Facing the Challenges in Vaccine Upstream Bioprocessing - 25

Facing the Challenges in Vaccine Upstream Bioprocessing * rAAV Production in Suspension CAP GT® Cells in BioBLU® 3c and 10c Single-Use Vessels

proteins, the other is the CAP GT cell platform for
stable and transient industrial-scale production
of recombinant adeno-associated viruses (rAAV),
lentiviral, and adenoviral gene therapy vectors. In
this study, researchers at Cevec aimed at scalingup
a rAAV transient production process using CAP GT
cells. When scaling up, they maintained constant
P/V between vessels, which is one of the most
prevalent strategies for scale-up.

Material and Methods
Cell line and medium
Human CAP GT cells were cultivated in a chemically defined, animal component-free medium
compatible with transient transfection.
The thawing, seedtrain and amplification of
the CAP GT cells was performed in shake flasks
in suspension, to generate sufficient biomass
for seeding the final production vessel
(stirred-tank bioreactor). The cell culture in the
bioreactor was inoculated to an initial cell
density of 5 x 105 cells/mL.
25

| GENengnews.com

Bioprocess system and process parameters
The bioprocess engineers at Cevec used a
BioFlo 320 bioprocess control station equipped
with BioBLU 3c or 10c Single-Use Vessels (Figure 1).
BioBLU Single-Use Vessels have an industrial, rigidwall design. They provide fast and efficient mixing
with magnetically coupled overhead drives.

tip speed of 0.69 m/s); for the BioBLU 10c
with a 10 L working volume, it was 175 rpm
(corresponding to a tip speed of 0.84 m/s).

The vessel geometry is similar across scales,
among others in terms of the ratio of impeller
diameter to vessel inner diameter and the ratio of
maximum liquid height to vessel inner diameter.
This makes it easy to use the BioBLU vessels at
small scale during process development and then
scale up the process to larger working volumes.
The researchers set the temperature to 37 °C. The
pH was regulated with CO2 (acid) and sodium
bicarbonate (base). The vessels were equipped
with a macrosparger and one pitched-blade
impeller. The gassing strategy was an automatic
gas mix, which automatically controls air, oxygen,
nitrogen, and CO2 , depending on the pH and
dissolved oxygen (DO) set points. The agitation
speed of the BioBLU 3c vessel with a 2 L working
volume was set to 200 rpm (corresponding to a

1. Expansion phase: CAP GT cells were expanded
in suspension in BioBLU Single-Use Vessels.

Virus production process
The bioprocess for rAAV production was divided
into four phases.

2. Transient transfection: At 72 hours into the
process, cells were transiently transfected with
a two-plasmid system from PlasmidFactory®
encoding for rAAV-GFP. Transfection was
mediated by Polyethylenimine (PEI). With the
transfection the production phase was started.
3. Production phase: During this phase the cells
produced rAAV-GFP.
4. Harvest phase: The culture medium was
collected and processed to harvest the virus
particles. Phase 4 is not further described in
this application note.

http://www.GENengnews.com

Facing the Challenges in Vaccine Upstream Bioprocessing

Table of Contents for the Digital Edition of Facing the Challenges in Vaccine Upstream Bioprocessing