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New technology is designed to improve production efficiency by taking advantage of the properties of single-use bags.
Single-use technology for bioreactors has come a long way during the past 25 years, yet some of its capabilities remain to be exploited. Equipment manufacturers have adopted the technology as if it were an evolutionary step, but it is, in fact, revolutionary. Current offerings in single-use technologies often are not presented this way, however.
Single-use bioreactors currently follow one of two general formats. In one of these, the single-use components are used as linings for stainless-steel tanks. In a second model, a flexible bag is affixed to a rocker system that helps aerate and mix components inside the bag (1–3). Both of these models limit the value of single use systems, however.
Equipment manufacturers conduct extensive exercises to chart the future of bioprocessing methods, but the real judge of what is needed is the consumer. The development of large-scale bioreactors for the manufacture of commercial quantities of monoclonal antibodies and vaccines at an affordable cost and with a short development time would fill an unmet need. Therapeutic Proteins is looking at ways to meet this demand by incorporating a comprehensive bioprocessing unit capable of upstream and downstream processing inside a single bag without any moving parts. The company has filed or received dozens of US and worldwide patents for these inventions. In this way, the company hopes to spur the further evolution of single-use technologies.
In this new unit, mixing is achieved by gentle pressing on the bag to create a wave. Figure 1 shows a bioreactor with a flapper that pushes down on the bag to create a wave motion inside the bag. The bag itself lies flat and does not move. The Navier–Stokes equations describe the motion of fluid substances, such as liquids and gases (4). These equations state that changes in the momentum (i.e., force) of fluid particles depend only on the external pressure and internal viscous forces (which are similar to friction) acting on the fluid. The equations also describe the balance of forces acting at any given region of the fluid. A force applied to any portion of a fluid would thus be transferred to the rest of the fluid (4). A flexible bag never needs to be shaken or rocked. All that is needed is to apply a minimal force, a pressure on any part of the bag, to start the motion of liquid. Rocking and shaking technologies generally fail to account for the physical constraints on the amount of stress that can be applied to the bag. Bags used in the rocking model cannot hold more than 500 L of media because the bag would break when rocked at a larger size.
Figure 1: A 400-L bioreactor for bacterial fermentation used by Therapeutic Proteins to manufacture filgrastim.
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 inletâoutlet, (2) exhaust, (3) media sample, (4) flexible 2D bag, (5) polyethylene perforated septum, (6) heatingâcooling 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.
This invention is intended to reduce the time and cost of drug manufacturing. Two major steps, both requiring expensive equipment and substantial time to achieve the same goal, are eliminated. It is anticipated that in the manufacturing of monoclonal antibodies, this new unit saves a process time of approximately 50 h for a 2000-L batch. In addition, the limited handling of proteins can improve the final yield substantially, sometimes as much as 20–30% (5).
Figure 5: Air-scrubbed filtration system for nutrient media perfusion, cell removal and volume reduction.
Proteins can be purified in the bag by using it as a chromatography column. Although the idea of using a flexible bag as a chromatography column appears alien, nothing prevents a process from being developed by taking into account the geometry and the physical state of resin suspension in the bag. The elution may include a step elution, a gradient elution, or a programmed elution. An example is washing the bound resin to remove cells, and then equilibrating the protein–resin conjugate in a buffer to elute the target drug. A buffer that would break down the binding can be used to collect a highly purified solution of the target protein. Even if this process of purification does not achieve the quality that traditional methods do, the possibility of eliminating a few steps in downstream processing would have a great effect on the cost of purification because no equipment needs to be installed for large volumes to be fed through the purification column. An AKTA Pilot liquid-chromatography system (GE Healthcare) might do the job of an AKTA Processor (GE Healthcare), for example.
Other uses of the new bioreactors include media and buffer preparation and sterile transfer to final containers. The unit also may be used as a pressure vessel in pharmaceutical manufacturing. The air-septum bioreactor is suited to performing many functions. As a complete system with no moving parts and the ability to be pressurized, this invention fulfills the bioprocessing industry's needs for manufacturing recombinant proteins, monoclonal antibodies, and vaccines.
Other uses of the perfusion filter include concentration of slurries, reduction of volume of a bacterial nutrient media, water purification, and sterile liquid transfers. The filter can be made in several shapes and combinations to fulfill the need for a particular flow rate from a specific mixture. Using air to scrub a filter and keep the pores open enables new filtration methods. This filter system requires a solid base to keep the filter from collapsing. The base can be layered with fine membranes, such as a 0.22-µm filter, to separate bacteria and sterilize a solution. The filter system can be sterilized in situ and placed inside a bag for an unlimited time of operation.
The new technology described above is designed to take advantage of the properties of a flexible bag. By incorporating a bioreactor inside the bag, the technology offers a transportable system that does not require extensive validation when manufacturing sites are changed. Because users can link the units together to produce batches of practically any size, the technology could expand the adoption of single-use systems for the commercial production of biological drugs.
A significant advantage of the new technology developed is its low capital and operational costs. The flexible bags are placed on a heating or cooling platform (see Figure 1). The system monitors the nutrient media for dissolved oxygen, pH, and glucose levels either by remote sensors or by direct sampling. Although other methods, such as fluorescence-based monitoring, are available, Therapeutic Proteins believes that, in the long-term, the wired sensors inside the bags are the most appropriate tools.
The systems described above are routinely used at Therapeutic Proteins's cGMP compliant facility to manufacture large-scale cytokine and monoclonal-antibody production batches. Although the technology requires substantial modification and validation of the process, the systems operate smoothly once these efforts have been completed because they contain few components.
Sarfaraz K. Niazi, PhD, is executive chairman of Therapeutic Proteins, 3440 S. Dearborn St., Chicago, IL 60616, firstname.lastname@example.org.
1. S. Niazi, Disposable Bioprocessing Systems (CRC Press, Boca Raton, FL, 2011).
2. Xcellerex, "XDR Single-Use Bioreactors," (Marlborough, MA), www.xcellerex.com/platform-xdr-single-use-bioreactors.htm, accessed Oct. 4, 2011.
3. GE Healthcare, "WAVE Bioreactor Systems," (Chalfont St Giles, UK), www.gelifesciences.com/aptrix/upp01077.nsf/Content/wave_bioreactor_home, accessed Oct. 4, 2011.
4. R. Temam, Navier-Stokes Equations: Theory and Numerical Analysis (AMS Chelsea Publishing, Providence, RI, 2000).
5. J. Liderfelt, G. Rodrigo, and A. Forss, "The Manugfacture of mABS—A Comparison of Performance and Process Time between Traditional and Ready-to-Use Disposable Systems," in Single-Use Technology in Biopharmaceutical Manufacture, R. Eibl and D. Eibl, Eds. (John Wiley and Sons, Hoboken, NJ, 2011).