come with the threshold values by EMA and FDA for residual
contaminants. For gene therapy vectors, a batch release requires
no “admissible levels” of host cell protein (HCP) or host cell DNA
(hcDNA) according to Ph. Eur. 5.14. In literature, an hcDNA
content of <5 pg per 10
À11 virus particles is recommended
[73]. For cell culture-derived vaccines (Ph. Eur. 6.3), the residual
hcDNA (>200 bp) must not exceed 10 ng/dose, and the amount
of residual bovine serum albumin should be <50 ng per dose. It is
advised that the quantity of HCP is monitored and minimized
throughout DSP, and reported using an appropriate assay. HCP
ranges for biological products are typically between 1 and 100 ppm
(<100 ng/mL) [74, 75]. A current draft guideline for gene therapy
vectors of the FDA recommends to follow the limits applied to
vaccines [76–78]. Useful guidelines for the quantification of the
product and the contaminants can be found elsewhere [72, 79].
Clarification and concentration processes are usually performed
via filtration methods [72] (in lab scale centrifugation [80]),
whereas purification and polishing are relevant for manifold systems. Focusing on purity, contaminant removal, and high yields of a
desired product [72, 79, 81], different strategies have been
employed for the BV, relying on the key technologies of centrifugation, filtration, and chromatography [65]. Centrifugation techniques are based on ultracentrifugation methods which are solely
used for lab-scale applications [30]. Chromatographic purification
has been exploited by size exclusion chromatography [82], ion
exchange chromatography [83–86], affinity chromatography [87–
89] and affinity-binding to magnetic beads [90], and steric exclusion chromatography (see Chap. 12) [91]. Filtration processes for
the BV purification usually involve tangential flow filtration (TFF),
also called cross-flow filtration [71, 92, 93]. Membrane-based
filtration is beneficial for handling a large-scale production whilst
simultaneously yielding high virus concentrations of sustained
infectivity and purity [84].
In the following methods chapter, we describe in detail the use
of depth filtration for a primary clarification, and a subsequent TFF
for the purification of the BV. Depth filtration is a dead-end
method, using pore size ratings between 0.1 μm and 10 μm
[94]. This procedure is very well suited for the filtration of highcell-density cultivation broths, for an easy scaling of the parameters,
and for employing single-use cartridges [95]. Recovery rates of
virus particles and VLPs have been reported to lie above 85%
using depth filtration [71, 96]. After cell separation, remaining
contaminants like HCP and hcDNA are removed via a purification
step. TFF is routinely applied in concentration mode or as diafiltration to further purify the BV. Due to the tangential flow (retentate),
the buildup of a filter cake on the membrane surface is reduced,
whilst a smaller fraction flows through the membrane (permeate).
Baculovirus-Mediated Gene Transfer to Stem Cells
371
contaminants. For gene therapy vectors, a batch release requires
no “admissible levels” of host cell protein (HCP) or host cell DNA
(hcDNA) according to Ph. Eur. 5.14. In literature, an hcDNA
content of <5 pg per 10
À11 virus particles is recommended
[73]. For cell culture-derived vaccines (Ph. Eur. 6.3), the residual
hcDNA (>200 bp) must not exceed 10 ng/dose, and the amount
of residual bovine serum albumin should be <50 ng per dose. It is
advised that the quantity of HCP is monitored and minimized
throughout DSP, and reported using an appropriate assay. HCP
ranges for biological products are typically between 1 and 100 ppm
(<100 ng/mL) [74, 75]. A current draft guideline for gene therapy
vectors of the FDA recommends to follow the limits applied to
vaccines [76–78]. Useful guidelines for the quantification of the
product and the contaminants can be found elsewhere [72, 79].
Clarification and concentration processes are usually performed
via filtration methods [72] (in lab scale centrifugation [80]),
whereas purification and polishing are relevant for manifold systems. Focusing on purity, contaminant removal, and high yields of a
desired product [72, 79, 81], different strategies have been
employed for the BV, relying on the key technologies of centrifugation, filtration, and chromatography [65]. Centrifugation techniques are based on ultracentrifugation methods which are solely
used for lab-scale applications [30]. Chromatographic purification
has been exploited by size exclusion chromatography [82], ion
exchange chromatography [83–86], affinity chromatography [87–
89] and affinity-binding to magnetic beads [90], and steric exclusion chromatography (see Chap. 12) [91]. Filtration processes for
the BV purification usually involve tangential flow filtration (TFF),
also called cross-flow filtration [71, 92, 93]. Membrane-based
filtration is beneficial for handling a large-scale production whilst
simultaneously yielding high virus concentrations of sustained
infectivity and purity [84].
In the following methods chapter, we describe in detail the use
of depth filtration for a primary clarification, and a subsequent TFF
for the purification of the BV. Depth filtration is a dead-end
method, using pore size ratings between 0.1 μm and 10 μm
[94]. This procedure is very well suited for the filtration of highcell-density cultivation broths, for an easy scaling of the parameters,
and for employing single-use cartridges [95]. Recovery rates of
virus particles and VLPs have been reported to lie above 85%
using depth filtration [71, 96]. After cell separation, remaining
contaminants like HCP and hcDNA are removed via a purification
step. TFF is routinely applied in concentration mode or as diafiltration to further purify the BV. Due to the tangential flow (retentate),
the buildup of a filter cake on the membrane surface is reduced,
whilst a smaller fraction flows through the membrane (permeate).
Baculovirus-Mediated Gene Transfer to Stem Cells
371