Biology Reference
In-Depth Information
with HAdVs vectors are also under evaluation. One of these
approaches is the use of nonhuman adenovirus vectors, being
Canine adenovirus type 2 (CAV-2) probably the best described
[
6
-
9
]. The paucity of neutralizing antibodies and memory T cells
in humans and efficient gene delivery obtained for CAV-2 vectors
in the central nervous system, make these viruses promising tools
for human gene therapy [
6
,
9
]. The extraordinary ability to prefer-
entially transduce neurons combined with a remarkable capacity of
axonal transport, make CAV-2 vectors candidates for the treatment
of neurodegenerative diseases.
Most of HadV production processes reported use HEK 293 cells as
the cell line of choice. However, the risk of recombinant competent
adenovirus (RCA) generation in those cells led to the development
of other alternatives, such as PER.C6 or CAP cells, that overcome
this problem by not having any overlapping sequences between E1
(expressed by cell line) and viral vector genome [
10
,
11
]. In order to
facilitate culture manipulation and process scale-up, cells grown in
suspension are preferred and maximal viable cell densities, up to
3-9 × 10
6
cells/mL for HEK 293 and PER.C6 are routinely achieved
in bioreactor cultures [
12
-
15
]. For anchorage-dependent cell lines,
like MDCK-E1 used for CAV-2 production, it is required the use of
microcarrier technology for production in scalable stirred tanks. The
most common microcarriers used for cell growth and virus produc-
tion are Cytodex™-1, but other types are available and it selection
depends on the cell line and culture conditions used [
16
,
17
]. Stirred
tank bioreactors (STB) allow for a fine control of cell environment
(nutrients, pH, O
2
, and temperature) permitting different modes of
operation (batch, fed-batch, and perfusion). The majority of HAdV
production processes reported in the literature are performed using
suspension cells cultured in bioreactor using batch mode with cul-
ture medium exchanged at the time of infection. Other operation
modes have been explored for AdV production at high cell densities
in order overcome the “cell density effect” phenomenon related
with limitation in key nutrient depletion or inhibitor byproducts
accumulation (
see
Fig.
1
). The infection of the cells, by a well-char-
acterized viral seed, is normally performed with cell concentrations
at infection (CCI) of 1-3 × 10
6
cells/mL and a multiplicity of infec-
tion (MOI) of 10-200 total virus particles/cell. AdV infections are
rapid and the harvest is normally done between 36 and 72 h post
infection (hpi). At the end of the process it is usually achieved an
amplification ratio higher than 200 with titers of AdV ranging from
10
4
to 10
5
total particles per cell which corresponds normally to
10
3
-10
4
infectious particles/cell (for detailed information see
reviews [
3
,
18
,
19
]).
This chapter describes methodologies for the scalable produc-
tion of two types of first generation (E1-deleted) adenoviral vectors
using STBs. The production of HAdV using HEK 293 cells grown
1.2 Production
Process
