and fibrosis [53]. This regenerative capacity of MSCs has been used
to treat the Alzheimer’s disease, bone and cartilage diseases, diabetes, myocardial infarction, and osteoarthritis [54]. Clinical investigations with genetically modified MSCs, analyzing the cell’s
potential to treat graft-versus-host disease [55] or ischemia [56],
repairing bone and cartilage, are still at the very beginning [57].
As already mentioned, the BV can be used to transduce MSCs.
An optimization of this process has been conducted concerning the
promoter. A recent promoter study, in which MSCs were transduced with a pseudotyped BV, recommends the human elongation
factor 1 alpha promoter for regulating the gene expression [58]. In
comparison, the CMV immediate early promoter/enhancer was
shown to be considerably weaker due to promoter silencing
[59, 60]. Others optimized the BV transduction of MSCs by constructing a hybrid vector of the baculovirus and the adenoassociated virus [61].
Due to the long history of applications using the baculovirus,
several industrially attuned production systems are at hand [62]:
They are the basis for the production of the BV in sufficient
quantity and quality for gene therapy products, or for genetically
modified cell therapeutics. As baculoviruses display a lytic life cycle,
they are produced in a batch-mode in bioreactors (e.g., stirred tank
reactor) [63, 64]. A production requires the consideration of the
GMP guidlines. Usually, baculovirus/BV is amplified in serum-free
medium [65] using a cell concentration at infection (CCI) of
1 Â 10
6 cells/mL [66]. Subsequent infection is routinely completed with a multiplicity of infection (MOI) between 0.01 and
1 pfu/cell, taking into account that a low MOI reduces the amount
of defective virus particles [67, 68]. Certain critical process parameters must be considered during a BV production, which include
the time of harvest (TOH), time of infection (TOI), shear stress,
dissolved oxygen, pH, and temperature [69]. In this chapter, methods of virus propagation are of a subordinate role, as they are
intensively reviewed in Chap. 8.
After upstream processing has been conducted, the BV must
undergo an extensive purification process to clear the culture supernatant from cell debris, residual protein and DNA. Generically, the
downstream process (DSP) for viral vectors is split into the procedural steps of clarification, concentration, potential nuclease treatment, purification, polishing, and—for viruses of a smaller size—
sterile filtration [70]. For the BV, a sterile filtration step is possible,
but it can lead to high virus losses. As an alternative, a complete
aseptic downstream process is suitable [71]. The performance of
every DSP process step depends on the upstream process. Hence, it
is inevitable, to reconcile up- and downstream processes in a holistic
approach, while, at the same time, building robust and efficient
methods that are in line with the approach of Quality by Design,
requested by the FDA [72]. Additional regulatory obligations
370
Friederike Eilts et al.
to treat the Alzheimer’s disease, bone and cartilage diseases, diabetes, myocardial infarction, and osteoarthritis [54]. Clinical investigations with genetically modified MSCs, analyzing the cell’s
potential to treat graft-versus-host disease [55] or ischemia [56],
repairing bone and cartilage, are still at the very beginning [57].
As already mentioned, the BV can be used to transduce MSCs.
An optimization of this process has been conducted concerning the
promoter. A recent promoter study, in which MSCs were transduced with a pseudotyped BV, recommends the human elongation
factor 1 alpha promoter for regulating the gene expression [58]. In
comparison, the CMV immediate early promoter/enhancer was
shown to be considerably weaker due to promoter silencing
[59, 60]. Others optimized the BV transduction of MSCs by constructing a hybrid vector of the baculovirus and the adenoassociated virus [61].
Due to the long history of applications using the baculovirus,
several industrially attuned production systems are at hand [62]:
They are the basis for the production of the BV in sufficient
quantity and quality for gene therapy products, or for genetically
modified cell therapeutics. As baculoviruses display a lytic life cycle,
they are produced in a batch-mode in bioreactors (e.g., stirred tank
reactor) [63, 64]. A production requires the consideration of the
GMP guidlines. Usually, baculovirus/BV is amplified in serum-free
medium [65] using a cell concentration at infection (CCI) of
1 Â 10
6 cells/mL [66]. Subsequent infection is routinely completed with a multiplicity of infection (MOI) between 0.01 and
1 pfu/cell, taking into account that a low MOI reduces the amount
of defective virus particles [67, 68]. Certain critical process parameters must be considered during a BV production, which include
the time of harvest (TOH), time of infection (TOI), shear stress,
dissolved oxygen, pH, and temperature [69]. In this chapter, methods of virus propagation are of a subordinate role, as they are
intensively reviewed in Chap. 8.
After upstream processing has been conducted, the BV must
undergo an extensive purification process to clear the culture supernatant from cell debris, residual protein and DNA. Generically, the
downstream process (DSP) for viral vectors is split into the procedural steps of clarification, concentration, potential nuclease treatment, purification, polishing, and—for viruses of a smaller size—
sterile filtration [70]. For the BV, a sterile filtration step is possible,
but it can lead to high virus losses. As an alternative, a complete
aseptic downstream process is suitable [71]. The performance of
every DSP process step depends on the upstream process. Hence, it
is inevitable, to reconcile up- and downstream processes in a holistic
approach, while, at the same time, building robust and efficient
methods that are in line with the approach of Quality by Design,
requested by the FDA [72]. Additional regulatory obligations
370
Friederike Eilts et al.