The Potential Application of Hollow Fiber Bioreactors to Large-Scale Production - A hollow fiber matrix allows for efficient harvest of secreted proteins. - BioPharm International


The Potential Application of Hollow Fiber Bioreactors to Large-Scale Production
A hollow fiber matrix allows for efficient harvest of secreted proteins.

BioPharm International Supplements
Volume 24, Issue 5, pp. s21-s26


The system included the following components:

  • A disposable, preassembled, and sterilized package including the cartridge, two media bags with fittings, and two disposable sensor "windows" with reactive pads bound inside.
  • Gravity pump with motorized cartridge angle controller.
  • Environmental enclosure.
  • Control unit with monitoring devices.

Figure 4: Hollow fiber cartridge on Fibercell Systems prototype large-scale HFBR.
The diameter of the fibers has been increased to reduce the resistance to flow while still presenting a large surface area to volume ratio. This allows the use of a gravity pumping mechanism that can generate a flow of 5–10 L of media per minute through the interior of the hollow fiber bundle. The gravity pump will then simply reverse direction. The bags contain both medium and gas, and when the liquid is finished flowing through the cartridge, the gas is drawn through the interior of the fibers, directly oxygenating the cartridge from within. The disposable bags (Thermo Scientific HyClone BPCs employing CX5-14, a pentalaminate animal component-free film) are gas-impermeable, and the gas composition inside the bags is tightly controlled for optimum cell culture performance. The environmental enclosure is a simple, non-pressurized, temperature controlled incubator, but can be modified to include further environmental control such as HEPA filters to create a Class 100 clean room environment within the system for cGMP operation. Sensors for pH, O2, and potentially real-time glucose monitor the culture environment. The absence of cells in the medium bags to be monitored supports more robust and sensitive measurements. These sensors will support the control of medium and gas in the media bags. More advanced monitoring is possible using any one of a number of new approaches, including capacitance based monitoring probes. This exciting new technology employs passive electrical (dielectric) radio frequency-based sensors to measure the overall capacitance, and therefore viability, of the cell mass within the cartridge. The low volume in both the cartridge and the medium present in the system at any one time makes both monitoring and process control more precise and responsive, even though the system may consume 50–100 L/day. Stacking like-sized cartridges with their media bags on top of each other can provide additional scale.

The FiberCell Systems HFBR allows the production of about 20–200 mg of antibody per week using the 20 mL cartridge (FiberCell Systems catalog # C5011). The product is harvested in a concentrated form 500–5000 g/mL in a volume of 20–40 mL and production of the antibody can be accomplished in a standard CO2 incubator.

Figure 5: Raw supernatant harvest was briefly dialyzed to reduce sample tonicity. No other purification was performed. The heavily loaded samples demonstrate low levels of contaminating proteins. (Data courtesy of Dr. Erin Bromage, U. Mass., Amherst)
As an example, two monoclonal antibodies produced in DMEM + CDM-HD are shown in Figure 5. Length of culture was three weeks, total antibody produced was in excess of 140 mg each, and average concentration was 3.0 mg/mL. The dialyzed supernatants had their protein concentration quantified and purity check on a 12% sodium dodecyl sulfate polyacrylamide gel. A total of 12 L of medium per antibody was consumed.

Extrapolating this data to a 1 L sized hollow fiber bioreactor cartridge would result in total antibody production of approximately 3 g/day in a volume of 1 L, consuming 50 L of DMEM/CDM-HD per day.

473 mg of purified recombinant protein recovered from an rCHO cell line was harvested from the FiberCell Systems 20kd MWCO cartridge (FiberCell Systems catalog # C2018). Medium was Dulbecco's Modified Eagles Medium (DMEM) with 2% fetal bovine serum, each harvest was 70 mL in volume, and total harvest volume was 4.8 L, for an average protein concentration of approximately 100 g/mL/d. The protein was a very complex, hexamerized immunoglobulin G (IgG) consisting of 6 IgG1 subunits held together with three IgA tails. The variable fragment (Fv) region was modified to contain the CD4 receptor (2). The cartridge consumed an average of 2 L of medium per day over a 60–day period of production.

Figure 6: Gel filtration chromatography of a hexeramized recombinant immunoglobulin (IgG). When cultured in flasks (top trace), approximately 40% of the protein is expressed as an improperly folded monomeric subunit (B) rather than the hexamer (A). These cells when transferred to a hollow fiber bioreactor exhibit 95% complete folding ( C ) as a result of the improved cell-culture conditions.
An interesting observation was the comparison of protein produced using T–flasks versus the hollow fiber cartridge (see Figure 6). When produced in flasks, approximately 40 % of the protein was secreted as an unfolded monomeric subunit. Placing the same cells into the HFBR cartridge resulted in nearly 95 % of the protein being produced as a properly folded hexamer (3). Better cell culture conditions resulted in better protein expression fidelity.

Expression levels were significantly below those found in commercially optimized CHO cell lines, although the results are still impressive. A 1 L sized HFBR cartridge would have a total harvest volume of 100 L, total protein recovered would be 10 g and total medium consumed would be 2,400 L.

Figure 7: Harvest from bioreactor rCHO cells. Raw supernatant was briefly dialyzed to reduce sample tonicity, no further purification was performed.
246 mg of purified recombinant IgG1 from a CHO (DG44) cell line was harvested from the FiberCell Systems 20kd MWCO cartridge (FiberCell Systems catalog #C2011). Medium was a serum-free, protein-free formulation similar to CDM-HD. Each harvest was 20 mL in volume; total harvest volume was 320 mL for an average concentration of over 800 g/day/mL. The cartridge consumed 1 L of medium every three days and the culture was maintained for a total of 35 days. For technical reasons, the cell viability in this run dropped rather low, however, the harvested protein produce was remarkably uncontaminated, as demonstrated by the gel of the unpurified harvest in Figure 7. In a 1 L sized HFBR, the total protein recovered would be 12.5 g in a volume of 16 L with 500 L of medium consumed.

To extrapolate these results to a commercially prepared recombinant CHO cell line with expression in the range of 1 g/L in a standard stirred reactor, the 1 L sized HFBR cartridge should be capable of producing 10 g or more of protein per day, while consuming 100 L of medium.

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