TECHNOLOGICAL FIELD

This disclosure provides a general purpose particle settling device with enhanced settling on the multilayered inclined curved surfaces, such as a number of inclined and curved plates attached to vertical cylindrical tubes of different sizes, stacked inside a cyclone. This device has applications in numerous fields, including (i) high cell density biological (mammalian, microbial, plant or algal) cell cultures secreting polypeptides, hormones, proteins or glycoproteins, or other small chemical products, such as ethanol, isobutanol, isoprenoids, etc., (ii) separating and recycling porous or non-porous solid catalyst particles catalyzing chemical reactions in liquid or gas phase surrounding solid particles, (iii) separating and collecting newly formed solids in physical transformations such as crystallization, flocculation, agglomeration, precipitation, etc., from the surround liquid phase and (iv) clarifying process water in large scale municipal or commercial waste water treatment plants by settling and removing complex biological consortia or activated sludge or other solid particles.

DESCRIPTION OF RELATED ART

Of all the above-mentioned fields of a pplication for settling devices, the most modern and challenging field is the production of biological proteins, polypeptides or hormones secreted from suspension cultures of recombinant microbial or mammalian cells. Most common methods of producing biological proteins in recombinant

mammalian and microbial cells rely on fed-batch cultures, wherein cells are grown to high cell densities and then typically exposed to an induction medium or inducer to trigger the production of proteins. If the desired proteins are secreted out of the cells, it is more profitable to switch from a fed-batch culture to a continuous perfusion culture, which can maintain high cell density and high productivity over a much longer duration of culture. During continuous perfusion cultures, live and productive cells are retained or recycled back to the bioreactor while the secreted proteins are continuously harvested from the bioreactor for downstream purification processes.

Some key advantages of continuous perfusion cultures over fed-batch cultures are: (1) the secreted protein products are continuously removed from the bioreactor, without subjecting these products to potential degradation by proteolytic and/or glycolytic enzymes released into the culture medium from dead cells, (2) live and productive cells are retained or recycled back to achieve high cell densities in continuous perfusion bioreactors, where they continue to produce valuable proteins inside the controlled bioreactor environment for much longer culture duration, rather than being removed from the bioreactor at the end of each fed-batch culture, (3) the perfusion bioreactor environment can be maintained at much closer to a steady state (maintaining a constant product quality by design) with fresh nutrient media being continuously fed and waste products removed along with the harvest, unlike the dynamically changing concentrations of nutrients and waste products in a fed-batch culture, and (4) with a subset of cell retention devices, smaller dead or dying cells can be selectively removed from the perfusion bioreactor before these cells lyse and release their intracellular enzymes, thereby maintaining a high viability fraction of cells and high quality of the secreted protein products as they are harvested.

Many cell retention devices have been developed in the mammalian cell culture industry, such as the internal spin filter devices (Himmelfarb et al., Science 164: 555-557, 1969), external filtration modules (Brennan et al., Biotechnol. Techniques, 1 (3): 169-174, 1987), hollow fiber modules (Knazek et al., Science, 178: 65-67, 1972), gravitational settling in a cyclone (Kitano et al., Appli. Microbiol. Biotechnol. 24, 282-286, 1986), inclined settlers (Batt et al., Biotechnology Progress, 6: 458-464, 1990), continuous centrifugation (Johnson et al., Biotechnology Progress, 12, 855-864, 1999), and acoustic filtering (Gorenflo et al., Biotechnology Progress, 19, 30-36, 2003). The cyclones were found to be incapable of producing enough centrifugal force for sufficient cell separation at the device sizes and harvest flow rates used in the mammalian cell culture

experiments (Kitano et al., 1986) and mammalian cells are seriously damaged at higher flow rates (and centrifugal forces) necessary for efficient cell separation (Elsayed, et al., Eng. Life Sci., 6: 347-354, 2006). While most of the other devices adequately retain all mammalian cells from the harvest, these devices are unable to separate dead cells from the live cells desired in the bioreactor. Consequently, dead cells keep accumulating inside the perfusion bioreactor and the membrane filters get clogged, necessitating the termination of the continuous perfusion bioreactor, typically in less than a week. Among all the cell retention devices available today, only the inclined settlers (Batt et al., 1990, and Searles et al., Biotechnology Progress, 10: 198-206, 1994) enable selective removal of smaller dead cells and cell debris in the overflow or harvest stream, while bigger live and productive mammalian cells are continually recycled via the underflow back to the perfusion bioreactor. Therefore, it is feasible to continue the perfusion bioreactor operation indefinitely at high viability and high cell densities while the protein product is continuously harvested from the top of the inclined settler. The inclined settler has previously been scaled up as multi-plate or lamellar settlers

(Probstein, R. F.; Yung, D. US. Patent 4151084, Apr 1979) and used extensively in several large-scale industrial processes such as wastewater treatment, potable water clarification, metal finishing, mining and catalyst recycling (e.g. Odueyngbo et al., US Patent

application 2004/0171702, September 2004).

Citing the present inventor's first demonstration of a single plate inclined settler (Batt et al., 1990) to enhance productivity of secreted proteins in mammalian cell culture applications, a multi-plate or lamellar settler device has been described for the scale up of inclined settlers for use in hybridoma cell culture (Thompson and Wilson, US Patent 5,817,505, October 1998). Such lamellar inclined settler devices have been used to culture recombinant mammalian cells in continuous perfusion bioreactors at high bioreactor productivity (due to high cell density) and high viability (>90%) for long durations (e.g. several months without any need to terminate the perfusion culture). U.S. Patent Publication No. 2011/0097800 to Kauling et al., describes a scaled up version of inclined settlers that uses cylindrical tubes wrapped at inclined angles. The device is described as useful in the culturing of larger mammalian cells, such as CHO, BHK, HEK, HKB, hybridoma cells, ciliates, and insect cells.

None of these cell retention devices have been demonstrated for harvesting secreted protein products in perfusion bioreactor cultures of the smaller and hence more challenging microbial cells. Lamellar settlers have been tested with yeast cells to investigate cell settling with limited success (Bungay and Millspaugh, Biotechnology and Bioengineering, 23:640-641, 1984). Hydrocyclones have been tested in yeast

suspensions mainly to separate the yeast cells from beer, again with limited success

(Yuan et al., Bioseparation, 6: 159-163, 1996, Cilliers and Harrison, Chemical Engineering Journal, 65: 21-26, 1997). SUMMARY

The particle separation devices of the present disclosure have numerous fields of application and provide a large improvement over all of the separation devices listed above.

The settler devices of this disclosure include a cyclone (often referred to as a

"hydrocyclone") housing, a series of concentric cylinders or vertical tubes inside the cyclone housing, joined at their bottom with conical surfaces tapering down to an opening. The inclined settling surfaces are provided by numerous annular strips, or 'ramps', of metal stretched and aligned at an angle between about 30 degrees and about 60 degrees (preferably about 45 degrees) from vertical, and joined to the outer surface of each cylinder or tube. The horizontal spacing between the successive parallel ramps in each annular region between the cylinders can be varied between about 5 mm to about 15 mm.

The inclined settling strips significantly enhance the settling efficiency of the particles from the bulk fluid as the bulk fluid moves upward in the annular settling zones created between the vertical tubes. As the harvest moves up through the annular inclined settling zones, bigger particles (e.g., live and productive cells) settle on the strips, slide down, exit at the outer edges of the strips and fall down vertically into the conical section of the cyclone assembly. These devices can be scaled up or down to suit the separation needs of different industries or applications or sizes as the separation surface is scaled up or down volumetrically in three dimensions, compared to the more typical one- or two-dimensional scaling of previous settling devices.

Scale up of the devices of this disclosure can be performed simply by increasing the diameter of the cyclone housing (and correspondingly increasing the number of cylindrical tubes inside) and/or increasing the height of the cyclone. For example, using a 10-inch (25.4 cm) diameter cylinder, with a spacing of approximately 10 mm between successive ramps, about 80 ramps going up may be welded to the outside of the 10-inch (25.4 cm) diameter cylinder. For a 12-inch (30.5 cm) diameter cylinder, approximately 92 inclined settling ramps can be placed within the cylinder, and so on, in proportion. The effective projected area for cell settling increases proportional to the square of the diameter of cyclone housing and increases proportional to the height of internal cylinders. So the effective settling area of the compact settling device of this disclosure scales up proportional to the cube of cyclone diameter (assuming the height of the internal settler is also increased proportionally) or equivalently, to the volume of cyclone housing. This three dimensional or volumetric scale-up of the effective settling area makes the settling device of this disclosure much more compact compared to previous inclined settler devices.

The radial spacing in the annular regions between different cylinders can be between about 1 cm to about 10 cm, with an optimum around 2.5 cm. A small clearance of about 1 mm between the inclined settling ramps and the internal surface of the next successive cylinder provides useful space for settled particles (for example cells) to slide down the surface of the ramps and exit the ramps on the side, rather than sliding all the way down to the bottom of the ramp. The side-exiting cells settle vertically along the inside of each cylinder. When these settling cells reach the conical surface at the bottom of each cylinder, they slide down on the inclined surface on the cone to the central opening at the bottom of the cyclone housing. An advantage of the increasing fluid velocity while going down the inclined conical surface to the central opening is that the increasing number of settled cells sliding down the cone are swept down to the central opening, rather than being allowed to accumulate by the faster liquid velocities.

The material of construction of this device can be stainless steel alloy 316, or similar materials used for applications in microbial or mammalian cell culture, as well as other metals used for applications in chemical process industries, such as catalyst separation and recycle. Metal settling devices of this disclosure can be constructed by cutting out annular strips from a flat metal sheet, and stretching them in a perpendicular direction to reach an angle between about 30 degrees and about 60 degrees (preferably about 45 degrees) from vertical around an inner cylinder, and welding tabs at the ends of the metal annular strips to the outside of the cylinder.

The material of construction of these devices may include non-metals, including plastics for use in single-use disposable bioreactor bags, etc. A plastic settling device according to this disclosure can be fabricated continuously, as a single piece, using, for example, injection molding or three-dimensional printing technologies.

The angle of inclination for the conical spiral surfaces ('ramps') may be between about 30 degrees to about 60 degrees from the vertical. For use with stickier particles (typically mammalian cells), the angle of inclination may be closer to the vertical (i.e., around 30 degrees from vertical). The stickiness of such cells may be reduced by coating surfaces of the device with non-sticky plastic, teflon or silicone. For use with non-sticky solid catalyst particles, the angle of inclination can be further from vertical (for example, around 60 degrees from vertical).

All or some of the surfaces of these settling devices may be completely or partially coated with a non-sticky plastic or silicone. Additionally or alternatively, the metals (especially stainless steel) may be electropolished to provide a smooth surface.

The thickness of the cylindrical tubes and annular strips is preferably as thin as necessary to maintain the rigidity of shape and to minimize the weight of the concentric cylindrical settler assembly to be supported inside the cyclone housing. The radius and height of this device can be scaled up independently as much as needed for the large- scale processes as calculated from predictive equations provided for inclined settlers (Batt et al. 1990).

The major factor causing the particle separation is the enhanced sedimentation on the inclined surfaces, which has been successfully demonstrated by Boycott (Nature, 104: 532, 1920) with blood cells and Batt et al. (1990) with hybridoma cells producing monoclonal antibodies. Additional factors enhancing the cell/particle separation are the centrifugal force on the cells/particles during their travel up the annular regions between successive cylinders and the settling due to gravity in the vertical sedimentation columns of the spiral channel.

Thus, a particle settling device of this disclosure may include a cyclone housing and at least one vertical tube disposed inside the cyclone housing, the at least one vertical tube joined at one end with a conical surface tapering down to a first opening in the cyclone housing. At least one annular strip is attached to a vertical surface of the at least one vertical tube at an angle between about 30 degrees and about 60 degrees from vertical. There is at least one additional opening in the cyclone housing substantially opposite the first opening. The vertical tubes may include at least one material selected from the group consisting of a metal and a plastic. The vertical tubes may be stainless steel, and may be composed entirely of stainless steel. The vertical tubes, the annular strip, and the conical surfaces may all be metals joined by welding. Alternatively, the tubes may be composed entirely of plastic. At least one surface of the cyclone housing, the at least one vertical tube, the annular strip, and the spiral conical surface is coated with a non-sticky plastic or silicone.

The angle of inclination for the conical surfaces is about 45 degrees from vertical, or about 30 degrees from vertical, or about 60 degrees from vertical.

The width of an annular ringed channel formed between adjacent vertical tubes is between about 1 mm and about 50 mm. The number of vertical tubes within the settler device may be between about 2 and about 30.

The settler device may include a closure over at least a portion of the cyclone housing at an end of the cyclone housing opposite the first opening. At least one additional opening in the cyclone housing, may be configured to open from a side of the cyclone housing tangential to at least one vertical tube, in liquid communication with the outside and the inside of the cyclone housing.

A liquid harvest outlet may be formed in the closure, in liquid communication with the outside and the inside of the cyclone housing.

The annular strip is attached to the at least one vertical tube in a spiral that rises at an angle of about 45 degrees to vertical from an end of the tube adjacent the first opening spiraling around the at least one vertical tube up to the opposite end of the at least one vertical tube. The annular strip may be attached to the at least one vertical tube and may be of sufficient width to leave a gap of between about 0.5mm and about 10mm between an edge of the annular strip and the cyclone housing or an adjacent vertical tube.

Methods of settling particles in a suspension may therefore include introducing a liquid suspension of particles into a particle settling device of this disclosure, collecting particles from a first opening in the settler device, and collecting a liquid from another opening in the settling device. In these methods, the liquid suspension may include at least one of a recombinant cell suspension, an alcoholic fermentation, a suspension of solid catalyst particles, a municipal waste water, and industrial waste water. The liquid suspension may include at least one of mammalian cells, bacterial cells, yeast cells, plant cells. The liquid suspension may include at least one of biodiesel-producing algae cells, mammalian and/or murine hybridoma cells, and yeast in beer. The liquid suspension may include recombinant microbial cells selected from Pichia pastoris, Saccharomyces cerevisiae, Kluyveromyces lactis, Aspergillus niger, Escherichia coli, Bacillus subtilis. The step of introducing a liquid suspension may include directing a liquid suspension from a plastic disposable bioreactor bag into the particle settling device. The liquid collected may include at least one of biological molecules, organic or inorganic compounds, chemical reactants, and chemical reaction products. The liquid collected may include at least one of hydrocarbons, polypeptides, proteins, alcohols, fatty acids, hormones, carbohydrates, antibodies, terpenes, isoprenoids, polyprenoids, and beer. The liquid collected may include at least one of biodiesel, insulin or its analogs, brazzein, antibodies, growth factors, colony stimulating factors, and erythropoietin (EPO).

BRIEF DESCRIPTION OF FIGURES

Figure 1 shows a sectional view through a concentric cylindrical inclined settler device of this disclosure, including a vertical sight glass on the outer surface to show the inclined settling ramps in the outermost annular region.

Figure 2 shows a top view through a settler device of this disclosure, showing numerous inclined settling ramps welded to inner cylinders in the annular regions.

Figure 3 shows a typical schematic of the attachment of a compact cell/particle settler of this disclosure to any modular bioreactor.

Figure 4 shows results obtained from a perfusion bioreactor attached to a particle settling device of this disclosure, set up as depicted in Figure 3.

Figure 5 shows particle size analysis of samples taken from the bioreactor and settler effluent from the apparatus set up as depicted in Figure 3.

Figure 6 shows centrifuge vials containing samples of effluent from the settler device (tube labeled 'D'), and from within the bioreactor (tube labeled 'C'), and, following centrifugation, cell pellets from effluent from the settler device (tube labeled 'B'), and cells pelleted from within the bioreactor (tube labeled Ά').

DESCRIPTION OF EMBODIMENTS

The term "a" or "an" entity refers to one or more of that entity. As such, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably.

The phrases "at least one," "one or more," and "and/or" are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions "at least one of A, B and C", "at least one of A, B, or C", "one or more of A, B, and C", "one or more of A, B, or C" and "A, B, and/or C" means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.

The transitional term "comprising" is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

The transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim, but does not exclude additional components or steps that are unrelated to the invention such as impurities ordinarily associated therewith.

The transitional phrase "consisting essentially of" limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

Referring to Figure 1, a sectional view of a concentric cylindrical inclined settler device of this disclosure includes an outer wall (1) of cylindrical section of the cyclone assembly, shown in Figure 1 with an optional cooling water jacket (12), a conical portion (2) of the cyclone assembly, with the optional cooling water jacket (12) extending to this conical portion (2), a lid (3) on top of the assembly, a tangential port inlet (4) for a liquid (for example, a cell culture), entering near the top of conical portion (2), through the optional cooling water jacket (12), a bottom outlet port (5) for returning concentrated liquid (for example, a concentrated cell culture containing settled cells to a bioreactor), and a top outlet port (6) for harvesting the clarified liquid (for example, culture liquid containing very few cells, which are mostly smaller dead cells and cell debris). Concentric cylindrical tubes (7) are located within the outer wall (1). Annular strips (not shown) are attached to the concentric cylindrical tubes (7) at an angle between about 30 degrees to about 60 degrees (preferably about 45 degrees) from vertical. The annular strips are attached to the inner cylinder, but NOT to the outer cylinder. Concentric cones (8) channel settled particles (for example, cells) to the bottom outlet port (5). A cooling water inlet (9) enters the optional water jacket (12) on the outside of the cyclone assembly, and a cooling water outlet (10) exits the optional water jacket (12) near the top of the cylindrical section (1) of the cyclone assembly. As depicted in Figure 1, an optional sight glass (11) is provided showing the inclined settler strips attached to the inside cylinder in the outermost annular region between the cylindrical tubes. As noted above, annular strips are NOT attached to the outer cylinder, intentionally leaving a small (approximately 0.5mm - 10mm) gap between the strips and the outer cylinder, thereby allowing the settled particles to fall down through this gap.

As depicted in Figure 1, the settler devices of this disclosure may include a closure or lid over at least a portion of the settler device an end of the settler device opposite the bottom outlet port. The closure or lid may also include an outlet or port for removing liquids or entering liquids into the settler device. The opening and the additional ports or outlets in the lid are in liquid communication with the outside and the inside of the settler device to allow the passage of liquids into and/or out of the settler device, and in each instance of such opening or inlet/outlet, these passage ways into and out of the cyclone housing may include valves or other mechanisms that can be opened or closed to stop or restrict the flow of liquids into or out of the settler devices of this disclosure. The lid covering the settler device may be concave, rising to a central core point. The angle of rise in the concave top plate may preferably be between 1 degree and 10 degrees, more preferably between 1 degree and 5 degrees. Such concave top plate creates a tent-like space above the center of the settler device. Gas, bubbles, froth or the like may accumulate in this space and a tube may be inserted through an opening in the settler device or through an opening in the top plate to withdraw such gasses, etc. from the space beneath the top of the settler device. Similarly, fluid or gas may be pumped into the settler device through such tube that is inserted through an opening in the settler device or through an opening in the lid.

As depicted in Figure 2, a top view of the concentric cylindrical inclined settler device of this disclosure shows numerous annular strips attached to the outside of each cylinder. The strips may be attached to the vertical cylinders at an angle between about 30 degrees to about 60 degrees to the vertical (typically at an angle of about 45 degrees to the vertical). As shown in Figure 2, small (approximately 1mm) spacings (13) are provided between each inclined settler strip and the next successive outer cylinder of each annual region, to allow the settled particles to fall down along the outer cylindrical wall onto the concentric cones in the bottom section of the assembly.

These settler devices may include a means to control the temperature of the settler device, such as reservoir for cooling or heating fluids to be circulated around all or part of the outer wall of the settler device. Ports may be inlet or outlet ports for the circulation of heating or cooling fluids through the reservoir. A lid is optionally attached to the top of the settler device by one or more screws, and may be secured in place over the settler device over an o-ring.

Methods of Use and Operation of Processes

Referring now to the settling device depicted in Figures 1 and 2 of this disclosure, exemplary methods of using the settling devices are described.

A particle containing liquid (including, for example, cell culture liquid, waste water or reaction fluid containing solid catalyst particles, etc.) is introduced tangentially into a device of this disclosure though the port (4) near the top of the conical section of cyclone housing assembly. Approximately 50% - 99% of the entering liquid (typically about 90%) is removed through the bottom port (5), while the remaining 1% - 50% (typically about 10%) of the liquid is removed through the top port (6). A pump (such as a peristaltic pump) may be used to suck liquid out of this top port (6), while the concentrated liquid exiting the bottom may be allowed to exit the bottom outlet (5) of the cyclone housing due to gravity, without the need for a pump. Most of the entering cells (or particles) are pushed against the conical walls of this assembly (8) through centrifugal forces upon entry, settle down the conical portion through a gentle vortex motion initially, getting faster as the liquid and particles/cells go down and exit via the bottom port. The rest of the cells, which have not settled, will move up through the annual regions in between the numerous inclined settling strips attached to the inside cylinder. As the liquid moves slowly up the annular inclined channels, bigger particles (e.g., live cells) will settle on the ramp and either slide down the ramp or more likely fall down the small (approximately lmm) spacing provided between the ramps and the outer walls of each annular region. These settled particles fall down vertically along the outer cylindrical walls until they reach the bottom conical section of the assembly and proceed to slide down the conical section to the bottom port (5).

By increasing the liquid flow rate through top port (6), it is possible to reduce the residence time of liquid inside the inclined settling zones such that smaller particles (for example dead cells and cellular debris) will not have settled by the time the liquid reaches the top of the settling zone, and therefore these smaller particles exit the settling device via the top port (6). This feature provides a simple method to remove smaller particles (such as dead cells and cellular debris) selectively via the top port (6) into a harvest stream, while larger particles (such as live and productive cells) are returned from the bottom port (5) to another vessel (such as a bioreactor).

Thus, in these methods, the step of introducing a liquid suspension into the settler device includes directing a liquid suspension from a plastic bioreactor bag into the particle settling device.

Liquid may be directed into, or drawn out of, any of the ports or openings in the settling device by one or more pumps (for example a peristaltic pump) in liquid

communication with the port or opening. Such pumps, or other means causing the liquid to flow into or out of the settler devices, may operate continuously or intermittently. If operated intermittently, during the period when the pump is off, settling of particles or cells occurs while the surrounding fluid is still. This allows those particles or cells that have already settled to slide down the inclined conical surfaces unhindered by the upward flow of liquid. Intermittent operation has the advantage that it can improve the speed at which the cells slide downwardly, thereby improving cell viability and

productivity. In a specific embodiment, a pump is used to direct a liquid suspension of cells from a bioreactor or fermentation media into the settler devices of the present disclosure.

In certain embodiments of these methods, the clarified liquid collected from the settler device includes at least one of biological molecules, organic or inorganic compounds, chemical reactants, and chemical reaction products. In certain embodiments of these methods, the clarified liquid collected from the settler device includes at least one of hydrocarbons, polypeptides, proteins, alcohols, fatty acids, hormones,

carbohydrates, antibodies, isoprenoids, biodiesel, and beer. In certain embodiments of these methods, the clarified liquid collected from the settler device includes at least one of insulin or its analogs, monoclonal antibodies, growth factors, sub-unit vaccines, viruses, virus-like particles, colony stimulating factors and erythropoietin (EPO).

Each publication or patent cited herein is incorporated herein by reference in its entirety. The invention now being generally described will be more readily understood by reference to the following examples, which are included merely for the purposes of illustration of certain aspects of the settler devices of this disclosure. The examples are not intended to limit the disclosure, as one of skill in the art would recognize from the above teachings and the following examples that other techniques and methods can satisfy the claims and can be employed without departing from the scope of the claims.

EXAMPLES

Example 1: Yeast or other microbial cells secreting protein products

Recombinant microbial cells, such as yeast or fungal (Pichia pastoris,

Saccharomyces cerevisiae, Kluyveromyces lactis, Aspergillus niger, etc.) or bacterial (Escherichia coli, Bacillus subtilis, etc.) cells, which have been engineered to secrete heterologous proteins (for example, insulin or brazzein) or naturally secreting enzymes (e.g. A. niger, B. subtilis, etc.) can be grown in bioreactors attached to a conical spiral settler device of this disclosure, to recycle live and productive cells back to the bioreactor, which will thereby achieve high cell densities and high productivities. Fresh nutrient media is continuously supplied to the live and productive cells inside the high cell density bioreactors and the secreted proteins or enzymes are continuously harvested in the clarified outlet from the top port, while the concentrated live and productive cells are returned back to the bioreactor. As dead cells and a small fraction of live cells are continuously removed from the bioreactor via the harvest outlet, cell growth and protein production can be maintained indefinitely, without any real need for terminating the bioreactor operation. I n operations using yeast Pichia cells with the conical settler devices of this disclosure, the perfusion bioreactor has been operated for over a month. As the microbial cells grow in suspension culture and the cell retention device can be scaled up to any desired size, this disclosure can be attached to suspension bioreactors of sizes varying from lab scale (<1 liter) to industrial scale (>50,000 liters) to achieve high cell density perfusion cultures.

Example 2: Removing yeast cells from beer

I n large-scale brewing operations, yeast cells are removed from the product beer by filtration devices, which regularly get clogged, or centrifugation devices, which are expensive high-speed mechanical devices. Previously, hydrocyclones were unsuccessfully tested for this application (Yuan et al., 1996; Cilliers and Harrison, 1997). These devices can be readily replaced by the settler devices of this disclosure to clarify beer from the top outlets and remove the concentrated yeast cell suspension from the bottom outlet. Due to the increased residence time in the spiral channels and enhanced sedimentation in the conical spiral settler zone of this disclosure, the inventor has achieved successful separation of yeast cells from cell culture liquid, harvesting the culture supernatant containing only about 5% of the cells entering the settler device in its first operation. As the device can be scaled up or down to increase or decrease its cell separation efficiency, it is feasible to obtain completely cell-free beer from the harvest port, if desired. Thus, the devices of this disclosure may be particularly useful in brewing beer, as well as clarifying beer, and in continuous brewing arrangements.

Example 3: Mammalian cell perfusion cultures

Enhanced sedimentation of recombinant mammalian cells and murine hybridoma cells in inclined settlers have already been demonstrated successfully (Batt et al., 1990 and Searles et al., 1994) and scaled up in lamellar settlers (Thompson and Wilson, US Patent No. 5,817,505, 1998). While the lamellar settlers are scaled up in three

dimensions independently, a conical spiral settler device of this disclosure can be scaled up in three dimensions simultaneously by simply increasing its radius, as discussed above. Further, the settlers of this disclosure benefit from an additional cell separating mechanism of increasing centrifugal forces as the cell culture liquid passes through decreasing radius of the vertical spiral section, followed by the proven enhanced sedimentation in the conical spiral settling zone. Thus, the settlers of this disclosure are more compact and more easily scalable cell retention devices with proven applications in mammalian cell cultures secreting glycoproteins, such as monoclonal antibodies (that may be captured on protein A linked to a resin or bead support, or antibody precipitates), and other therapeutic proteins. The clarified harvest output from the top port containing the secreted protein is harvested continuously from the cell retention device, while the concentrated cells from the bottom outlet are recycled back to the bioreactor, resulting in a high cell density perfusion bioreactor, that can be operated indefinitely, (i.e. over several months of continuous perfusion operation). The continuous high titer harvest from a single, 1000-liter, high cell density perfusion bioreactor can be more than the accumulated production from a large (>20,000 liter) fed-batch bioreactor on an annual basis.

Example 4: Solid catalyst particle separation and recycle

Separation of a solid catalyst particle for recycle into the reactor and reuse in further catalyzing liquid phase chemical reactions, such as Fischer-Tropsch synthesis, has been demonstrated before with lamellar settlers (US Patent No. 6,720,358, 2001). Many such two-phase chemical reactions, involving solid catalyst particles in liquid or gas phase reactions can be enhanced by the particle settling devices of this disclosure, which presents a more compact particle separation device to accomplish the same solids separation and recycle as demonstrated with lamellar settlers.

Example 5: Plant and algal cell harvesting

Recombinant plant cell cultures secreting valuable products, while not yet commercially viable, are yet another field of potential applications for the settling devices of this disclosure. Inclined settlers have been used in several plant cell culture applications. Such devices can be replaced by the more compact conical spiral settler devices of this disclosure. With the size of plant cells being much higher than those of yeast or mammalian cells, the cell separation efficiency will be much higher with single plant cells or plant tissue cultures.

A more immediate commercial application of the settler devices of this disclosure may be in the harvesting of algal cells from large scale cultivation ponds to harvest biodiesel products from inside algal cells. Relatively dilute algal cell mass in large (acre sized) shallow ponds converting solar energy into intracellular fat or fatty acid storage can be harvested easily through the conical spiral settler device of this disclosure, and the concentrated algal cells can be harvested from the bottom outlet.

Example 6: Municipal waste water treatment

Large scale municipal waste water treatment plants (using activated sludge or consortia of multiple bacterial species for degradation of biological and organic waste in sewage or waste water) commonly use large settling tanks and more modern versions of these plants use lamellar settlers to remove the clarified water from the sludge. The conical spiral settler devices of this disclosure can be scaled up to the larger sizes required in these plants, while remaining smaller in size than the large settling tanks or lamellar settlers currently used in these treatment plants.

Example 7: Industrial process water clarification

Large scale water treatment plants, cleaning either industrial waste water or natural sources of turbid water containing suspended solids, use large scale settling tanks or lamellar inclined settlers. These large scale devices can now be replaced with the more compact conical spiral settler devices of this disclosure to accomplish the same goal of clarifying water for industrial reuse or municipal supply of fresh water. Example 8: Experimental results from perfusion bioreactor culture of yeast Pichia pastoris cells

Yeast Pichia pastoris cells were grown in a 5-liter, computer-controlled bioreactor, initially in batch mode to grow the cells from the inoculum for the first 50 hours, then in fed-batch mode to fill up the attached 12-liter cell settler slowly for the next 100 hours, and then in continuous perfusion mode with a compact cell settler of this disclosure to remove the smaller dead cells and recycle the larger live cells back into the bioreactor. A typical schematic of the attachment of a compact cell/particle settler of this disclosure to any modular bioreactor is shown in Figure 3.

Referring to Figure 3, the yeast Pichia pastoris cells were grown in a perfusion bioreactor (38). Growth media was added to the bioreactor (38) from media reservoir (20) via pump (22). Dissolved oxygen content and pH were continuously monitored in the bioreactor (38) by dissolved oxygen monitor (26) and pH monitor (24). Yeast cell culture from the bioreactor (38) was delivered (32) to the 12-liter compact cell settler (28) via pump (34). Effluent (30) from the compact cell settler (28) contained smaller dead cells, while larger live cells were recycled back to the bioreactor (38) via pump (36). Media and cell culture levels in the bioreactor (38) were controlled by removing excess cell culture (42) via pump (40) to be captured or discarded.

Results obtained with this perfusion bioreactor set up with a compact cell/particle settler of this disclosure are shown in Figure 4. The diamonds show the optical density of bioreactor samples, measured at 600 nm, building up during the initial batch and fed- batch culture period of about 150 hours, followed by a slower increase during about 600 hours of continuous perfusion operation. These results show that as the cell settler is gradually filled up during the fed-batch mode (for about 100 hours), along with continuous partial recycle of concentrated cells from the settler to the bioreactor, the bioreactor achieves a very high cell density of over 800 OD. At about 150 hours, the cell settler is filled up and the clarified culture harvest emerges from the top of the settler at a rate equivalent to the difference in pumped inlet rate from the bioreactor and the pumped bottom recycle from the settler to the bioreactor. The settler effluent or harvest rate is adjusted by manipulating either settler inlet pump setting and/or settler recycle pump setting. The cell concentration (as measured by OD at 600 nm) and the size distribution are determined by the harvest flow rate and cell size distribution of the cells entering from the bioreactor and other factors such as the recycle ratio from the settler. As the bioreactor OD fell during the initial perfusion rate setting of above 2000 ml/day, the two pumps were manipulated to reduce the harvest or settler effluent rate to about 1000 ml/day. At this lower perfusion flow rate maintained approximately over the next 500 hours, the bioreactor OD gradually increased to over 900 OD and the settler effluent OD stabilized to around 280 OD. These results demonstrate that very high cell density was obtained and maintained in the bioreactor to the recycle of most of the live cells back to the bioreactor and selective removal of smaller dead cells and cell debris. At this lower perfusion rate, the bioreactor can be operated indefinitely at high cell density without any reason to terminate the bioreactor, such as clogged membranes in competing membrane based cell retention devices.

Samples from the bioreactor and settler effluent taken at the same time point were analyzed with a particle size analyzer. The normalized cell size distribution results shown in Figure 5 clearly indicate that the settler effluent contains a significantly smaller cell size distribution compared to that found for the cells in the bioreactor. These results demonstrate that the settler removed the smaller dead cells and any cell debris preferentially in the effluent, while the larger live cells are preferentially returned to the bioreactor. Thus, the bioreactor is continuously cleaned by selective removal of dead cells and cell debris by the settler effluent and consequently there is no accumulation of dead cells and cell debris within the bioreactor, as happens routinely with all other cell retention devices.

The bioreactor and settler effluent samples from an early time point during the perfusion culture were collected and centrifuged in small 2 ml vials. Figure 6 shows centrifuge vials containing samples of effluent from the settler device (tube labeled 'D') and from within the bioreactor (tube labeled 'C') and the cell pellets following centrifugation: cells pelleted from effluent from the settler device (tube labeled 'B') and cells pelleted from within the bioreactor (tube labeled Ά'). The pelleted cells from the bioreactor occupying almost 50% of the wet packed cell volume in the vial, while the pelleted cells in the settler effluent occupy only about 5% of the wet packed cell volume. These results again confirm that only a very small fraction of the intact smaller cells from the bioreactor are removed in settler effluent while most of the larger intact cells are preferentially returned to the bioreactor.

The foregoing examples of the present disclosure have been presented for purposes of illustration and description. These examples are not intended to limit the disclosure to the form disclosed herein. Consequently, variations and modifications commensurate with the teachings of the description of the disclosure, and the skill or knowledge of the relevant art, are within the scope of the present disclosure. The specific embodiments described in the examples provided herein are intended to further explain the best mode known for practicing the disclosure and to enable others skilled in the art to utilize the disclosure in such, or other, embodiments and with various modifications required by the particular applications or uses of the present disclosure. It is intended that the appended claims be construed to include alternative embodiments to the extent permitted by the prior art.