Production In Disposable Bioreactors
In the same bag, cell suspension was partially removed and replaced by fresh medium to keep same seeding density between bioreactors.
Cells were maintained at 37 °C for a few days, then a temperature switch was performed and production temperature was applied
until production day 7.
Oxygen was regulated at 50% with air and oxygen at a flow rate scaled-down from MSB 1,250 L production bioreactor for paddle
and stirred mixing bioreactors and at a flow rate fixed by supplier and MSB according to their experiences with the orbital
shaking bioreactor. The pH was maintained between 6.8 and 7.2 with sodium hydroxide 1N and CO2 with a flow rate fixed by supplier.
Agitation speed was fixed by the supplier for the paddle bioreactor. Agitation speed for stirred and orbital-shaking bioreactors
was fixed by the supplier and increased by MSB to avoid large oxygen variations.
Feed was added twice at the same amount per L, and sampling was performed every day at 1/200 of working volume.
In-Process and Quality Controls
Every day, pH was checked with an external pH-meter and corrected if the difference was above 0.05 pH unit; cell density and
viability were measured. During the production process, biochemistry analyses and molecule titration were performed in addition
to previous in-process controls. At the end of the process, a capture was performed to check quality attributes (e.g., glycosylation,
Oxygen transfer coefficient, KLa, was measured at maximum working volume in water at 37 °C by sparging air at the working flow rate applied during production
process and at the maximum flow rate allowed by the equipment.
Mixing time was measured at maximum working volume in phosphate saline buffer between pH 6.8 and 7.4 at 37 °C by injecting
a small amount of sodium hydroxide 5N to increase the pH by 0.20–0.30 units. The pH was adjusted initial level with 8M acetic
Temperature mapping was performed at maximum working volume in water with six calibrated temperature probes at different places
inside each disposable bag. Temperature increase was followed between room temperature up to 37 °C and temperature decrease
to 37 and 29 °C.
RESULTS AND DISCUSSION
One of MSB's objectives is to provide grams of high quality recombinant proteins for preclinical and clinical studies. This
production can be performed at different scales depending on needs. Thus, process performances in terms of cell growth, but
especially protein concentration and quality, should be representative to MSB bioreactors.
Cell Growth During Cell Amplification Phase
Cell growth at minimum and maximum working volume allowed MSB to evaluate seeding performances of disposable bioreactors.
Cell growth was compared between disposable bioreactors and 250 L production seeding bioreactor in terms of doubling time
or population doubling level (Figure 1).
Figure 1. Population doubling level (PDL) from inoculation to production phase. For disposable bioreactors, it represents
growth at minimum working volume (from working days 0 to 2) and at maximum working volume (from working days 3 to 5). Date
for seeding bioreactors were from 1,250 L seeding bioreactors presented later. Data are the means from two bioreactors, with
errors bars representing minimum and maximum levels.
Linear regressions of these curves gave cell doubling time (see Table 1).
Table 1. Regression coefficients and slopes of Figure 1 curves
Regression coefficients of all curves were above 0.99; therefore, cells exhibited a regular growth either at minimum working
volume or at maximum working volume, whichever bioreactors were used.
These results indicate that flexibility of disposable bioreactors as the minimum working volumes was between half and 1/5
of maximum working volumes. Doubling times were similar between stainless steel and disposable bioreactors. They are all compatible
with production bioreactor seeding application.