Preparation of buffers and membranes
14. Instead of using a PBS buffer, theoretically any buffer of choice
may be used depending on the requirements of the virus to be
produced. Be aware of the buffer osmolarity and ionic strength
to avoid virus aggregation.
15. PEG can be dissolved by slightly heating up the solution. Overheating must be avoided.
16. All solutions and buffers need to be filtered and degassed, in
order to increase the shelf life of the buffers and to prevent the
gassing-out of the liquids during the chromatographic run.
17. Cellulose membranes can be autoclaved in the respective buffer
without PEG, shortening the swelling process of the cellulose
membranes and, thus, improving the process robustness.
18. Store unused membranes in 20% ethanol and at 4
C.
Preparation of the sample
19. In order to prevent virus dilution during sample preparation,
buffer stocks containing elevated PEG concentrations of up to
32% PEG 8000 are recommended.
20. When preparing the sample to be loaded onto the chromatography column, the sample volume for the off-line analytics must
be considered.
21. Sample preparation should be done under slight constant stirring in an appropriate beaker, and for larger scale processes an
inline preparation is recommended.
22. The PEG concentration and the PEG molecular weight must
be adjusted individually for each virus. As an initial condition,
8% PEG 8000 are recommended to test the principal applicability of the method. However, for an optimal chromatographic
process, the critical process parameters must be optimized,
preferably by a Design-of-Experiment (DoE) approach. In
general, it can be said, that the smaller the nanoplexes to be
purified are, the higher is the required PEG concentration, or
PEG molecular weight. PEG concentrations above 14% should
not be used, in order to prevent the risk of virus precipitation.
Performing the SXC
23. The minimum requirements for the chromatographic system
are two independent pumps and a UV 280 -detector. It is even
possible, to load the sample and sequentially push the appropriate buffers through the filter holder by a syringe, and to
collect the product in the final elution step. However, for a
better process control, the system pressure and the conductivity
should be monitored. An additional light scattering detection
increases the detection possibilities of nanoplexes significantly
240
Keven Lothert et al.
14. Instead of using a PBS buffer, theoretically any buffer of choice
may be used depending on the requirements of the virus to be
produced. Be aware of the buffer osmolarity and ionic strength
to avoid virus aggregation.
15. PEG can be dissolved by slightly heating up the solution. Overheating must be avoided.
16. All solutions and buffers need to be filtered and degassed, in
order to increase the shelf life of the buffers and to prevent the
gassing-out of the liquids during the chromatographic run.
17. Cellulose membranes can be autoclaved in the respective buffer
without PEG, shortening the swelling process of the cellulose
membranes and, thus, improving the process robustness.
18. Store unused membranes in 20% ethanol and at 4
C.
Preparation of the sample
19. In order to prevent virus dilution during sample preparation,
buffer stocks containing elevated PEG concentrations of up to
32% PEG 8000 are recommended.
20. When preparing the sample to be loaded onto the chromatography column, the sample volume for the off-line analytics must
be considered.
21. Sample preparation should be done under slight constant stirring in an appropriate beaker, and for larger scale processes an
inline preparation is recommended.
22. The PEG concentration and the PEG molecular weight must
be adjusted individually for each virus. As an initial condition,
8% PEG 8000 are recommended to test the principal applicability of the method. However, for an optimal chromatographic
process, the critical process parameters must be optimized,
preferably by a Design-of-Experiment (DoE) approach. In
general, it can be said, that the smaller the nanoplexes to be
purified are, the higher is the required PEG concentration, or
PEG molecular weight. PEG concentrations above 14% should
not be used, in order to prevent the risk of virus precipitation.
Performing the SXC
23. The minimum requirements for the chromatographic system
are two independent pumps and a UV 280 -detector. It is even
possible, to load the sample and sequentially push the appropriate buffers through the filter holder by a syringe, and to
collect the product in the final elution step. However, for a
better process control, the system pressure and the conductivity
should be monitored. An additional light scattering detection
increases the detection possibilities of nanoplexes significantly
240
Keven Lothert et al.