area (or volume) in relation to the viral load. For
an initial method development and optimization, a 13 mm
device will be sufficient.
25. Setting up the membrane stack within the membrane holder,
use forceps with rounded (not sharp!) edges and avoid air
bubbles between the individual layers.
26. If necessary, a layer of PTFE-band can be used to properly seal
the filter holder.
27. The chromatography system is commonly stored in 20% ethanol solution. Before flushing the system with buffer, the system
needs to be rinsed with water to remove the ethanol.
28. Loading the sample loop by a syringe can be challenging for
larger loop sample volumes. Thus, it might be easier to directly
prefill the loop under the bench for sample volumes between
50 and 150 mL. For sample volumes above 150 mL, an external sample pump should be used.
29. Prior to the loading of the virus particles, the system needs to
be equilibrated with at least ten column volumes, until all
relevant signals are constant.
30. Directly before the sample is loaded onto the column, the
UV 280 -detector and the light scattering detector must be set
to zero.
31. After sample loading, the subsequent washing step should be
completed after five to ten column volumes, or once the detector signals reach the baseline.
32. Priming the system tubing with the elution buffer, allows for a
faster elution and higher concentrated product fraction.
33. Elution is usually completed after ten column volumes. However, for small column volumes, in relation to the system
volume, a small fraction of the virus particles may still be in
tubing of the system. Here, a decision must be made between a
complete virus elution and a higher virus concentration.
34. The buffer system for the virus elution does not have to correspond with the sample buffer, allowing for a buffer exchange
within the chromatography run according to the requirements
of the subsequent unit operation.
Viral Nanoplex Vaccine Production
241