Hollow fiber bioreactors have been in use for more than 30 years for the production of secreted proteins and antibodies from mammalian cells. Advances in fiber construction and methods for oxygenation have the potential to bring the advantages of hollow fiber cell culture to biomanufacturing.
In order to more closely approximate in vivo cell growth conditions, Richard Knazek developed the hollow fiber bioreactor (HFBR) in 1972 (1). The HFBR system is a high-density, continuous-perfusion culture system. It is a well-established system for the production of secreted proteins from hybridoma, Chinese hamster ovary cells (CHO), human embryonic kidney (HEK) 293 and other mammalian and insect cells. The primary advantage is that it is possible to culture large numbers of cells at high density, resulting in product accumulating to very high concentrations. Up until now, the application of HFBR technology has been limited to laboratory scale applications. HFBRs provide some features that are advantageous in biopharmaceutical manufacturing, such as continuous, modular, flexible manufacturing formats employing compact and disposable systems. There is also growing interest in perfusion-based bioproduction protocols (2). FiberCell Systems has developed a large-scale HFBR system that brings the benefits of hollow fiber cell culture to the bio-production arena (see Figure 1).
Figure 1: Cells growing at high density in Fibercell Systems hollow-fiber bioreactor (HFBR).
HFBRs consist of thousands of semi-permeable hollow fibers in a parallel array within a tubular housing or cartridge, fitted with inlet and outlet ports. These fiber bundles are potted at each end so that any liquid entering the ends of the cartridge will necessarily flow through the interior of the fibers. Cells are generally seeded within the cartridge, but outside of the hollow fibers in what is referred to as the extra capillary space (ECS) (see Figure 2). Culture media is pumped inside the hollow fibers, allowing dissolved gas, nutrients, and waste products to diffuse across the fiber walls. Once having passed through the cartridge, the culture medium is oxygenated and returned to the cartridge.
Figure 2: Schematic of the growing conditions within a HFBR.
Unlike current popular batch-style manufacturing formats, including such disposable systems as rocked bags and bags supported by stainless steel containers, HFBRs do not subject cells to significant variations in primary and secondary metabolites (such as glucose and lactate), oxygen, or product levels. A HFBR significantly cuts the operating footprint and reduces the product harvest volume by factor of 10 to 100 times. Furthermore, it avoids such scale-determined discrepancies as impeller and sparge-induced hydrodynamic forces, and reactor column height-induced differential gas solubility. The high cell density and low-sheer environment permits the use of simplified serum-free, protein-free media that can be less expensive and that does not require such components as synthetic membrane protective surfactants or antifoams. Attached cultures are supported, therefore no lengthy adaptation of cells to suspension growth is required. The 10–100 times more concentrated secreted product, the absence of high molecular weight medium components, and the greatly reduced levels of host-cell proteins and DNA facilitate downstream processing, a major cost in bioproduction.
SCALING UP A HBFR
Bringing the advantages of HFBRs to large-scale bio-manufacturing has long been a goal for those familiar with hollow fiber systems. The operational scale for HFBR systems has been limited by several factors:
- Solubility of gas in cell culture medium decreases as the temperature is increased to 37° C.
- It is difficult to design a pump that will provide 6–10 L/min flow rate without denaturing biological components or generating significant backpressure.
- The gross filtration rate of cellulosic hollow fibers (i.e., the rate of nutrient and waste product exchange) have typically been too low to effectively support the culture of adherent cell lines such as CHO and HEK293.
- Fiber geometry is such that the fibers were not uniformly distributed within the cartridge housing, resulting in areas where a large inter-fiber distance led to necrotic areas within the cartridge.
FiberCell Systems has developed a unique approach to address these issues. Instead of using pumps and systems to perform gas exchange with the cell culture medium, a gravity-based system for medium flow that also directly oxygenates the bioreactor cartridge from the inside out has been developed (see Figure 3). A new generation of high flux fibers is employed, and they are also cast with "waves" in them to evenly distribute the fiber bundle within the hollow fiber cartridge. With a cartridge volume of 1 L, this fully disposable system has the potential to replace up to 10,000 L stirred tank reactors for mammalian protein production over a 100–day period of culture. This large-scale system offers all of the advantages outlined above, along with others that are specific to bio-production in a disposable system.
Figure 3: Fibercell Systems prototype large-scale HFBR.