System and method for cultured cell meat production

Abstract

A system for processing growth medium and a method for processing growth medium are disclosed. The system includes a vessel, a first separation device constructed to receive fluid flow from the vessel, a second separation device constmcted to receive the liquid from the first separation device, and a third separation device constructed to receive fluid flow from the permeate side of the second separation device and to separate one or more small molecules or ions from the fluid flow. The method includes culturing cells in a mixture of growth medium and cultured cells; separating a portion of the mixture into cultured cells and spent medium in a first separation device; filtering the spent medium in a second separation device having a molecular weight cut-off of 1 to 20 kDa; and selectively separating small molecule waste products from the permeate in a third separation device, forming cleaned medium.

Description

[0001] SYSTEM AND METHOD FOR CULTURED CELL MEAT PRODUCTION

[0002] Cross Reference to Related Application

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 607,708, filed December 8, 2023, which is incorporated herein by reference in its entirety.

[0004] Field

[0005] The present disclosure relates to a system for processing growth medium. The present disclosure also relates to a method for processing growth medium. The present disclosure further relates to systems and method for processing growth medium from cultured meat production.

[0006] Introduction

[0007] The application of cell cultured meats has become an increasingly attractive market. The field of cell cultured meat products has the potential to mitigate the large environmental impacts of traditional meat production. A current limitation to success and competitiveness in the application of cell cultured meat products is the cell growth medium used in the cultured meat cell proliferation step. This ingredient is a major contributor to the overall and total cost of the cell cultured meat product. Improvements to this process regarding the volume required of cell growth medium are desired. It would be desirable to reuse at least some components of used growth medium. It would further be desirable to remove certain waste products from used growth medium before reuse. Further improvements that allow lowering the cost of supplying cell growth medium are desired.

[0008] Summary

[0009] The present disclosure describes a system for processing growth medium and a method for processing growth medium.

[0010] In one aspect, the present disclosure describes a system for processing growth medium. The system includes a vessel comprising a fluid and cultured cells; a first separation device constructed to receive fluid flow from the vessel; a second separation device constructed to receive the liquid from the first separation device; and a third separation device includes a selective separator constructed to receive fluid flow from the permeate side of the second separation device and to separate one or more small molecules or ions from the fluid flow. The first separation device is constructed to separate cultured cells from a liquid. The second separation device has a retentate side and a permeate side and having a molecular weight cut-off in a range of 1 kDa to 20 kDa.

[0011] In another aspect, the present disclosure describes a method for processing growth medium. The method includes culturing cells in a mixture of growth medium and cultured cells; separating a portion of the mixture into cultured cells and spent medium in a first separation device; filtering the spent medium in a second separation device and forming a permeate and a retentate comprising proteins, the second filtration device having a molecular weight cut-off in a range of 1 kDa to 20 kDa; and selectively separating small molecule waste products from the permeate in a third separation device, forming a waste stream and a cleaned medium.

[0012] Reference throughout this specification to “one aspect,” “an aspect,” “aspects,” “one embodiment,” “an embodiment,” “certain embodiments,” or “some embodiments,” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment or aspect is included in at least one embodiment or aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment or aspect of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments or aspects.

[0013] The above summary of the present disclosure is not intended to describe each disclosed embodiment or every implementation of the present disclosure. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the disclosure, guidance is provided through lists of examples, which examples may be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive or exhaustive list. Thus, the scope of the present disclosure should not be limited to the specific illustrative structures described herein, but rather extends at least to the structures described by the language of the claims, and the equivalents of those structures. Any of the elements that are positively recited in this specification as alternatives may be explicitly included in the claims or excluded from the claims, in any combination as desired. Although various theories and possible mechanisms may have been discussed herein, in no event should such discussions serve to limit the claimable subject matter.

[0014] The complete disclosure of all patents, patent applications, and publications, and electronically available material cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the present disclosure and the disclosure(s) of any document incorporated herein by reference, the present disclosure shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

[0015] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0016] Brief Description of Figures

[0017] FIG. 1 is schematic diagram of the system for processing growth medium according to an embodiment.

[0018] FIG. 2 is schematic diagram of the system for processing growth medium according to an embodiment.

[0019] FIG. 3 is a graphical representation of the results of Example 1.

[0020] Definitions

[0021] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0022] The term “substantially” as used here has the same meaning as “significantly,” and can be understood to modify the term that follows by at least about 90 %, at least about 95 %, or at least about 98 %. The term “not substantially” as used here has the same meaning as “not significantly,” and can be understood to have the inverse meaning of “substantially,” i.e., modifying the term that follows by not more than 25 %, not more than 10 %, not more than 5 %, or not more than 2 %.

[0023] The term “about” is used here in conjunction with numeric values to include normal variations in measurements as expected by persons skilled in the art, and is understood to have the same meaning as “approximately” and to cover a typical margin of error, such as ±5 % of the stated value.

[0024] Terms such as “a,” “an,” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration.

[0025] The terms “a,” “an,” and “the” are used interchangeably with the term “at least one.” The phrases “at least one of’ and “comprises at least one of’ followed by a list refers to any one of the items in the list and any combination of two or more items in the list.

[0026] As used here, the term “or” is generally employed in its usual sense including “and / or” unless the content clearly dictates otherwise. The term “and / or” means one or all of the listed elements or a combination of any two or more of the listed elements.

[0027] The recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc. or 10 or less includes 10, 9.4, 7.6, 5, 4.3, 2.9, 1.62, 0.3, etc.). Where a range of values is “up to” or “at least” a particular value, that value is included within the range.

[0028] As used here, “have,” “having,” “include,” “including,” “comprise,” “comprising,” or the like are used in their open-ended sense, and generally mean “including, but not limited to.” It will be understood that “consisting essentially of,” “consisting of,” and the like are subsumed in “comprising” and the like. As used herein, “consisting essentially of,” as it relates to a composition, product, method, or the like, means that the components of the composition, product, method, or the like are limited to the enumerated components and any other components that do not materially affect the basic and novel character! stic(s) of the composition, product, method, or the like. The words “preferred” and “preferably” refer to embodiments that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the disclosure, including the claims.

[0029] Any direction referred to here, such as “top,” “bottom,” “left,” “right,” “upper,” “lower,” and other directions and orientations are described herein for clarity in reference to the figures and are not to be limiting of an actual device or system or use of the device or system. Devices or systems as described herein may be used in a number of directions and orientations.

[0030] Detailed Description

[0031] The present disclosure relates to a system for processing growth medium. The present disclosure also relates to a method for processing growth medium. The present disclosure further relates to systems and method for processing growth medium from cultured meat production.

[0032] During cell culturing, as the cells proliferate, they use nutrients in the growth medium, and produce waste products. Thus, during culturing, the nutrients are depleted and waste products accumulate in the growth medium. Waste products can include, for example, debris, and compounds such as ammonia, acetate, lactate, growth inhibition factors, and excess salts or ions. The accumulating waste products inhibit and eventually stop cell proliferation. To maintain the ability of the cells to proliferate, at least some of the waste products are removed and fresh growth medium is added. However, in current systems, removal of the waste products also results in removal of unused nutrients from the system. This adds expense to the production of cells. Estimates of the cost attributable to growth medium range from 40 % to 80 % of the total cost of cultured cell meats. Due to the high cost of growth medium, it would be desirable to recycle the unused nutrients back into the system while removing the waste products that inhibit cell proliferation.

[0033] It would be beneficial to lower the cost attributed to cell growth medium used in the cell proliferation. It would therefore be desirable to provide a system capable of removing waste products from the used growth medium, enabling the reuse of at least some of the medium. The selective removal of waste products would lessen the need to use a high volume of growth medium in the process. It follows, then, that a decrease in the amount of growth medium needed would lessen the cost of overall production of cultured cells or cultured meat.

[0034] The system of the present disclosure selectively filters and removes at least some of the waste products in used growth medium, while retaining and recycling key nutrients for further cell proliferation. The system of the present disclosure allows for the reuse of at least some of the cell growth medium in the cultured meat production process. The system of the present disclosure thereby enables lowering the cost of overall production of cultured meat due to a lower volume of cell growth medium consumption during the proliferation process.

[0035] According to an embodiment, a system for processing growth medium includes a vessel containing a fluid and cultured cells. The vessel may be a bioreactor, a tank, or a holding vessel.

[0036] According to an embodiment, the system for processing growth medium includes a bioreactor. The bioreactor is capable of maintaining an environment suitable for cell culturation. Bioreactors typically include a vessel and means for maintaining a desired cell growth environment within the vessel. For example, bioreactors typically include means for mixing, maintaining a desired temperature, maintaining a desired pH, a source of gas (e.g., oxygen, carbon dioxide, air, and the like), and a source of nutrients. A bioreactor may be used to culture cells in cell growth medium. Growth medium is typically an aqueous solution of amino acids, vitamins, inorganic acids, salts (e.g., salts of sodium, potassium, calcium, and the like), sugars (e.g., glucose, galactose, hexose, fructose, and the like), other sources of carbon (e.g., glycerol, pyruvate, glutamine, etc.), growth factors, hormones, etc. Here, these compounds are collectively referred to as nutrients. Growth medium is typically buffered. Examples of buffered media include phosphate-buffered saline (PBS), tri s(hydroxymethyl)aminom ethane (TRIS), phosphate citrate buffer, Sorensen’s phosphate buffer, sodium citrate buffer, 4-(2-hydroxyethyl)l-piperazine ethanesulfonic acid (HEPES), and the like. Growth medium is sometimes also referred to as cell culture medium or simply medium. The system may alternatively include cell producing environments other than a bioreactor. The system of the present disclosure is not particularly limited by the configuration of the cell producing environment, whether a bioreactor or another vessel.

[0037] In some embodiments, the vessel is a bioreactor or other vessel or system configured for culturing cells. The cells that are cultured in the bioreactor (or other vessel) may be referred to as cultured cells. In some cases, the cultured cells may be cultured meat cells. For example, the vessel may be bioreactor in a system configured for production of cultured meat, such as cultured salmon, cultured pork, cultured venison, cultured beef, cultured poultry, and the like. The cultured meat may also be referred to as lab-grown meat, cell-based meat, in-vitro meat, cell- cultured meat, and cell -agri culture meat. Cultured meat cells may be used as a food source. For example, cultured salmon cells may be used as a replacement for salmon, and cultured beef cells may be used as a replacement for beef. The bioreactor can alternatively be used for production of cellular products, including compounds produced by cells. For example, the bioreactor may be used to produce cells or cell products for cell therapies and gene therapies, biopharmaceuticals, and the like.

[0038] The system may operate using a perfusion cycle for continuous liquid flow. Perfusion may increase cell density of cultured cells by over ten times the rate. The primary reason for the increase in cell density of the cultured cells is that the concentration of waste products is controlled in the cell growth medium.

[0039] The vessel (e.g., bioreactor) has one or more ports or openings that provide access to the interior or the vessel. Such ports may provide the vessel with an outlet. The vessel may also have a separate inlet, or the same port or opening may act as both an inlet and an outlet. The vessel may have multiple ports for different inputs, such as medium, nutrition replacement, gas (e.g., air or oxygen), and the like. The vessel may include ports for instrumentation, such as a mixer, pH meter, thermometer, glucose meter, and the like. The one or more inlets and ports may be used to control the environment within the vessel by allowing for the desired inputs and taking measurements within the vessel.

[0040] The outlet or one or more ports may be used to extract medium and cultured cells from the vessel. The vessel can also receive recycled growth medium via the one or more ports from a mixing tank, as explained in further detail below. The vessel can also receive at least some of the cells from a first separation device, also explained in further detail below.

[0041] According to an embodiment, the system includes various separations devices that work in combination to selectively remove at least some of the waste products from used growth medium, for example from a cultured meat production process. The system allows for separation of waste products, including small molecule waste products, from the growth medium that increase in concentration throughout the cell culturing process. The system also allows for reintroduction of components, such as nutrients, for a desired make-up of the growth medium. The system enables the reuse of at least some of the growth medium. The system may include one or more separation devices to separate and retain cells from growth medium.

[0042] According to an embodiment, the system for processing growth medium includes a first separation device constructed to receive fluid flow from the vessel (e.g., a bioreactor). The first separation device may be constructed to separate cultured cells from the fluid flow received from the vessel. The first separation device may include one or more cell retention devices. The one or more cell retention devices may be housed within the first separation device. Examples of suitable cell retention devices include, for example, spin filters, settling tank systems, centrifuges, cyclone separators, hydrodynamic separators, cross-flow filters, and tangential flow filters. The separated fluid may be further directed to a second separation device for a further separation.

[0043] The first separation device may optionally have the capability to separate cells from the fluid flow based on viability. A cell may be considered inviable when it is dead or diseased. The separation based on viability in the first separation device may occur by selectively concentrating cells of a given size and eliminating cells of larger or smaller size comparatively to the given size. Generally, viable cells may be larger than inviable cells. Thus, size separation may be used to separate viable cells from inviable cells or to concentrate viable cells by removing more inviable cells than viable cells. The specific size may depend on the particular cell species that are being cultivated. For example, the first separation may have a cutoff size in a range of 0.2 pm to 30 pm. The first separation device may be used to selectively remove inviable cells and to return at least some of the viable cells to the vessel.

[0044] The system may allow for harvesting at least some of the cultured cells after the cells are separated from growth medium in the first separation device (e.g., by the one or more cell retention devices). Another option is that the system may recirculate at least some of the cells back to the vessel (e.g., bioreactor) for further proliferation. The system can optionally allow the recirculation of viable cells back into the vessel. A return line may connect the first separation device to the vessel for the return of cells to the vessel. The separated fluid from the first filtration device may include larger molecules, such as left-over nutrients, proteins, etc., and small molecules, such as cell culture waste products. It may be desirable to recycle the left-over nutrients, proteins, etc. back into the vessel (e.g., bioreactor), while removing the small molecules that could inhibit cell proliferation. However, it may be challenging to separate out the small molecules without removing the larger molecules from the fluid. According to an embodiment, the system includes filtration devices that first separate the filtered fluid into a stream that includes at least some of (or a majority of, or substantially all of) the larger molecules, and a stream that includes at least some of (or a majority of, or substantially all of) the small molecules. The stream with the small molecules can then be treated to remove at least some of (or a majority of, or substantially all of) the small molecules. The remaining medium, with at least some of (or a majority of, or substantially all of) the small molecules removed, can then be recycled back into the vessel and reused as growth medium.

[0045] According to an embodiment, the system for processing growth medium includes a second separation device constructed to receive separated fluid from the first separation device. The separated fluid received from the first separation device may be free or substantially free of cultured cells. The second separation device may be an ultrafiltration device. The second separation device (e.g., ultrafiltration device) may have a molecular weight cut-off in a range of about 1 kDa to about 20 kDa. Molecules, ions, cell fragments, and other contaminants that are larger than the molecular weight cut-off are separated from the fluid within the second separation device. The second separation device may further separate the fluid into a retentate (e.g., a first waste stream) and a permeate stream (e.g., an ultrafiltered stream). The first waste stream may be further processed or may be discarded. The first waste stream may also be recycled back into the second filtration device. The ultrafiltered permeate stream may be directed into a third filtration device for further processing.

[0046] The recycling of the retentate, and thus unused nutrients, replaces some of the need for new growth medium and effectively reduces the overall cost of the cell culturing process.

[0047] The second separation device may be any suitable separation device that achieves the size separation at the desired size cut-off. Conveniently, the second separation device may be an ultrafiltration device. Any suitable configurations of ultrafiltration may be used in the second filtrations device. Examples of suitable configurations of ultrafiltration used in the system can be crossflow filtration, dead end filtration, spin filtration, batch filtration, and the like. In some embodiments, the ultrafiltration device is arranged in a crossflow mode. The second separation device may include two or more ultrafiltration devices. The second separation device may include two or more ultrafiltration devices arranged in different configurations. The second separation device may additionally include flocculation, chromatographic separation, dialysis, tangential flow filtration system, alternative tangential system, or a combination of two or more thereof.

[0048] The second separation device may have a molecular weight cut-off of 1 kDa or greater, 1.25 kDa or greater, 1.5 kDa or greater, 2 kDa or greater, 2.5 kDa or greater, 3 kDa or greater, 3.5 kDa or greater, or 4 kDa or greater. The second separation device has a molecular weight cutoff of 20 kDa or lower, 15 kDa or lower, 12 kDa or lower, 10 kDa or lower, 9 kDa or lower, 8 kDa or lower, 7 kDa or lower, 6 kDa or lower, or 5 kDa or lower. The molecular weight cut-off may be in the range of 1 kDa to 20 kDa, 1.5 kDa to 20 kDa, 2 kDa to 20 kDa, 3 kDa to 20 kDa, 1 kDa to 15 kDa, 1.5 kDa to 15 kDa, 2 kDa to 15 kDa, 3 kDa to 15 kDa, 1 kDa to 12 kDa, 1.5 kDa to 12 kDa, 2 kDa to 12 kDa, 1 kDa to 10 kDa, 1.5 kDa to 10 kDa, 2 kDa to 10 kDa, 1 kDa to 8 kDa, 1.5 kDa to 8 kDa, or 2 kDa to 8 kDa. In embodiments where the second separation device includes two or more ultrafiltration devices, the ultrafiltration devices may have different molecular weight cut-offs. For example, the ultrafiltration devices may be arranged in series where a first ultrafiltration device may have a higher molecular weight cut-off than a second ultrafiltration device.

[0049] The fluid flow from the first separation device may include cell debris, cell fragments, and other larger contaminants. It may be desirable to remove such contaminants because they could cause fouling of the system if returned into the vessel with the recycle flow. The system may optionally include a prefilter disposed in fluid communication between the first separation device and the second separation device. The prefilter may be configured to remove cell debris, cell fragments, and other larger contaminants. The prefilter may be a microfilter. The microfilter may include any suitable filtration media, such as wet-laid media, spun bound media, electro spun media, extruded membrane, expanded membrane, phase-inverted membrane, or a combination of two or more thereof. The filtration media may be made of any suitable material, such as polyester, nylon, polyether sulfone, polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), cellulose or its derivatives (such as cellulose acetate), and the like, or a combination of two or more thereof. The microfilter may have a pore size of 0.1 pm or greater, 0.2 pm or greater, 0.3 pm or greater, 0.5 pm or greater, 0.8 pm or greater, 1 pm or greater, 2 pm or greater, 5 pm or greater, 10 pm or greater, 20 pm or greater, 50 pm or greater, or 100 pm or greater. The microfilter may have a pore size of 500 pm or less, 250 pm or less, 200 pm or less, 150 pm or less, 100 pm or less, or 50 pm or less. The pore size may be in the range of 0.5 pm to 500 pm, 0.8 pm to 250 pm, 1 pm to 200 pm, 1 pm to 150 pm, or 1 pm to 100 pm. The retentate from the prefilter (e.g., microfilter) may be discarded. The permeate from the prefilter (e.g., microfilter) is directed to the second filtration device.

[0050] According to an embodiment, the system includes a third separation device. The third separation device may be configured to separate small molecule and ionic waste products from the fluid flow. The third separation device receives fluid flow from the permeate side of the second separation device. That is, the input into the third separation media constitutes growth medium that has had all or substantially all cell debris, cell fragments, proteins, and other compounds above the molecular weight cut-off of the second separation device removed.

[0051] The third separation device may be constructed to remove small molecule waste products, ionic waste products, or both, from the fluid flow. Preferably, the third separation device is configured to selectively remove undesired waste products. The third separation device may be capable of removing ammonium, ammonia, salts, carboxylic acids or conjugate bases thereof, amino acids, nucleotides, nucleic acids, ketone bodies, calcium, potassium, and combinations of two or more thereof. The specific combination of waste products may depend on the types of cells being cultured, and the growth conditions.

[0052] The third separation device may operate based on ion exchange, adsorption, absorption, chemical reaction, filtration, or a combination of two or more thereof.

[0053] In some embodiments, the third separation device includes an ion exchange separator which includes ion exchange resin. The ion exchange resin may be arranged in a column, a packed bed, or any other suitable configuration. The ion exchange resin may be a cation exchange resin, anion exchange resin, or zwitterion exchange resin. In some embodiments, the third separation device includes at least a cation exchange resin. The cation exchange resin may be a strong acid cation exchange resin. The cation exchange resin may be a sodium based resin. The cation exchange resin may be a potassium based resin.

[0054] The third separation device may include two or more different ion exchange resins to selectively remove two or more different waste products. In some embodiments, the third separation device includes a first separator with a cation exchange resin and a second separator with anion exchange resin. The third separation device may include two or more different types of cation exchange resins. The third separation device may include two or more different types of anion exchange resins.

[0055] In some embodiments, the third separation device includes a selective absorbent. Examples of selective absorbents include silica gel, activated alumina, ion-exchange resins (such as strong acid, strong base, weak acid, or weak base resin), affinity chromatography absorbents, metal-organic frameworks, hydrogel polymers, biopolymers like chitosan, biosorbents such as fungi or bacteria and combinations of two or more thereof. A selective absorbent may be capable of removing inorganic salts, organic acids (such as acetic acid, citric acid, pyruvic acid, lactic acid, and the like), amino acids, sugars, glycans, fatty acids, lipids, phosphoric acid, ketones, sulfur compounds, hormones, growth factors, antibiotics, ammonium, urea, and the like, and combinations of two or more thereof .

[0056] In some embodiments, the third separation device includes a selective adsorbent. Examples of selective adsorbents include activated carbon, biochar, zeolite, ion-exchange resins (such as strong acid, strong base, weak acid, or weak base resin), metal-organic frameworks, porous polymers, molecularly imprinted polymers, and combinations of two or more thereof. A selective adsorbent may be capable of removing inorganic salts, organic acids (such as acetic acid, citric acid, pyruvic acid, lactic acid, and the like), amino acids, sugars, glycans, fatty acids, lipids, phosphoric acid, ketones, sulfur compounds, hormones, growth factors, antibiotics, ammonium, urea, and the like, and combinations of two or more thereof.

[0057] In some embodiments, the third separation device includes a catalytic separator. Examples of catalytic separator include transition metals and metal oxides, metal and metal oxide doped ceramics, zeolites, metal and metal-oxide nanotubes, graphene, metal and metal oxide nanoparticles, and combinations of two or more thereof. The catalytic separator may operate by photocatalytic oxidation or reduction; thermal catalytic oxidation or reduction, microwave assisted catalysis, ozonation, hydrogenation, enzymatic catalysis (peroxidases, oxidases, catalases, proteases, lipases), sono-catalysis, fenton and fenton-like catalysis, or a combination of two or more thereof. A catalytic separator may be capable of removing inorganic salts, organic acids (such as acetic acid, citric acid, pyruvic acid, lactic acid, and the like), amino acids, sugars, glycans, fatty acids, phosphoric acid, ketones, sulfur compounds, hormones, growth factors, antibiotics, ammonium, urea, and the like, and combinations of two or more thereof.

[0058] The third separation device, via its one or more ion exchange resins, selectively separates small molecule waste products from the permeate. A wash medium can be added to the third separation device to aid this part of the process. Examples of appropriate wash medium include water or, optionally, water with one or more nutrients. The one or more nutrients can be or can include, for example, salts, sugars, or a combination of two or more thereof.

[0059] The third separation device, as a result from the one or more ion exchange devices, forms a waste stream and a cleaned medium. The waste stream includes small molecule and ionic waste products. The waste products may include ammonium, ammonia, salts, carboxylic acids or conjugate bases thereof, amino acids, nucleotides, nucleic acids, ketone bodies, calcium, potassium, or a combination of two or more thereof. The salts may include one or more ionic species, such as sodium, potassium, calcium, magnesium, iron, or the like. The carboxylic acids or their conjugate bases may include lactic acid, acetic acid, uric acid, pyruvic acid, or any other carboxylic acids that may have accumulated in the cell culture. The amino acids may include glutamic acid or glutamate, methionine, tryptophan, cysteine, asparagine, tyrosine, phenylalanine, or a combination of two or more thereof .

[0060] High levels of ammonia or ammonium in the growth medium may lead to inhibition of cell proliferation and growth or an overall decreased maximum cell density in cultures. At the pH conditions of the growth medium, the ammonia may be in the form of ammonium. It has been found that ammonium may be challenging to remove from growth medium due to interference or unintended interactions of the separation media with other compounds present in growth medium, and the lower volatility of ammonium as compared to ammonia. For example, testing with vacuum distillation, zeolites, dialysis, and membrane chromatography demonstrated that these techniques were not able to adequately remove waste products from the growth medium without the use of the second and third filtration devices described herein. Specifically, these types of other filtration methods failed to adequately filter out ammonia or ammonium or both.

[0061] In some embodiments, the system is used to culture meat cells, such as salmon cells, and the waste products include ammonium. The third separation device may be constructed for filtering waste from a system used to culture salmon cells may be capable of removing ammonium from the fluid stream.

[0062] The waste stream of the third separation device may optionally be further treated to remove waste products. Such treatment may include, for instance, reverse osmosis or other waste-water treatment techniques. Once the waste stream is treated, the treated water may be cycled back into the third separation device and used as a wash medium.

[0063] The system may include a mixing tank constructed to receive the cleaned medium from the third separation device and the retentate from the second separation device. The mixing tank combines the retentate, which desirably contains nutrients, such as proteins, growth factors, and the like, and the cleaned medium from the third separation device. By mixing the retentate and cleaned growth medium, the mixing tank creates a recycle medium that may be cycled back to the vessel (e.g., bioreactor). One or more additional nutrients may optionally be added to the mixing tank to bring the level of nutrients to a desired level for further cell proliferation. For example, nutrients that may be added to the mixing tank may include calcium, proteins, growth factors, amino acids, salts, vitamins, sugars (e.g., glucose, galactose, hexose, fructose, and the like), fatty acids, trace elements (zinc etc.), nucleotides, nucleosides, cholesterol, antioxidants, or a combination of two or more thereof. Calcium may be added to replenish calcium ions lost in the third separation device.

[0064] According to an embodiment, the system includes one or more metering devices constructed for monitoring various parameters at various points in the system. For example, the metering devices may be used to monitor the accumulation or removal of the waste products in the vessel, in the fluid streams, in the fdtration units, in the mixing tank, or a combination thereof. The one or more metering devices may be disposed upstream, downstream, or both upstream and downstream of the third separation device. The metering devices may include one or more sensors configured to detect O2, CO2, glucose, ammonia, ammonium, pH, temperature, turbidity, pressure, optical density, glucose, lactate, redox potential, or a combination of two or more thereof.

[0065] Referring now to FIGURE 1, the system includes a vessel 10. The vessel 10 may be a bioreactor, a holding vessel, or other suitable vessel. In embodiments, where the vessel 10 is a bioreactor, it may be used to cultivate cells that are suspended in the growth medium. The vessel 10 may include means for maintaining a desired cell growth environment within the vessel 10. For example, the vessel 10 may include a means for mixing 15, maintaining a desired temperature, maintaining a desired pH, a source of gas (e.g., oxygen or air), and a source of nutrients. The vessel 10 may include one or more inlet ports 11, 12 and one or more outlet ports 13. The vessel 10 is in fluid communication with a first separation device 20. It should be noted that the system 1 may include more than one vessel 10. Each of the vessels 10 may be independently in fluid communication with the first separation device 20. The first separation device 20 is arranged to receive a fluid flow from the vessel 10 along line 110. The first separation device 20 may be a cell retention device, constructed to separate cells from the fluid flow. The separated cells from the cell growth medium can either flow back to the bioreactor 10 via line 121 or be harvested 122. The remaining fluid is directed to the second separation device 30 along line 123.

[0066] The second separation device 30 may be an ultrafiltration device with a retentate side 310 and a permeate side 320. The retentate side 310 and the permeate side 320 are separated by a separation membrane 315. The ultrafiltration device may be arranged in crossflow mode. The second separation device 30 may include more than one ultrafiltration device, such as two, three, four, or more ultrafiltration devices. The retentate from the retentate side 310 of the second separation device 30 may be directed to a mixing tank 50 via line 131.

[0067] The second separation device may have a molecular weight cut-off in a range of about 1 kDa to about 20 kDa, as discussed in more detail above. The permeate from the second separation device 30 preferably is free or substantially free of cells and large molecules, such as proteins. The permeate from the second separation device 30 is directed to a third separation device 40 via line 132. The third separation device 40 may be constructed as an ion exchange separation device containing ion exchange resin. In some embodiments, the ion exchange resin of the third separation device 40 is a strong acid cation ion exchange resin. The cleaned medium stream is conducted from the third separation device 40 back to the vessel 10, optionally via a mixing tank 50. The cleaned medium may be flown to the mixing tank along line 141 continues to travel through the system to a mixing tank 50.

[0068] A waste steam may be discarded along line 143. Alternatively, as shown in FIGURE 2, at least some of the waste stream may be conducted into a waste processing unit 70 along line 144. The waste processing unit 70 may include one or more devices that can produce clean water, such as reverse osmosis, and the like.

[0069] Optionally, a wash medium may be introduced into the third separation device 40 via line 142. The wash medium may be water or water with added nutrients. The added nutrients in the wash medium 45 may include one or more salts, sugars, or a combination thereof. If the waste stream is further processed in a waste processing unit 70 (FIGURE 2), the cleaned stream (which may consist substantially of water) may be used as the wash medium directed to the third separation device via line 142. Additional water, with or without nutrients, may be added.

[0070] The retentate stream (line 131) from the second separation device 30 and the cleaned medium (line 141) from the third separation device 40 may be combined in the mixing tank 50. The mixing tank 50 may also optionally receive additional inputs, such as water, additional nutrients, growth regulators, pH adjusting agents, and the like, along input line 55 to achieve a desired growth medium consistency. The mixture from the mixing tank 50 may be cycled back into the vessel 10 along line 90, 111 and inlet 11. Additional inputs, such as water and additional nutrients, growth regulators, pH adjusting agents, and the like, may be added along input line 112.

[0071] The system may further include additional components, such as storage tanks, mixing tanks, feed lines, bleed lines, outlets, valves, pumps, heaters, coolers, sensors, flow meters, controls, etc.

[0072] In one or more embodiments, the system may be described as including a control unit. The control unit may be operably coupled with one or more user interfaces, sensors, metering devices, valves, pumps, heaters, coolers, and other units. The control unit may be configured to receive information from the one or more user interfaces, sensors, metering devices, valves, pumps, heaters, coolers, and other units. The control unit may be configured to send signals and to control various units, such as valves, pumps, heaters, and coolers, based on information received from the unit or another unit, such as the one or more user interfaces, sensors, or metering devices. Such control units may include one or more programmable processors that include processing capabilities (for example, microcontrollers or programmable logic devices), data storage (for example, volatile or non-volatile memory or storage elements), input devices, and output devices. Program code, or logic, described herein may be applied to input data to perform functionality described herein and generate desired output information. The output information may be applied as input to one or more other devices or processes as described herein or as would be applied in a known fashion. The exact configuration of the control unit is not limiting and essentially any device capable of providing suitable computing capabilities and control capabilities to implement the method may be used.

[0073] According to an embodiment, a method for processing growth medium includes culturing cells in a growth medium. The system may be set up as a perfusion system. The cells may be cultured in a vessel, such as a bioreactor. The vessel includes a mixture of the growth medium and cultured cells. The growth medium may be tailored to the specific cell type being cultured. The growth medium may be an aqueous nutrient solution. The growth medium may include, for example, an aqueous solution of amino acids, vitamins, inorganic acids, glucose, growth factors, hormones, and the like. The vessel may be provided with air or oxygen. The temperature and pH of the growth medium may be measured using appropriate sensors, and may be controlled to desired levels. The vessel may include other sensors that determine, for example, the use of nutrients, the accumulation of waste products, the accumulation of cells, or a combination thereof. For example, the method may include measuring the amount of glucose, the amount of organic acids (e.g., lactic acid, acetic acid, uric acid, and the like), pH, the accumulation of cells, or a combination thereof. When one or more of the parameters reach a triggering level, at least a portion of the mixture of growth medium and cultured cells may be drawn out of the vessel into the first separation device.

[0074] The first separation device may be a cell retention device. The mixture may be separated in the first separation device into cultured cells and spent medium. The cultured cells may be harvested. Some of the cells may be returned back into the vessel. The spent medium may be flown into a second separation device, where it may be separated into a permeate and a retentate. In some embodiments, the spent medium is pre-filtered prior to separating in the second filtration device. For example, the spent medium may be microfiltered prior to separating in the second filtration device.

[0075] The second separation device may be or may include an ultrafiltration device. The second separation device may have a molecular weight cut-off in a range of 1 kDa to 20 kDa. The second separation device may be capable of separating large molecules, such as proteins, from the fluid stream.

[0076] The permeate of the second separation device is further flown to a third separation device. The third separation device may be constructed to selectively separate small molecule waste products from the permeate, forming a waste stream and a cleaned medium.

[0077] The method may further include adding wash medium into the third separation device. The wash medium may include water and optionally nutrients, such as one or more salts, sugars, or a combination of two or more thereof.

[0078] The third separation device may include ion exchange resin. The ion exchange resin may include cation exchange resin, anion exchange resin, zwitterionic ion exchange resin, or a combination of two or more thereof. The spent medium may be filtered through multiple ion exchange separation devices (e.g., ion exchange columns or beds), where each separation device includes a different ion exchange resin to target specific waste products. The third separation device may include a selective absorbent. The third separation device may include a catalytic separator.

[0079] The small molecule waste products separated from the spent medium in the third separation device may include one or more of ammonium, ammonia, salts, carboxylic acids or conjugate bases thereof, amino acids, nucleotides, nucleic acids, ketone bodies, calcium, potassium, or a combination of two or more thereof. In some embodiments, the amino acids include glutamate. In some embodiments, the carboxylic acids include lactic acid, acetic acid, uric acid, pyruvic acid, a conjugate base thereof, or a combination of two or more thereof.

[0080] The cleaned medium from the third separation device and the retentate from the second separation device may be mixed into a recycle medium. The cleaned medium from the third separation device and the retentate from the second separation device may be mixed in a mixing tank.

[0081] The recycle medium may be added back into the vessel. Additional nutrients or fresh growth medium may further be added to the recycle medium or directly into the vessel. The recycle medium and fresh growth medium may be added at a ratio of 1 or more parts to 10 parts; 2 or more parts to 10 parts; 3 or more parts to 10 parts; 4 or more parts to 10 parts; or 5 or more parts to 10 parts. The recycle medium and fresh growth medium may be added at a ratio of 50 or fewer parts to 10 parts; 40 or fewer parts to 10 parts; 30 or fewer parts to 10 parts; 20 or fewer parts to 10 parts; 15 or fewer parts to 10 parts; or 10 or fewer parts to 10 parts. In some embodiments, the recycle medium is added without adding fresh growth medium. In some embodiments, the recycle medium is modified (e.g., pH adjusted, nutrients added, or both) added with or without adding fresh growth medium.

[0082] The method may further include monitoring the amount of ammonium, ammonia, an organic acid or its conjugate base, or a combination thereof in one or more locations in the system. For example, such parameters may be monitored in the vessel, the mixing tank, one or more lines between the unit operations, within one or more unit operations. The parameters may be monitored in the growth medium in the vessel, the spent medium, the cleaned medium, or the recycle medium.

[0083] EXEMPLARY EMBODIMENTS

[0084] Embodiment l is a system for processing growth medium, the system comprising: a vessel comprising a fluid and cultured cells; a first separation device constructed to receive fluid flow from the vessel, the first separation device being constructed to separate cultured cells from a liquid; a second separation device constructed to receive the liquid from the first separation device, the second separation device comprising a retentate side and a permeate side and having a molecular weight cut-off in a range of 1 kDa to 20 kDa, 1.5 kDa to 20 kDa, 2 kDa to 20 kDa, 3 kDa to 20 kDa, 1 kDa to 15 kDa, 1.5 kDa to 15 kDa, 2 kDa to 15 kDa, 3 kDa to 15 kDa, 1 kDa to 12 kDa, 1.5 kDa to 12 kDa, 2 kDa to 12 kDa, 1 kDa to 10 kDa, 1.5 kDa to 10 kDa, 2 kDa to 10 kDa, 1 kDa to 8 kDa, 1.5 kDa to 8 kDa, or 2 kDa to 8 kDa; and a third separation device comprising a selective separator constructed to receive fluid flow from the permeate side of the second separation device and to separate one or more small molecules or ions from the fluid flow.

[0085] Embodiment 2 is the system of embodiment 1, wherein the vessel is a bioreactor.

[0086] Embodiment 3 is the system of any one of embodiments 1 or 2, wherein the system further comprising a mixing tank constructed to receive fluid flow from the third separation device and the retentate of the second separation device.

[0087] Embodiment 4 is the system of any one of embodiments 1 to 3, wherein the system further comprising a conduit from the mixing tank to the vessel.

[0088] Embodiment 5 is the system of any one of embodiments 1 to 4, wherein the system comprises forms a perfusion cycle.

[0089] Embodiment 6 is the system of any one of embodiments 1 to 5, wherein the first separation device comprises a cell retention device comprising a spin filter, a settling tank system, a centrifuge, a cyclone separator, a hydrodynamic separator, a cross-flow filter, a tangential flow filter, or a combination thereof.

[0090] Embodiment 7 is the system of any one of embodiments 1 to 6, wherein the first separation device is constructed for harvesting cultured cells.

[0091] Embodiment 8 is the system of any one of embodiments 1 to 7, wherein the system further comprising a return line from the first separation device to the vessel.

[0092] Embodiment 9 is the system of any one of embodiments 1 to 8, wherein the system further comprising a microfilter disposed in fluid communication between the first filtration device and the second filtration device.

[0093] Embodiment 10 is the system of any one of embodiments 1 to 9, wherein the second separation device comprises an ultrafiltration device, optionally wherein the second separation device comprises two or more ultrafiltration devices.

[0094] Embodiment 11 is the system of any one of embodiments 1 to 10, wherein the second separation device has a molecular weight cut-off of 1 kDa or greater, 1.25 kDa or greater, 1.5 kDa or greater, 2 kDa or greater, 2.5 kDa or greater, 3 kDa or greater, 3.5 kDa or greater, or 4 kDa or greater.

[0095] Embodiment 12 is the system of any one of embodiments 1 to 11, wherein the second separation device has a molecular weight cut-off of 20 kDa or lower, 15 kDa or lower, 12 kDa or lower, 10 kDa or lower, 9 kDa or lower, 8 kDa or lower, 7 kDa or lower, 6 kDa or lower, or 5 kDa or lower.

[0096] Embodiment 13 is the system of any one of embodiments 1 to 12, wherein the second separation device is arranged as in a crossflow configuration, dead end configuration, batch configuration, or spin configuration.

[0097] Embodiment 14 is the system of any one of embodiments 1 to 13, wherein the third separation device comprises an ion exchange separator comprising ion exchange resin, optionally wherein the ion exchange resin comprises cation exchange resin, anion exchange resin, zwitterionic ion exchange resin, or a combination of two or more thereof.

[0098] Embodiment 15 is the system of any one of embodiments 1 to 14, wherein the system further comprising one or more meters constructed for monitoring the amount of ammonium, ammonia, an organic acid or its conjugate base, or a combination thereof, wherein the one or more meters are disposed upstream, downstream, or both upstream and downstream of the third separation device.

[0099] Embodiment 16 is a method for processing growth medium, the method comprising: culturing cells in a mixture of growth medium and cultured cells; separating a portion of the mixture into cultured cells and spent medium in a first separation device; filtering the spent medium in a second separation device and forming a permeate and a retentate comprising proteins, the second filtration device having a molecular weight cut-off in a range of 1 kDa to 20 kDa, 1.5 kDa to 20 kDa, 2 kDa to 20 kDa, 3 kDa to 20 kDa, 1 kDa to 15 kDa, 1.5 kDa to 15 kDa, 2 kDa to 15 kDa, 3 kDa to 15 kDa, 1 kDa to 12 kDa, 1.5 kDa to 12 kDa, 2 kDa to 12 kDa, 1 kDa to 10 kDa, 1.5 kDa to 10 kDa, 2 kDa to 10 kDa, 1 kDa to 8 kDa, 1.5 kDa to 8 kDa, or 2 kDa to 8 kDa; and selectively separating small molecule waste products from the permeate in a third separation device, forming a waste stream and a cleaned medium.

[0100] Embodiment 17 is the method of embodiment 16, wherein the method further comprising mixing the cleaned medium and the retentate into a recycle medium.

[0101] Embodiment 18 is the method of any one of embodiments 16 or 17, wherein the method further comprising adding the recycle medium to the mixture of growth medium and cultured cells, optionally further comprising adding nutrients to the recycle medium.

[0102] Embodiment 19 is the method of any one of embodiments 16 to 18, wherein the method wherein the method comprises perfusion.

[0103] Embodiment 20 is the method of any one of embodiments 16 to 19, wherein the first separation device comprises a cell retention device comprising a spin filter, a settling tank system, a centrifuge, a cyclone separator, a hydrodynamic separator, a cross-flow filter, a tangential flow filter, or a combination thereof.

[0104] Embodiment 21 is the method of any one of embodiments 16 to 20, wherein the separating comprises harvesting at least some of the cultured cells.

[0105] Embodiment 22 is the method of any one of embodiments 16 to 21, wherein the method further comprising returning at least some of the separated cultured cells to the mixture.

[0106] Embodiment 23 is the method of any one of embodiments 16 to 22, wherein the method further comprising microfiltering the spent medium prior to the second filtration device.

[0107] Embodiment 24 is the method of any one of embodiments 16 to 23, wherein the second separation device comprises an ultrafiltration device, optionally wherein the second separation device comprises two or more ultrafiltration devices.

[0108] Embodiment 25 is the method of any one of embodiments 16 to 24, wherein the second separation device has a molecular weight cut-off of 1 kDa or greater, 1.25 kDa or greater, 1.5 kDa or greater, 2 kDa or greater, 2.5 kDa or greater, 3 kDa or greater, 3.5 kDa or greater, or 4 kDa or greater.

[0109] Embodiment 26 is the method of any one of embodiments 16 to 25, wherein the second separation device has a molecular weight cut-off of 20 kDa or lower, 15 kDa or lower, 12 kDa or lower, 10 kDa or lower, 9 kDa or lower, 8 kDa or lower, 7 kDa or lower, 6 kDa or lower, or 5 kDa or lower.

[0110] Embodiment 27 is the method of any one of embodiments 16 to 26, wherein the second separation device is arranged as in a crossflow configuration, dead end configuration, batch configuration, or spin configuration.

[0111] Embodiment 28 is the method of any one of embodiments 16 to 27, wherein the method further comprising adding wash medium into the third separation device.

[0112] Embodiment 29 is the method of any one of embodiments 16 to 28, wherein the wash medium comprises water and optionally nutrients, optionally wherein the nutrients comprise one or more salts, sugars, or a combination of two or more thereof.

[0113] Embodiment 30 is the method of any one of embodiments 16 to 29, wherein the third separation device comprises ion exchange resin, optionally wherein the ion exchange resin comprises cation exchange resin, anion exchange resin, zwitterionic ion exchange resin, or a combination of two or more thereof.

[0114] Embodiment 31 is the method of any one of embodiments 16 to 30, wherein the third separation device comprises a selective absorbent.

[0115] Embodiment 32 is the method of any one of embodiments 16 to 31, wherein the third separation device comprises a catalytic separator.

[0116] Embodiment 33 is the method of any one of embodiments 16 to 32, wherein the small molecule waste products comprise one or more ammonium, ammonia, salts, carboxylic acids or conjugate bases thereof, amino acids, nucleotides, nucleic acids, ketone bodies, calcium, potassium, or a combination of two or more thereof.

[0117] Embodiment 34 is the method of any one of embodiments 16 to 33, wherein the amino acids comprise glutamate, methionine, tryptophan, cysteine, asparagine, tyrosine, phenylalanine, or a combination of two or more thereof.

[0118] Embodiment 35 is the method of any one of embodiments 16 to 34, wherein the carboxylic acids comprise lactic acid, acetic acid, uric acid, pyruvic acid, a conjugate base thereof, or a combination thereof. Embodiment 36 is the method of any one of embodiments 16 to 35, wherein the method further comprising monitoring the amount of ammonium, ammonia, an organic acid or its conjugate base, or a combination thereof in the cleaned medium.

[0119] EXAMPLES

[0120] Example 1: 14-day Cell Culture Filtration Evaluation

[0121] These examples are merely for illustrative purposes only and are not meant to be limiting on the scope of the appended claims. All parts, percentages, ratios, etc. in the examples and the rest of the specification are by weight, unless noted otherwise.

[0122] The performance of spent growth medium and filtered growth medium were tested and evaluated against a control sample. The samples had a volume of 50 mL of growth medium and were seeded with 1.7 • 106cells / mL, in triplicate. The cells were salmon cells to simulate a salmon meal culturing process.

[0123] At day 14 of culturing, the control samples C1-C3 received 100 % fresh growth medium. Samples Al -A3 received 50 % of fresh growth medium, mixed with 50 % of spent growth medium. Samples B1-B3 received 50 % of fresh growth medium, mixed with 50 % filtered spent growth medium.

[0124] TABLE 1, Samples.

[0125] The filtered spent growth medium was filtered using ion exchange filtration with a strong acid cation exchange resin treated with a NaCl solution. The resin had the following relative selectivity for various ions, shown in TABLE 2:

[0126] TABLE 2, Resin selectivity The filtered spent growth medium was replenished with calcium, potassium, magnesium, other additives, and the pH was adjusted to the level of the fresh growth medium.

[0127] The cell counts of each of the samples were calculated at day 14. The results were normalized to the cell count of the control. The results are shown in FIGURE 3 and TABLE 3.

[0128] TABLE 3. Sample performance.

[0129] It was observed that three different factors— potassium, ammonium, and positively charged cell waste-affected cell growth. The total cell count of Sample B (50% Spent Medium) was 67 % of the control, while the total cell count of Sample C (50 % Filtered Medium) was 79 % of the control. This amounted to a 12 % restoration of the ability of the growth medium to support a cell culture. It was concluded that the ion exchange resin is an effective strategy to use for at least partially restoring the conditions of the cell growth medium.

[0130] All references and publications cited herein are expressly incorporated herein by reference in their entirety into this disclosure, except to the extent they may directly contradict this disclosure. Although specific embodiments have been illustrated and described herein, it will be appreciated by those of ordinary skill in the art that a variety of alternate and / or equivalent implementations can be substituted for the specific embodiments shown and described without departing from the scope of the present disclosure. It should be understood that this disclosure is not intended to be unduly limited by the illustrative embodiments and examples set forth herein and that such examples and embodiments are presented by way of example only with the scope of the disclosure intended to be limited only by the claims set forth here.

Claims

Claims1. A system for processing growth medium, the system comprising: a vessel comprising a fluid and cultured cells; a first separation device constructed to receive fluid flow from the vessel, the first separation device being constructed to separate cultured cells from a liquid; a second separation device constructed to receive the liquid from the first separation device, the second separation device comprising a retentate side and a permeate side and having a molecular weight cut-off in a range of 1 kDa to 20 kDa; and a third separation device comprising a selective separator constructed to receive fluid flow from the permeate side of the second separation device and to separate one or more small molecules or ions from the fluid flow.

2. The system of claim 1, wherein the vessel is a bioreactor.

3. The system of claim 1 or 2, further comprising a mixing tank constructed to receive fluid flow from the third separation device and the retentate of the second separation device.

4. The system of any one of claims 1 to 3, further comprising a conduit from the mixing tank to the vessel.

5. The system of any one of claims 1 to 4, wherein the system comprises forms a perfusion cycle.

6. The system of any one of claims 1 to 5, wherein the first separation device comprises a cell retention device comprising a spin filter, a settling tank system, a centrifuge, a cyclone separator, a hydrodynamic separator, a cross-flow filter, a tangential flow filter, or a combination thereof.

7. The system of any one of claims 1 to 6, wherein the first separation device is constructed for harvesting cultured cells.

8. The system of any one of claims 1 to 7, further comprising a return line from the first separation device to the vessel.

9. The system of any one of claims 1 to 8, further comprising a microfilter disposed in fluid communication between the first filtration device and the second filtration device.

10. The system of any one of claims 1 to 9, wherein the second separation device comprises an ultrafiltration device, optionally wherein the second separation device comprises two or more ultrafiltration devices.

11. The system of any one of claims 1 to 10, wherein the second separation device has a molecular weight cut-off of 1.5 kDa or greater, 2 kDa or greater, 2.5 kDa or greater, 3 kDa or greater, 3.5 kDa or greater, or 4 kDa or greater.

12. The system of any one of claims 1 to 11, wherein the second separation device has a molecular weight cut-off of 20 kDa or lower, 15 kDa or lower, 12 kDa or lower, 10 kDa or lower, 9 kDa or lower, 8 kDa or lower, 7 kDa or lower, 6 kDa or lower, or 5 kDa or lower.

13. The system of any one of claims 1 to 12, wherein the second separation device is arranged as in a crossflow configuration, dead end configuration, batch configuration, or spin configuration.

14. The system of any one of claims 1 to 13, wherein the third separation device comprises an ion exchange separator comprising ion exchange resin, optionally wherein the ion exchange resin comprises cation exchange resin, anion exchange resin, zwitterionic ion exchange resin, or a combination of two or more thereof.

15. The system of any one of claims 1 to 14, further comprising one or more meters constructed for monitoring the amount of ammonium, ammonia, an organic acid or its conjugate base, or a combination thereof, wherein the one or more meters are disposed upstream, downstream, or both upstream and downstream of the third separation device.

16. A method for processing growth medium, the method comprising: culturing cells in a mixture of growth medium and cultured cells; separating a portion of the mixture into cultured cells and spent medium in a first separation device;filtering the spent medium in a second separation device and forming a permeate and a retentate comprising proteins, the second filtration device having a molecular weight cut-off in a range of 1 kDa to 20 kDa; and selectively separating small molecule waste products from the permeate in a third separation device, forming a waste stream and a cleaned medium.

17. The method of claim 16, further comprising mixing the cleaned medium and the retentate into a recycle medium.

18. The method of claim 16 or 17, further comprising adding the recycle medium to the mixture of growth medium and cultured cells, optionally further comprising adding nutrients to the recycle medium.

19. The method of any one of claims 16 to 18, wherein the method comprises perfusion.

20. The method of any one of claims 16 to 19, wherein the first separation device comprises a cell retention device comprising a spin filter, a settling tank system, a centrifuge, a cyclone separator, a hydrodynamic separator, a cross-flow filter, a tangential flow filter, or a combination thereof.

21. The method of any one of claims 16 to 20, wherein the separating comprises harvesting at least some of the cultured cells.

22. The method of any one of claims 16 to 21, further comprising returning at least some of the separated cultured cells to the mixture.

23. The method of any one of claims 16 to 22, further comprising microfiltering the spent medium prior to the second filtration device.

24. The method of any one of claims 16 to 23, wherein the second separation device comprises an ultrafiltration device, optionally wherein the second separation device comprises two or more ultrafiltration devices.

25. The method of any one of claims 16 to 24, wherein the second separation device has a molecular weight cut-off of 1.5 kDa or greater, 2 kDa or greater, 2.5 kDa or greater, 3 kDa or greater, 3.5 kDa or greater, or 4 kDa or greater.

26. The method of any one of claims 16 to 25, wherein the second separation device has a molecular weight cut-off of 20 kDa or lower, 15 kDa or lower, 12 kDa or lower, 10 kDa or lower, 9 kDa or lower, 8 kDa or lower, 7 kDa or lower, 6 kDa or lower, or 5 kDa or lower.

27. The method of any one of claims 16 to 26, wherein the second separation device is arranged as in a crossflow configuration, dead end configuration, batch configuration, or spin configuration.

28. The method of any one of claims 16 to 27, further comprising adding wash medium into the third separation device.

29. The method of any one of claims 16 to 28, wherein the wash medium comprises water and optionally nutrients, optionally wherein the nutrients comprise one or more salts, sugars, or a combination of two or more thereof.

30. The method of any one of claims 16 to 29, wherein the third separation device comprises ion exchange resin, optionally wherein the ion exchange resin comprises cation exchange resin, anion exchange resin, zwitterionic ion exchange resin, or a combination of two or more thereof.

31. The method of any one of claims 16 to 30, wherein the third separation device comprises a selective absorbent or adsorbent.

32. The method of any one of claims 16 to 31, wherein the third separation device comprises a catalytic separator.

33. The method of any one of claims 16 to 32, wherein the small molecule waste products comprise one or more of ammonium, ammonia, salts, carboxylic acids or conjugate bases thereof, amino acids, nucleotides, nucleic acids, ketone bodies, calcium, potassium, or a combination of two or more thereof.

34. The method of any one of claims 16 to 33, wherein the amino acids comprise glutamate , methionine, tryptophan, cysteine, asparagine, tyrosine, phenylalanine, or a combination of two or more thereof.

35. The method of any one of claims 16 to 34, wherein the carboxylic acids comprise lactic acid, acetic acid, uric acid, pyruvic acid, a conjugate base thereof, or a combination thereof.

36. The method of any one of claims 16 to 35, further comprising monitoring the amount of ammonium, ammonia, an organic acid or its conjugate base, or a combination thereof in the cleaned medium.

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