Evaluation of Single-Use Fluidized Bed Centrifuge System for Mammalian Cell Harvesting - This article discusses the evaluation of a novel single-use fluidized bed centrifuge for harvesting of antibodi

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Evaluation of Single-Use Fluidized Bed Centrifuge System for Mammalian Cell Harvesting
This article discusses the evaluation of a novel single-use fluidized bed centrifuge for harvesting of antibodies.


BioPharm International
Volume 25, Issue 11, pp. 34-40

MATERIALS AND METHODS

Materials

All studies used CHO cells cultured in shake flasks (Corning) followed by expansion into WAVE bioreactor (GE Healthcare). Cells were cultured in Janssen R&D's proprietary, chemically-defined medium. 1X phosphate-buffered saline (PBS, Gibco) was used for priming and rinsing during the runs.

The FBC (model kSep 400) and the single-use sets were purchased from kSep Systems, Inc. Cells were placed on an orbital shaker (VWR International) set at 100 RPM during operations to keep cells in suspension.

Cell culture

Because of the simulated nature of the study, CHO cells were only cultured up to four days and up to a cell density of approximately 5x106 cells/mL. To simulate cell cultures with lower viabilities such as those found in 18–20 day cultures to harvest antibodies or therapeutic proteins, a second batch of cells were intentionally starved/asphyxiated by turning off nutrient and air supply after day four and then mixed with healthy cultures to lower the viability.

Determination of maximum chamber capacity

The maximum number of cells each chamber can hold is of great significance because, in most applications, the total number of cells from a bioreactor will exceed what the four chambers can hold. The maximum capacity can also vary from cell line to cell line and is dependent on factors such as cell size. It is, therefore, crucial to determine the maximum chamber capacity at which breakthrough of the cell bed will occur. For this experiment, one chamber was used and the centrifugal force was maintained at 1000g. Cell suspensions were continuously processed by the FBC at 140 mL/min/chamber and samples were taken from the centrate to examine the amount of cells escaping from the chamber. Samples were taken until the breakthrough threshold of the chamber capacity had been reached. Cell count was performed using Cedex cell counter (Roche).

Determination of initial feed flow rate

The initial flow rate when cell suspensions are first introduced into the FBC was determined. It is hypothesized that prior to the formation of the cell bed in the chambers, the flow rate should be kept lower to minimize potential escape of cells from the chambers due to initial instability of the cell bed. To test this hypothesis, 100 mL/min/chamber and 80 mL/min/chamber were compared in two separate runs. Two chambers were used in each of the runs, and the centrifugal force was maintained at 1000g. Cell counts samples were taken and performed on the Cedex to determine the cell density.

Determination of optimal process feed flow rate

Once the fluidized bed is established in the single-use chamber and is stable, the feed flow rate can then be increased. Consecutive runs were performed where process flow rates were increased incrementally from 140 to 225 mL/min/chamber while maintaining the centrifugal force at the maximum of 1000g. Cell counts samples were taken from the centrate to determine the cell density. One chamber was used in each of the runs.

Cell harvesting with FBC

Five total cell-harvesting runs were completed using the FBC. The first three runs were conducted using CHO cell cultures with > 90% viability, followed by two remaining runs using CHO cell cultures with < 50% viability. For all runs, the centrifugal force was kept at the maximum g-force of 1000g. The initial feed flow rate was 80 mL/min/chamber during initial establishment of the cell bed, and increased to 140 mL/min/chamber after 5 min into the process. Samples were taken to measure cell density, turbidity (NTU), LDH level, and residual DNA content.




Cell density and viability were determined using the Cedex cell counter. Turbidity was measured using the HACH 2100AN Turbidimeter. Cell density and turbidity were also used to calculate the FBC's clarification efficiency using Equations 4 and 5:

where CDCentrate is the final cell density in the final harvest vessel, CDBioreactor is the starting cell density in the bioreactor, NTUCentrate is the turbidity in the final harvest vessel, and NTUBioreactor is the starting turbidity in the bioreactor.

LDH levels and residual DNA from samples before and after the FBC process were used as a measure of shear stress and cell lysis during the process. LDH and residual DNA were measured using Johnson & Johnson Vitros Chemistry System DTSC Module and Applied Biosystems Prism 7500 Sequence Detection System, respectively. Antibody titer was also determined using Agilent 1100 Series HPLC.

Theoretical calculations to estimate cell harvesting process time

Finally, because the studies discussed in this article were simulations on a small scale, theoretical calculations were performed to estimate the total process time required for larger bioreactor scales. Calculations were performed using both the kSep 400 and the process scale version, kSep 4000, for 50-L, 25-0L, and 1000-L bioreactors, assuming all four single-use chambers were run at 1000g and at various feed-flow rates.


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