Air-septum mixing is another efficient method employed by the new system. Figure 2 shows a model of an air septum that pushes
air from the bottom of the bioreactor to create mixing throughout the bag. The bag design incorporates three layers of polyethylene.
The middle polyethylene layer has fine holes and is joined to the bottom layer at various points to create an upper chamber
and a lower chamber. Gas is passed through the bottom chamber to create a sparging system that extends to the entire base
of the bag. This system allows extensive mixing, thus removing the need for moving parts in the bioreactor.
Figure 2: A stationary bioreactor, including (1) a flexible 2D bag, (2) gas intake, (3) gas sterilizing filter, (4) sparging
rod, (5) exhaust, (6) media inlet, (7) flapper, (8) heating and cooling element, (9) support frame, and (10) support base.
Aeration is provided either by a ceramic sparging rod or by a perforated septum. (see Figures 2 and 3). Aeration levels of
6 vvm are easily reached, thus allowing every type of cell and organism to grow in flexible bags. The KLa values are comparable
with or higher than those achieved in stainless-steel bioreactors. Until now, it was not possible to manufacture bacterial
products in flexible bags. The new invention, combining a sparging system with a proprietary exhaust system, broadens the
uses of this technology. The GE WAVE system uses surface aeration, which limits it to cell-culture work. Other products use
traditional mixing systems that add substantial cost to the design and almost inevitably limit the size of the bioreactor.
Figure 3: A separative bioreactor, including (1) liquid inlet–outlet, (2) exhaust, (3) media sample, (4) flexible 2D bag,
(5) polyethylene perforated septum, (6) heating–cooling element, (7) gas sterilizing filter, (8) gas flow valve, (9) source
of gas, (10) drain, (11) drain control valve, (12) lower chamber, (13) upper chamber, (14) nutrient media or chromatography
media, (15) support stand, (16) support base, (17) septum tufting point, (18) buffer inlet, and (19) mixing plenum.
The size of the new bioreactor is less limited because the bag remains stationary, which eliminates stress on the seam. Because
the mixing and aeration systems in the new invention are part of the bag, a flexible bag can take any size, from a few liters
to thousands of liters. The flappers are arranged along the longer edge of the flexible bag, and, in the case of the air-septum
design, mixing and aeration are fully integrated. In addition, because the bag is not pressurized or bloated, the volume of
nutrient medium can be as much as 70–80% of the bag volume. This feature further reduces the cost of manufacturing.
Batch size is varied by a gravity-driven system that mixes the contents of multiple bags to meet the 21 CFR definition of a batch without the need for transferring the nutrient media to a larger container. This design eliminates
the need for validating multiple batch sizes. This invention, though not unique to the new bioreactor, confirms the idea that
it is not necessary to validate large bioreactors. Instead, manufacturers can save costs by validating a single size and making
a daisy chain of bioreactors to produce large batches. The gravity system (see Figure 4) reduces stress on the biological
culture and requires no equipment other than a moving platform. The collection bag has no moving parts for mixing, which is
achieved through a venturi effect as the media enters the bag.
Figure 4: A gravity-driven mixing system, including (1) vertical moving stand, (2) bioreactors, (3) drain tube, (4) support
base, (5) transitory vessel, (6) and venture mixing vent.
Perfusion of culture is made possible by installing a ceramic filter. An air-scrubbing method prevents the filter from clogging.
(see Figure 4). The nutrient media is drawn through filters that are continuously scrubbed by a constant stream of fine air
bubbles. This filter can be used in many other stages of bioprocessing that require the concentration of nutrient media, thus
making cross-flow filtration redundant. No equipment currently available can perform the function of this filter. It can be
positioned inside the bag and used indefinitely.
Secreted proteins can be harvested by binding them to a resin in the bioreactor, thus eliminating the need for cell separation
and cross-flow filtration. The resin is added to the bag after the completion of the upstream cycle in the upper chamber of
the air-septum bioreactor (see Figure 3). Once the binding is complete, the nutrient medium and cell culture are drained out.
The protein–resin complex can be eluted or packed into columns for further purification. This method works on the principle
that it is unnecessary to remove the cells and reduce the volume of nutrient media if the purpose is to separate a protein.
The binding resin can be a specific resin, such as protein A, that can be reused hundreds of times, or a mixture of inexpensive
resins, including hydrophobic and ion-exchange resins. The nutrient media's properties can be adjusted to maximize the binding.