35. Membranes are intended for a single-use application. However,
it is possible to reuse membrane stacks several times after
washing them with 0.5 M NaOH in buffer.
36. SXC works best near the isoelectric point of the virus particle.
Thus, the buffer’s pH may need an adjustment for an optimal
product recovery and purity. However, it is crucial to test the
virus stability of the respective virus for the applied pH, particularly in the case of a pH below 7.
37. The virus recovery can be improved by increasing the ionic
strength (e.g., addition of NaCl) in the elution buffer. NaCl
concentrations of up to 1 M show a positive effect. However, as
for the pH above, the stability of the respective virus needs to
be confirmed for these conditions.
38. It is strongly recommended to evaluate the capacity of the
membrane stack for the respective nanoplex. This can be
done by using an online light scattering monitor, or by
performing offline virus quantifications from fractions during
sample loading.
39. A storage of the chromatographic samples should be done at
À80
C. However, virus infectivity can be impaired by the
freezing step; hence, protective agents (e.g., sucrose) may be
added, or infectivity assays must be done directly after the
chromatographic experiment.
Quantification and characterization of the purified virus
particles
40. If the virus infectivity is not relevant for the anticipated product, other quantification methods, such as qPCR, ELISA, or
particle counting techniques, are suitable alternatives.
41. For virus titration via the FACS assay, it is essential to have a
calibration standard based on an alternative quantification
method. The assay must be completely validated for each individual type of virus, cells, and culture conditions.
42. The host cells for the assay must be chosen with regard to the
infectivity of the targeted virus. Furthermore, the virus must
carry either the genetic information of a fluorescent protein to
be subsequently expressed in the infected cells for a possible
detection, or the viral proteins on the surface of infected cells
must be labeled by fluorescently tagged antibodies.
43. Before removing the supernatant from the centrifuged plate,
visually check for cell pellets. The supernatant may then be
removed by pipetting or by flipping the plate. This requires
practice, and the presence of pellets should be checked before
and afterward.
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Keven Lothert et al.
it is possible to reuse membrane stacks several times after
washing them with 0.5 M NaOH in buffer.
36. SXC works best near the isoelectric point of the virus particle.
Thus, the buffer’s pH may need an adjustment for an optimal
product recovery and purity. However, it is crucial to test the
virus stability of the respective virus for the applied pH, particularly in the case of a pH below 7.
37. The virus recovery can be improved by increasing the ionic
strength (e.g., addition of NaCl) in the elution buffer. NaCl
concentrations of up to 1 M show a positive effect. However, as
for the pH above, the stability of the respective virus needs to
be confirmed for these conditions.
38. It is strongly recommended to evaluate the capacity of the
membrane stack for the respective nanoplex. This can be
done by using an online light scattering monitor, or by
performing offline virus quantifications from fractions during
sample loading.
39. A storage of the chromatographic samples should be done at
À80
C. However, virus infectivity can be impaired by the
freezing step; hence, protective agents (e.g., sucrose) may be
added, or infectivity assays must be done directly after the
chromatographic experiment.
Quantification and characterization of the purified virus
particles
40. If the virus infectivity is not relevant for the anticipated product, other quantification methods, such as qPCR, ELISA, or
particle counting techniques, are suitable alternatives.
41. For virus titration via the FACS assay, it is essential to have a
calibration standard based on an alternative quantification
method. The assay must be completely validated for each individual type of virus, cells, and culture conditions.
42. The host cells for the assay must be chosen with regard to the
infectivity of the targeted virus. Furthermore, the virus must
carry either the genetic information of a fluorescent protein to
be subsequently expressed in the infected cells for a possible
detection, or the viral proteins on the surface of infected cells
must be labeled by fluorescently tagged antibodies.
43. Before removing the supernatant from the centrifuged plate,
visually check for cell pellets. The supernatant may then be
removed by pipetting or by flipping the plate. This requires
practice, and the presence of pellets should be checked before
and afterward.
242
Keven Lothert et al.