Facing the Challenges in Vaccine Upstream Bioprocessing - 32

Facing the Challenges in Vaccine Upstream Bioprocessing * Vero Perfusion, Packed-Bed Vessels Intensify Vaccine Production

of CO2 (acid) and 0.45 M sodium bicarbonate
(base). The culture was agitated at 100 rpm.
We added Antifoam C Emulsion (Sigma-Aldrich)
when it was needed.

Feeding and Perfusion Control
We started with a perfusion rate of 0.2 vessel
volumes per day (VVD) and gradually increased
it to 1.5 VVD at the end of the run. We determined
the perfusion rate by monitoring metabolite
levels with a Cedex® Bio Analyzer (Roche Diagnostics). The goal was to keep the ammonium
concentration below 4 mM and the glucose
concentration between 2 and 4 g/L.
In addition to perfusion, we performed extra
glucose bolus feeding (200 g/L glucose stock
solution) based on the glucose level in the
bioreactor at the end of every day. The aim
was to bring the glucose level in the bioreactor
close to the glucose concentration in the perfusion medium (4.7 g/L) at the beginning of the
next day. We also determined lactate and glutamine levels daily.
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Cell Growth
In the packed-bed bioreactor, the Vero cells
cannot be counted directly during the culture
because the cells adhere to the Fibra-Cel disks.
To obtain an indirect measure of cell growth, we
analyzed the glucose consumption of the culture.
The rate of glucose consumption (R) in grams
per day can be calculated based on the total
glucose added to the bioreactor minus the
residual glucose. The daily total amount of
glucose added to the bioreactor is therefore
equal to the amount of glucose in the vessel
at the start of the day (Gvessel-start) combined
with the glucose supplied through perfusion
(Gperfusion) and the extra glucose added via
bolus feed (Gbolus). By subtracting the amount
of glucose remaining in the vessel at the end
of the day (Gvessel-end) as well as the amount of
glucose remaining in the harvested perfusate
(Gharvest), we arrive at the amount of glucose
consumed (g) per day (24 h). The daily glucose
consumption rate is represented by the
following equation:

R = (Gvessel-start + Gperfusion + Gbolus −
Gvessel-end − Gharvest) / day
The glucose amount in the vessel at the start and
the end of the day can be calculated by multiplying the glucose concentration in the medium
by the working volume. The amount of glucose
added by bolus feed can be calculated based on
the concentration of the glucose stock solution
and the volume of the bolus feed. The amount of
glucose supplied through perfusion can be calculated based on the glucose concentration in the
perfusion medium and the perfusion volume. The
amount of glucose remaining in the harvested
perfusate can be calculated based on its volume.
In addition, we directly measured the cell number
at the end of the cell culture run by counting
nuclei using a crystal violet nuclei counting assay
(Chemglass Life Sciences, CLS-1332-01). We cut the
vessel open below the head plate and collected
Fibra-Cel samples from two different locations in
the basket. We extracted the cell nuclei, stained
them with crystal violet, and counted them using
a Vi-CELL® XR Cell Viability Analyzer (Beckman
Coulter) as described previously.2
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Facing the Challenges in Vaccine Upstream Bioprocessing

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