increased temperature [98]. Virus stocks can be used for
6 months before their expression is reduced.
7. Usually, a baculovirus/BV harvest is performed between 50%
and 90% of cell viability. Influencing factors are proteases from
the cells, an increase in the production of defective interfering
particles, an increased protein load-impeding purification processes, as well as the viruses’ concentration and functionality.
8. The harvesting procedure should be initiated before the culture enters the stationary phase. Insect cells produce strong
viral proteases [99, 100], which lead to a degradation of the
virus, and to defective viral particles. The processing of harvested baculoviruses should be performed quickly. Any further
cell lysis needs to be evaluated with regard to an increased viral
concentration and protein load.
9. This means, that all outgoing volume flows are reunited in the
feed tank.
10. The ratio of retentate to permeate flow is approx. 1:1 for the
Sartocon
® Slice 200,300 kDa cassette.
11. A pressure increase, especially at the beginning, is expected for
depth filtration. Keep in mind, that the maximum back pressure of 2 bar for the series connection should not be exceeded.
12. Flow rates may be varied to increase throughput. Desirable are
high recovery rates at high filtration rates. The optimization of
these two factors depends on the permeate flux, the capacity of
the module, and the yield for the target particles. The permeate
flux is limited by the retention of particles by the membrane,
which build a polarization boundary layer. Additionally, the
flux and the membrane capacity are impaired by membrane
fouling [101]. Fouling is detectable as a rapid flux decline,
followed by steady-state conditions [102]. Fouling is a major
problem when filtering biological feeds, due to strongly heterogeneous components, which cause pore clogging and filter
cakes. The amount of cell debris and proteins can be reduced
by optimized cell harvesting and cell lysis. For this reason, the
alignment of upstream and downstream procedures is very
important.
13. A typical pressure gradient is described as follows [101]. At the
beginning of the filtration process, the flux declines rapidly, due
to fouling. Fouling pool mechanisms are pore clogging, pore
constriction, and cake formation. After this initial decline, an
extended period of a quasi-steady-state can be observed. Here,
fouling and cleaning by crossflow are in balance. Due to the
concentration of the virus in the feed, viscosity increases, which
in turn leads to a persisting pressure increase.
Baculovirus-Mediated Gene Transfer to Stem Cells
383
6 months before their expression is reduced.
7. Usually, a baculovirus/BV harvest is performed between 50%
and 90% of cell viability. Influencing factors are proteases from
the cells, an increase in the production of defective interfering
particles, an increased protein load-impeding purification processes, as well as the viruses’ concentration and functionality.
8. The harvesting procedure should be initiated before the culture enters the stationary phase. Insect cells produce strong
viral proteases [99, 100], which lead to a degradation of the
virus, and to defective viral particles. The processing of harvested baculoviruses should be performed quickly. Any further
cell lysis needs to be evaluated with regard to an increased viral
concentration and protein load.
9. This means, that all outgoing volume flows are reunited in the
feed tank.
10. The ratio of retentate to permeate flow is approx. 1:1 for the
Sartocon
® Slice 200,300 kDa cassette.
11. A pressure increase, especially at the beginning, is expected for
depth filtration. Keep in mind, that the maximum back pressure of 2 bar for the series connection should not be exceeded.
12. Flow rates may be varied to increase throughput. Desirable are
high recovery rates at high filtration rates. The optimization of
these two factors depends on the permeate flux, the capacity of
the module, and the yield for the target particles. The permeate
flux is limited by the retention of particles by the membrane,
which build a polarization boundary layer. Additionally, the
flux and the membrane capacity are impaired by membrane
fouling [101]. Fouling is detectable as a rapid flux decline,
followed by steady-state conditions [102]. Fouling is a major
problem when filtering biological feeds, due to strongly heterogeneous components, which cause pore clogging and filter
cakes. The amount of cell debris and proteins can be reduced
by optimized cell harvesting and cell lysis. For this reason, the
alignment of upstream and downstream procedures is very
important.
13. A typical pressure gradient is described as follows [101]. At the
beginning of the filtration process, the flux declines rapidly, due
to fouling. Fouling pool mechanisms are pore clogging, pore
constriction, and cake formation. After this initial decline, an
extended period of a quasi-steady-state can be observed. Here,
fouling and cleaning by crossflow are in balance. Due to the
concentration of the virus in the feed, viscosity increases, which
in turn leads to a persisting pressure increase.
Baculovirus-Mediated Gene Transfer to Stem Cells
383