RESULTS AND DISCUSSION
Determination of maximum chamber capacity
Figure 4: Determination of maximum chamber capacity: cell density and estimated total cells.
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In Figure 4, the measured cell density in the centrate is displayed in blue, and the estimated total cell number based on the flow rate
and time is displayed in red. The cell densities in the centrate were initially low until approximately 9–10 billion cells
were retained in the chamber. After this point, cell density in the centrate became exponential, as the cell density began
to approach the cell density of the bioreactor (~2.6x106 cells/mL). The study was completed before the centrate cell density actually reached the cell density of the bioreactor.
Otherwise, it would have been observed where the cells entering the chamber would directly exit into the centrate, and the
cell density in the centrate would be equal to the cell density of the bioreactor. Based on the results of this study, to
minimize the amount of cells from escaping into the centrate, the maximum amount of cells per chamber should be kept below
10 x 109. This limit was factored into the remainder of the studies.
Determination of initial feed flow rate
Based on cell count results from the Cedex cell counter, when running the FBC at 100 mL/min/chamber initially, a sharp spike
(result not shown) was observed in the first few minutes of the run. When the initial flow rate was reduced to 80 mL/min/chamber,
the spike was not observed. Hence, a slower flow rate of 80 mL/min/chamber was more optimal and was used in remaining studies
during the formation of the cell bed.
Determination of optimal process feed flow rate
Figure 5: Centrate cell density over time at various process flow rates.
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Cell densities, both viable and total, that correspond to the amount of cells in the centrate at various flowrates are shown
in Figure 5. As the flow rates increased from 140 mL/min/chamber to 225 mL/min/chamber, both viable and total cell density in the centrate
increased exponentially as the centrifugal force alone became increasingly insufficient to retain cells inside the chamber.
The trend exhibited in Figure 5 suggests that the process feed flow rate can potentially be set at 160 or 180 mL/min/chamber with only minimal amount of
cells lost into the centrate. The authors elected to use 140 mL/min/chamber as the process feed flow rate for all studies
reported in this article because this flow rate is adequate to accomplish all of the objectives in the studies. Increasing
the process feed flow rate up to 160 or 180 mL/min/chamber can be a part of future studies if reducing process time becomes
critical.
Cell harvesting with FBC
Table I: Starting bioreactor and final centrate parameters for cell harvesting runs. LDH is lactate dehydrogenase. NTU is
turbidity.
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Table I lists the pre-FBC (bioreactor) and post-FBC (centrate) data from three cell-harvesting runs with high cell viability (> 90%)
and two cell-harvesting runs with low cell viability (< 50%).
Cell density data collected from the centrate indicated that the FBC was efficient in separating cells from the supernatant
as shown by the low cell counts. Starting with cell densities of 2.3x106 cells/mL (Run #1), 5.4x106 cells/mL (Run #2), 4.8x106 cells/mL (Run #3), 4.3x106 cells/mL (Run #4), and 4.0x106 cells/mL (Run #5) in the bioreactor, none of the cell densities measured from the centrate exceeded 0.215x106 cells/mL. Comparing the cell counts in the final harvest vessels to the starting bioreactor cell counts for each run, the
efficiencies of cell removal were in the range of 95.7–98.7% for all runs.
Turbidity data of the centrate samples, measured in NTU, also reflected a similar outcome as the cell densities. It was shown
by the large reduction in NTUs that the FBC was efficient in separating cells from the supernatant. Starting with NTUs of
38.4 (Run #2), 33.3 (Run #3), 63.7 (Run #4), and 57.4 (Run #5) in the bioreactor, the FBC effectively separated cells from
the supernatant, resulting in significantly reduced NTUs in the range of 2.47–6.91.
The clarification efficiencies for each of the runs, based on NTU measurements, were 93.4%, 90.2%, 89.7%, and 88.2% for runs
#2 through #5, respectively. Similar results on clarification efficiencies have been reported using the disc-stack centrifuge
technology (7).
Figure 6: LDH level comparison between bioreactor and centrate.
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LDH levels measured pre- and post-FBC are shown in Figure 6. The average LDH levels for > 90% viability cultures were 3027±313 U and 3459±785 U for pre-FBC and post-FBC samples, respectively,
and the LDH levels for the < 50% viability culture were 43,611±2782 U and 37,287±10,419 U for pre-FBC and post-FBC samples,
respectively. Post-FBC LDH levels did not increase compared to pre-FBC levels, a clear indication and confirmation that no
cell lysis occurred during the process. This is a unique advantage of using the FBC, because cells are suspended in a fluidized
bed rather than having a high g-force packing them against the centrifuge wall. A similar outcome was confirmed by the analysis
of residual DNA content. Residual DNA for the > 90% viability culture were 12.9 mg and 12.1 mg for pre-FBC and post-FBC samples,
respectively, and the residual DNA for the < 50% viability culture were 49.6 mg and 51.4 mg for pre-FBC and post-FBC samples,
respectively, again showing minimal cell lysis in the FBC.
Centrate samples from both Run #3 and Run #5 were analyzed to determine the antibody titers. Minimal antibody titer loss or
dilution was observed after processing with FBC due to FBC's efficient washing capabilities.
Theoretical calculations to estimate cell harvesting process time
Table II: Theoretical calculations showing estimated total process time using fluidized bed centrifuges.
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Table II shows the estimated time for a typical cell harvesting process based on theoretical calculations using both scales of the
FBCs. Assuming all four single-use chambers are used at 1000g and 180 ml/min/chamber, approximately 1.2 hr and 5.8 hr are
required to harvest a 50-L and 250-L bioreactor, respectively, using kSep 400. The estimated times of 42 min and 2.8 hr are
required at 1.5 L/min/chamber to harvest a 250 L and 1000-L bioreactor, respectively, using kSep 4000. Combined with the advantages
the FBC possesses over other cell harvesting alternatives, the FBC is emerging as a promising option for cell harvesting.
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