Mixing It Up
“Bioprocess is all about mixing and mass transfer,” says Hans van den Berg, director of business development at CELLution Biotech. The first-generation single-use bioreactors relied on a rocking motion to mix the contents of a pillow-shaped bag. They represented a major advance and opened the door to the development of a disposable bioprocess workflow.
However, claims van den Berg, “with wave-type bioreactors the mixing is poor and the mass transfer limited. With stirred (agitated) single-use bioreactors, in theory, it is possible to improve this dramatically. However the properties of plastics are such that a good, reliable, and robust agitator still cannot be introduced.”
Available stirred single-use bioreactors still face some problems, in van den Berg’s view: “sterility (dynamic seals) and limited mass transfer (due to a plastic agitator sealing).”
CELLution patented the CELL-tainer® disposable bioreactor, which combines horizontal and vertical movement to yield a 70-fold increase in mass transfer, according to van den Berg. Compared to a mass transfer coefficient (kLa) of <15/h for a wave-type reactor, and about 30/h for a typical stirred tank reactor, the CELL-tainer can achieve a kLa <200/h and with oxygen up to 700/h in a pillow-shaped bag at a working volume of 10 L, van den Berg says.
With a higher kLa the reactor “can support higher cell densities; the higher mass transfer rate also reduces CO2 buildup,” which increases the cell density potential and productivity of the process, he adds. In a recent poster, CELLution demonstrated growth of PER.C6® cells up to densities as high as 20–25 x 106 cells/mL in fed-batch mode with minimal process control, making it possible to produce 60–80 g of IgG per batch in a 10 L single-use bioreactor.
The CELL-tainer can also support microbial fermentation, and the company reports rising interest in the system for process development and inoculants preparation for fermentation applications.
To allow for low-volume operations, the company mounted the disposable sensors for pH and dissolved oxygen in the bottom of the bag. The bioreactor bag can be clamped, dividing it into three compartments to allow for starting volumes (inoculants) as low as 150 mL. When sufficient cell density is achieved, the clamps can be removed, facilitating scale-up in the same bag from 150 mL to 15 L. “The microenvironment remains the same,” says van den Berg.
CELLution is developing new online sensors and plans to introduce a glucose and lactate sensor during the fourth quarter of 2009. “New technologies are needed to measure viable biomass,” and van den Berg reports that the company is exploring the use of near-infrared sensing devices under single-use conditions.
Microbial Culture Market
Until recently, Xcellerex focused its capabilities on single-use mammalian cell culture technology, but the company has expanded into the microbial culture market with the launch of a 30 L fermentor; a 200 L fermentor is in the prototype stage of development.
In addition to strong interest in the new small-scale, disposable fermentor, the company reports growing demand for its mammalian cell systems, according to Geoff Hodge, vp of process technology and development. Xcellerex recently introduced 2,000 L and 50 L units, rounding out the range of its bioreactor product line.
Xcellerex developed its biomolecule manufacturing platform based on the modular FlexFactor™ systems and XDR single-use bioreactors. The company recently sold a FlexFactory production line to Crucell for use in its manufacturing facility in Leiden, The Netherlands, for vaccine and monoclonal antibody production.
According to Andrew Bulpin, Ph.D., vp for upstream processing at Millipore, “single-use stirred tank bioreactors have been gaining favor for pilot- up to commercial-scale production in volumes ranging from 50–2,000 L. Disposable technologies eliminate sterilize-in-place/clean-in-place steps, offer multipurpose and process flexibility, and enable streamlined validation and reductions in overall turnaround times. “Single-use STRs can be used within a fully disposable or hybrid disposable-reusable production scheme and are ideally suited for mid-scale (1,000–2,000 L) cGMP manufacturing,” adds Dr. Bulpin.
Millipore developed the Mobius® CellReady 3L stirred-tank bioreactor, which was launched at “ESACT,” in partnership with Applikon Biotechnology. Designed for cell culture applications, the unit operates with existing Applikon motors and controllers.
All parts of the rigid 1/4-inch thick reactor vessels, including the plastic head plate, are disposable. Larger size vessels will follow, according to Dr. Bulpin. The units are delivered preassembled and gamma irradiated and contain multiple fluid addition ports above and below the liquid level and an integrated harvest line at the bottom of the vessel to ensure sterile transfer and full removal of contents.
Applikon’s bioprocessing product line, which includes the AppliFlex line of single-use bioreactors, is available with 10 L, 20 L, or 50 L bags, and monitors pH, temperature, and dissolved oxygen using a choice of disposable or reuseable sensors. In the first quarter of 2010, the company will introduce a new form of shaker that uses acoustic energy to achieve high oxygen transfer rates and enhanced expression levels. For the biofuels market, Applikon recently introduced a six-pack, 500 mL fermentation system designed for feedstock selection applications.