Regenerative bioreactor with ion-concentration-polarization waste removal

Abstract

A method for processing spent cell-culture media from a bioreactor using an ion-concentration-polarization device includes providing an ion-concentration-polarization device comprising alternating layers of ion-selective membranes and spacer layers. An electric field is applied to the ion-concentration-polarization device; and a feed, including spent cell-culture media and nutrients, is introduced from the bioreactor into the ion-concentration-polarization device. A charged waste product is separated from the feed in the ion-concentration-polarization device. The remainder of the feed, including the spent cell-culture media, is removed from the ion-concentration-polarization device in a combined flow that is not differentiated via separate diluate and concentrate streams.

Description

[0001] Attorney Docket No. mit-25891 pct

[0002] REGENERATIVE BIOREACTOR WITH ION-CONCENTRATION-POLARIZATION

[0003] WASTE REMOVAL

[0004] RELATED APPLICATION

[0005] This application claims the benefit of U.S. Provisional Application No. 63 / 701,892, filed 1 October 2024, the entire content of which is incorporated herein by reference.

[0006] GOVERNMENT SUPPORT

[0007] This invention was made with government support under 70NANB21H086 awarded by the National Institute of Standards and Technology. The US government has certain rights in the invention.

[0008] BACKGROUND

[0009] The discussion of the background state of the art below may reflect hindsight gained from the disclosed invention(s), and these characterizations are not necessarily admitted to be prior art.

[0010] Monoclonal antibodies (mAbs) produced by mammalian cell culture represent a significant part of modern therapeutics, and mAbs are many of the largest global drugs by revenue. For a production-scale perfusion bioreactor, cell-culture media costs could total over $200,000 per run, and material costs tend to assume an increasingly larger part of the cost of goods as processes scale up. As patents expire and generic versions of many prominent therapies begin to enter the market, it is increasingly important to optimize antibody production by utilizing continuous manufacturing methods, such as perfusion culture.

[0011] Perfusion cell culture processes offer the potential for higher cell density and productivity compared to batch processes. Intensified perfusion cultures consume large amounts of media, sometimes exchanging multiple bioreactor volumes per day. Higher bioreactor productivity allows perfusion culture to hit a lower media cost per gram of mAb than batch processes. However, perfusion bioreactors produce a large volume of spent cell-culture media that is only partially depleted of nutrients and still contains expensive growth factors. Analysis comparing perfusion and fed-batch setups has indicated that perfusion has an economic advantage but is often worse for the environment due to water consumption and single-use consumables. A recent interest in reducing the environmental footprint of bioreactors has led to new metrics for measuring the efficiency of a process, such as process mass intensity (PMI). Calculating PMI is done by summing the mass of raw materials used in a process and dividing that sum by the mass of pharmaceutical products. Water is most of the input mass in perfusion culture, so reusing spent cell-culture media could further drive down the cost Attorney Docket No. mit-25891 pct per gram of mAbs and reduce the water-intensive environmental impact of the biomanufacturing process.

[0012] Efforts have been made in the past to recycle and reuse cell-culture media, either as a total replacement or by mixing it with fresh cell-culture media as a supplement. Mammalian cell-culture waste can be repurposed as growth media for bacteria, such as E. coli, with the goal of producing recombinant proteins, such as insulin. Many previous studies have explored methods of filtering leftover batch culture media and using it in subsequent batch culture.

[0013] Recently, there has been a demonstration of reusing media from completed perfusion cultures in subsequent mock-perfusion cultures, as a replacement for cellculture media or as a nutrient supplement. For example, S.R. Madabhushi, et al., “An innovative strategy to recycle permeate in biologies continuous manufacturing process to improve material efficiency and sustainability,” 38 Biotechnol. Prog. E3262 (16 April 2022), reported on recycling the flow-through from Protein-A antibody harvesting columns and demonstrated that 25-50% media recycling ratios produced decreases in productivity of only 13-30% (Madabhushi, et al.). Therefore, antibody production can be decreased by recycling, yet more efficient use of materials can lead to lower PMI, with a reduction of up to 9.8% achieved by Madabhushi, et al.

[0014] Though spent cell-culture media contains leftover nutrients that can be utilized, it also contains waste products that can inhibit growth and productivity. Ammonium (NH4+) is one of the key waste products of the Chinese hamster ovary (CHO) metabolism, and it has been demonstrated that minimizing ammonium concentration is important to achieving high cell densities and an ideal glycosylation profile. Lactate is another such waste product that can be harmful if allowed to accumulate. However, lactate can also be a source of carbon utilized by the tricarboxylic acid (TCA) cycle, and lactate consumption can even be adjusted to minimize ammonium production. The currently employed method for recycling cell -culture media (filtration) is not adequate for removing these two disadvantageous (and charged) waste products from the spent culture media, which would ultimately limit the utility of cell-culture media recycling. Accumulating waste that negatively impacts cell metabolism would undermine any benefit provided by recycling spent cell-culture media. Therefore, it would be ideal to develop methods for removing waste prior to reusing cell-culture media.

[0015] There has been prior exploration of strategies for removing waste from culture media in an effort to improve cell density and productivity. Waste removal during the culture has been explored by using electric fields to drive ammonium and lactate through permeable membranes and away from cells, yielding increased cell density [Y.D. Chang, et al., “In-situ removal of ammonium and lactate through electrical means for hybridoma cultures,” 47 Biotechnol. Bioeng. 308-318 (1995) and Y.D. Chang, et al., Attorney Docket No. mit-25891 pct

[0016] “Nutrient enrichment and in-situ waste removal through electrical means for hybridoma cultures,” 47 Biotechnol. Bioeng. 319-326 (1995)]. Removal through cation exchange membranes has been explored via dialysis, in which ammonium diffuses through the membrane into a gaseous phase [J. Thommes, etal., “Integrated Detoxification: Reduction of Ammonium Concentration by Dialysis with Cation Exchange Membranes,” Animal Cell Technology 171-175 (1992)].

[0017] SUMMARY

[0018] Systems and methods for processing spent cell-culture media from a bioreactor are described herein, where various embodiments of the systems and methods may include some or all of the elements, features, and steps described below.

[0019] These systems and methods involve a new use for ion-concentration-polarization (ICP) systems, in which spent cell-culture media from a bioreactor is processed by a device that removes waste products while retaining leftover nutrients. In a device with alternating layers of cation exchange membrane and millifluidic spacer layers, we can apply an electric field to move charged waste products, such as ammonia and lactate, to a concentrated waste stream while creating a harvest stream that contains low amounts of waste but retains nutrients, such as amino acids e.g., a majority of the charged waste products are separated into the concentrated waste stream while a majority of the nutrients are retained with the spent cell-culture medium in the harvest stream).

[0020] The systems and methods can remove waste produced, e.g., by Chinese hamster ovary (CHO) or other cell metabolisms from spent cell-culture media while retaining leftover nutrients, enabling the recycling of spent cell-culture media without accumulating waste products that inhibit cell growth and antibody production. A problem that can be solved by the systems and methods is the material waste / inefficiency that is common in perfusion bioreactor systems. Perfusion bioreactors create large amounts of waste that is typically disposed of after antibody harvest; but this waste contains many nutrients, especially if the rate of cell-culture media consumption for the bioreactor is high. There is a potential economic and environmental benefit to removing waste from spent cell-culture media and recycling it. Using cell-culture media that would otherwise be discarded to produce valuable monoclonal antibodies has the potential to reduce the production cost (i.e., cost / mass, e.g., $ / g) for some drugs. Additionally, recycling the cell-culture media would reduce the amount of purified water and other raw materials involved in perfusion, thereby reducing the environmental impact.

[0021] The systems and methods differ from existing solutions because there are currently two known options associated with spent cell-culture media produced by perfusion bioreactors. First, the primary current practice is to simply dispose of the Attorney Docket No. mit-25891 pct spent cell-culture media, although it contains leftover nutrients; our systems and methods can reduce this wasteful practice. Second, some research is being conducted into recycling spent cell-culture media without any additional processing; our process differs because we remove waste products, preventing the accumulation of molecules, such as ammonia, that inhibit cell metabolism.

[0022] Challenges were encountered in adapting the operation of the ion concentration polarization device to work with cell-culture media. Previously, when ion concentration polarization devices were used for desalination, the operating parameters were optimized for minimizing the salt content of the filtrate stream while balancing energy efficiency. In comparison, when using ion concentration polarization on cell-culture media, the operating conditions are set to maximize ammonia and lactate removal while not significantly altering the pH of the filtrate stream. Large changes in pH may lead nutrients that are typically neutral to acquire charge and, therefore, begin to be removed by our device. Thus, our use of ion concentration polarization faces different challenges than previous desalination work because we are interested in not just the removal of waste but also the retention of nutrients.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 is a photograph of a fully assembled device with various features labeled.

[0025] FIG. 2 is an illustration describing the transport of waste products, metabolites, and electrolytes during the ICP separation process in one layer of the device. Black dashed lines represent a semi-permeable membrane. UF represents flow velocity, while Uc+ and UA- represent the flux of cations and anions, respectively. AA+ and AA- represent positive and negatively charged amino acids, respectively.

[0026] FIG. 3 plots measurements of the electrical current and voltage while processing fresh cell-culture media as a function of time.

[0027] FIG. 4 plots the measurements of the change in conductivity at the harvest outlet.

[0028] FIG. 5 plots viable cell density (VCD) over a fifteen-day-period

[0029] FIG. 6 plots cell viability for the microfluidic cell retention perfusion culture described in the section under Materials and Methods, subtitled, Perfusion culture of CHO cells.

[0030] FIGS. 7-12 shows measurements of ammonium-spiked fresh cell-culture media from the harvest and waste stream of sodium (FIG. 7), potassium (FIG. 8), ammonium (FIG. 9), glucose (FIG. 10), glutamate, (FIG. 11), and glutamine (FIG. 12). FIGS. 13-20 plot the results, over a six-day period of time, of a batch CHO culture using varying mixtures of fresh, regenerated, and spent cell-culture media. FIG. 13 plots the viable cell density. FIG. 14 plots cell viability (%). FIG. 15 plots ammonium concentration. FIG. 16 Attorney Docket No. mit-25891 pct plots lactate concentration. FIG. 17 plots glutamine concentration. FIG. 18 plots glucose concentration. FIG. 19 plots glutamate concentration. FIG. 20 plots antibody titer concentration. Closed markers represent mixtures with regenerated cell-culture media processed by the ICP separation device, open markers represent mixtures with unprocessed spent cell-culture media.

[0031] FIGS. 21-28 plot the results of mock-perfusion CHO culture, over six days, using varying mixtures of fresh, regenerated, and spent cell-culture media. FIG. 21 plots viable cell density. FIG. 22 plots cell viability (%). FIG. 23 plots ammonium concentration. FIG. 24 plots lactate concentration. FIG. 25 plots glutamine concentration. FIG. 26 plots glucose concentration. FIG. 27 plots glutamate concentration. FIG. 28 plots antibody titer concentration. Closed markers represent mixtures with regenerated cell-culture media processed by the ICP separation device, open markers represent mixtures with unprocessed spent cell-culture media.

[0032] FIGS. 29-32 plot normalized monoclonal antibody (mAb) harvest for recycling cultures with 25%, 50%, and 75% recycled cell culture. FIG. 29 plots the normalized monoclonal antibody (mAb) harvest for a batch process. FIG. 30 plots the normalized monoclonal antibody (mAb) harvest for mock perfusion, where normalized water process mass intensity (PMI) changes are compared with cultures generated with 100% fresh cell-culture media. FIG. 31 plots the change in PMI for a batch process. FIG. 32 plots the change in PMI for mock-perfusion.

[0033] FIG. 33 includes simplified schematics for a secondary batch culture.

[0034] FIG. 34 includes simplified schematics for a secondary perfusion culture.

[0035] FIG. 35 includes simplified schematics for self-recycling perfusion.

[0036] FIG. 36 and 37 plot measured levels of ammonium [FIG. 36] and lactate [FIG. 37] for spent cell-culture media and regenerated cell-culture media processed by ICP waste separation. Spent cell-culture media was generated by the microfluidic cell retention perfusion culture described in the section under Materials and Methods, subtitled, Perfusion culture of CHO cells.

[0037] FIG. 38 and 39 plot measured levels of ammonium [FIG. 38] and lactate [FIG. 39] for spent cell-culture media and regenerated cell-culture media processed by ICP waste separation. Low-ammonia spent cell-culture media was generated by a 1L perfusion culture of NIH VRC01 CHO Ki cells in AMBIC 1.1 media that lasted 56 days.

[0038] FIGS. 40-46 plot the results of mock-perfusion CHO culture using varying mixtures of fresh, regenerated, and spent cell-culture media over a four-day period. FIG. 40 plots viable cell density (VCD). FIG. 41 plots cell viability (%). FIG. 42 plots ammonium concentration. FIG. 43 plots lactate concentration. FIG. 44 plots glutamine concentration. FIG. 45 plots glucose concentration. FIG. 46 plots glutamate concentration. Attorney Docket No. mit-25891 pct

[0039] In the accompanying drawings, like reference characters refer to the same or similar parts throughout the different views; and apostrophes are used to differentiate multiple instances of the same item or different embodiments of items sharing the same reference numeral. The drawings are not necessarily to scale; instead, an emphasis is placed upon illustrating particular principles in the exemplifications discussed below. For any drawings that include text (words, reference characters, and / or numbers), alternative versions of the drawings without the text are to be understood as being part of this disclosure; and formal replacement drawings without such text may be substituted therefor.

[0040] DETAILED DESCRIPTION

[0041] The foregoing and other features and advantages of various aspects of the invention(s) will be apparent from the following more-particular description of various concepts and specific embodiments within the broader bounds of the invention(s). Various aspects of the subject matter introduced above and discussed in greater detail below may be implemented in any of numerous ways, as the subject matter is not limited to any particular manner of implementation. Examples of specific implementations and applications are provided primarily for illustrative purposes.

[0042] Unless otherwise herein defined, used, or characterized, terms that are used herein (including technical and scientific terms) are to be interpreted as having a meaning that is consistent with their accepted meaning in the context of the relevant art and are not to be interpreted in an idealized or overly formal sense unless expressly so defined herein. For example, if a particular composition is referenced, the composition may be substantially (though not perfectly) pure, as practical and imperfect realities may apply; e.g., the potential presence of at least trace impurities (e.g., at less than i or 2%) can be understood as being within the scope of the description. Likewise, if a particular shape is referenced, the shape is intended to include imperfect variations from ideal shapes, e.g., due to manufacturing tolerances. Percentages or concentrations expressed herein can be in terms of weight or volume. Processes, procedures, and phenomena described below can occur at ambient pressure (e.g., about 50-120 kPa— for example, about 90-110 kPa) and temperature (e.g., -20 to 5O°C— for example, about 10- 35°C) unless otherwise specified.

[0043] Although the terms, first, second, third, etc., maybe used herein to describe various elements, these elements are not to be limited by these terms. These terms are simply used to distinguish one element from another. Thus, a first element, discussed below, could be termed a second element without departing from the teachings of the exemplary embodiments. Attorney Docket No. mit-25891 pct

[0044] Spatially relative terms, such as “above,” “below,” “left,” “right,” “in front,” “behind,” and the like, may be used herein for ease of description to describe the relationship of one element to another element, as illustrated in the figures. It will be understood that the spatially relative terms, as well as the illustrated configurations, are intended to encompass different orientations of the apparatus in use or operation in addition to the orientations described herein and depicted in the figures. For example, if the apparatus in the figures is turned over, elements described as “below” or “beneath” other elements or features would then be oriented “above” the other elements or features. Thus, the exemplary term “above” may encompass both an orientation of above and below. The apparatus may be otherwise oriented (e.g., rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. The term “about” can mean within ±10% of the value recited. In addition, where a range of values is provided, each subrange and each individual value between the upper and lower ends of the range is contemplated and, therefore, disclosed.

[0045] Further still, in this disclosure, when an element is referred to as being “on,” “connected to,” “coupled to,” “in contact with,” etc., another element, it may be directly on, connected to, coupled to, or in contact with the other element, or intervening elements may be present unless otherwise specified.

[0046] Some of the terminology used herein is associated with particular embodiments and is not intended to limit more generic exemplifications of the invention. As used herein, singular forms, such as those introduced with the articles, “a” and “an,” are intended to include the plural forms as well, unless the context indicates otherwise. Additionally, the terms “includes,” “including,” “comprises,” and “comprising” specify the presence of the stated elements or steps but do not preclude the presence or addition of one or more other elements or steps.

[0047] Additionally, the various components identified herein can be provided in an assembled and finished form; or some or all of the components can be packaged together and marketed as a kit with instructions (e.g., in written, video, or audio form) for assembly and / or modification by a customer to produce a finished product.

[0048] In modern bioprocessing, cell-culture media is one of the largest cost drivers, even if all the nutrients and other critical factors in the cell-culture media are often not fully utilized. With the renewed emphasis on reducing the cost of bioprocessing, there is much interest in ways to reduce the overall use of cell-culture media.

[0049] In this work, we introduce a mesoscale microfluidic separation device [based on the ion concentration polarization process (ICP)J to regenerate the spent cell-culture media for reuse by removing critical waste products from the cell culture that are known to inhibit the growth of the cells. We demonstrated that up to 75% of spent cellculture media can be regenerated and reused without affecting the cell viability. Plus, a Attorney Docket No. mit-25891 pct detailed analysis of the materials consumed during antibody production indicated that one could improve the water process mass intensity by up to 33% by regenerating and recycling the cell-culture media. Given that ICP separation systems have already been scaled up to support large-volume processing, it would be feasible to deploy this technology for manufacturing-scale bioreactors e.g., 50L perfusion culture of CHO cells), reducing the overall cost.

[0050] We have drawn on previous waste removal methods to build a “milli-fluidic” device that uses electric fields to drive ammonium and lactate into a waste stream that can be discarded while producing a harvest stream that is lower in waste concentration but not depleted of nutrients. To our knowledge, this is the first demonstration of a continuous waste removal method that can be integrated into perfusion culture without modifying any part of the bioreactor setup because our device is placed in the waste stream that is typically discarded.

[0051] This study demonstrates an electrokinetic (EK) separation method using ion concentration polarization (ICP) to remove ammonium and lactate from spent CHO media, a method built upon previous ICP desalination and return-flow ICP devices [B. Kim, et al., “Purification of high salinity brine by multi-stage ion concentration polarization desalination,” Sci. Rep. 6 ( 2016); B. Kim, et al., “Partial desalination of hypersaline brine by lab-scale ion concentration polarization device,” 412 Desalination 20-31 (2017); J. Yoon et al., “Return flow ion concentration polarization desalination: A new way to enhance electro membrane desalination,” 159 Water Res. 501-510 (2019); J. Yoon, et al., “Portable Seawater Desalination System for Generating Drinkable Water in Remote Locations,” Environ. Sci. Technol. (2021); US 10,252,924 B2; and US 11,780,754 B2]. The effect of ICP separation on leftover nutrients, pH, and osmolarity was measured; and a process for conditioning regenerated cell-culture media was developed. ICP-Processed cell-culture media were mixed in varying ratios with fresh cell-culture media to characterize the impact on cell density and productivity in batch and mock perfusion cultures. This method of separation, conditioning, and reuse presents an opportunity to utilize cell-culture media that would otherwise be disposed of and, therefore, reduce the overall amount of material input involved in perfusion culture.

[0052] Materials and Methods:

[0053] Materials and reagents:

[0054] Sodium chloride, calcium chloride, potassium chloride, sodium sulfate, hydrochloric acid, and ammonium chloride were purchased from Sigma Aldrich in St. Louis, Missouri, US. Cation exchange membranes were purchased from Fujifilm in Valhalla, New York, US, and from Ralex International Corp, in Doral, Florida, US. Attorney Docket No. mit-25891 pct

[0055] Manufacturing and operation of ICP separation device:

[0056] The ICP separation device stacks alternating layers of cation exchange membranes (CEMs) and return-flow spacer layers. The spacer layers can be in the form of semi-permeable membranes that restrict the flow of fluid therethrough without significantly restricting the flow of ions through the membranes. Spacers used in return-flow configurations can be in the form of a middle plastic layer sandwiched between two semi-permeable membranes that restrict fluid flow but allow the movement of ions. Fluid flows from the inlet of the middle layer until it reaches the end of the channel, where there is a bifurcated path that allows the fluid to return on the other side of the semi-permeable membrane. Additional details of the spacer construction is described in previous work (e.g., Kim, et al., 2017; Yoon, et al., 2.019 US 2023 / 0416122 Al; and US 11,780,754 B2). Fujifilm type 10 CEMs were used between the spacer layers, and Ralex CMHPES layers were placed between the electrode rinsing layers and the rest of the device. The electrode layers contain titanium plates coated with Ru-Ir and are constantly rinsed at 0.8 L / min with a 1-M sodium sulfate solution. The return-flow spacer layers were laser cut to have a three-layered channel measuring approximately 15 cm by 5 cm, with each layer separated by a microporous membrane. Plastic mesh is placed in the intermediate channel, the harvest channel, and the waste channel for mechanical stability and to stabilize the electroosmotic flow generated near the CEM surface. Laser-cut silicone gaskets placed between the spacer layers and the CEM seal the device. The use of CEMs— but not anion exchange membranes (AEMs)— delays fouling, as CEMs tend to be less prone to fouling than AEMs.

[0057] Voltage and electrical current were supplied and measured using a BK Precision 9205B power supply, and the conductivity of the harvest stream was measured using an Orion Versa Star Pro Benchtop meter. Spent cell-culture media was pumped into the device using a Cole Parmer MASTERFLEX Peristaltic pump, and a 12V DC brushless motor pump was used for the rinse solution. Flowmeters and control valves were placed at the harvest and waste outlets and adjusted such that the flow rates were equivalent. Control of the power supply and automated measurements were handled using a Python program.

[0058] Perfusion culture of CHO cells:

[0059] To generate spent cell-culture media for regeneration and culture experiments, a 500-ml bioreactor (from Getinge Applikon of Delft, Netherlands) was employed for cultivating CHO cells that produce monoclonal antibodies (from Sartorius Stedim Biotech GmbH of Gottingen, Germany). A microfluidic cell retention device was utilized to facilitate perfusion culture (T. Kwon, et al., “Continuous removal of small nonviable suspended mammalian cells and debris from bioreactors using inertial microfluidics,” Attorney Docket No. mit-25891 pct

[0060] 18 Lab Chip 2829-2837 (2018); L. Yin, et al., “Miniature auto-perfusion bioreactor system with spiral microfluidic cell retention device,” 118 Biotechnol. Bioeng. 1951-1961 (2021); and WO 2021 / 188269 Al). The CHO cells were cultured in 4CELL SMARTCHO production medium (CQP3FB2150 production medium from Sartorius Stedim Biotech GmbH), supplemented with feed cell-culture media (CQP3FB3152 and CQP3FC4104 feed cell-culture media from Sartorius Stedim Biotech GmbH). Inoculation of the bioreactor occurred at a cell density of 0.5 x 106cells / ml. An integrated controller managed dissolved oxygen and pH, maintaining them at 60% of air saturation and 7.2 ± 0.1, respectively. Following two days of batch-mode culture, perfusion commenced at a rate of 0.5 vessel volume / day (WD) and was later increased to 1.5 WD. The perfusion rate was adjusted to sustain glucose concentration at 3-5 g / L and ammonium concentration below 5 mmol / L. For foam control, a solution of Antifoam C Emulsion (from Sigma-Aldrich) diluted to 0.2% with deionized water was added as needed. The bioreactor was run for 16 days with the harvest cell-culture media collected, filtered, and stored every 24 hours. Viable cell density and viability during the run are respectively shown in FIGS. 5 and 6.

[0061] Ammonium removal from fresh cell-culture media:

[0062] Ammonium chloride was mixed into fresh SMARTCHO production media (from Sartorius) at a level of 6 mM, a concentration chosen to be similar to the highest ammonium level typically measured in spent cell-culture media from bioreactors. For each operating condition, we waited for the electrical current and conductivity to reach a steady state before taking samples from the harvest and waste stream of the device. Metabolites, ammonium, and background electrolytes were measured using a FLEX 2 Cell Culture Analyzer (from Nova Biomedical, Waltham, MA).

[0063] Recycling in batch and mock perfusion CHO culture:

[0064] Spent cell-culture media from our perfusion bioreactor was processed at a flow rate of 10 mL / min using 0.75 A. The pH of the harvest was measured using an Orion Star Versa Star Pro benchtop meter, and hydrochloric acid was added to adjust the pH so that it matched fresh cell-culture media. Background electrolytes were measured using a FLEX 2 Cell Culture Analyzer, and sodium chloride, potassium chloride, and calcium chloride were added to processed cell-culture media to match concentrations found in fresh cell-culture media. Regenerated cell-culture media was sterilized with a 0.2-pm filter (from Thermo Fisher Scientific, Waltham, MA) prior to use in culture.

[0065] For mock-perfusion cultures, six-well plates (from Corning Inc., Corning, NY) were used for mAb-producing CHO cells inoculated at 1 x 106cells / mL in 5 mL per well and cultured at 36.8°C and 7.5% CO2 in an incubator with an orbital shaker. Cell density, viability, and metabolites were measured every 24 hours using a Flex 2 Cell Attorney Docket No. mit-25891 pct

[0066] Culture Analyzer. During mock perfusion culture, the plates were centrifuged at 1,000 rpm for 5 minutes, and then two-thirds of the cell-culture media was replaced with fresh cell-culture media before resuspending the pellet. The same parameters were used for batch cultures, except cells were inoculated at 0.2 x 106 cells / mL in 5 mL per well.

[0067] Results:

[0068] Device characterization with ammonium-spiked fresh cell-culture media:

[0069] The assembled ICP separation device 10, including 5 channel layers, is pictured in FIG. 1, which shows the cathode 12, anode 14, electrical contact 16, inlet 18, harvest outlet 20, and waste outlet 22. To characterize the effect of separation upon waste products, metabolites, and background electrolytes, we varied the flow rate and electrical current used to operate the device while measuring concentrations of analytes at the harvest and waste outlets of the device. For each set of operating conditions, we waited until a steady state was measured for the current 48, voltage 50, and conductivity measured at the harvest outlet, as seen in FIGS. 3 and 4. Flow rates of 5, 10, and lsmL / min were tested, and the range of electrical currents used at each flow rate was adjusted such that when the flow rate was doubled, the electrical current was also doubled to maintain a similar level of waste removal even at different flow rates.

[0070] With the inlet 34, harvest 33, and waste 31 flows on opposite sides of the internal membranes 28 shown in FIG. 2, it is expected that the efficiency of the removal for each ionic species is correlated to the electrophoretic mobility; and, therefore, molecules with higher electrophoretic mobility (smaller ions and metabolites) will generally be more significantly depleted than larger molecules and species with lower electrophoretic mobility. The internal membranes 28 are part of the spacers, which also include plastic layers 35 that define channels for fluid flow, as shown in FIGS. 53 and 54, on opposite sides of the internal membranes 28. As shown therein, positively charged amino acids (AA+), sodium (NH4+), and sodium (Na+) cations are drawn through the top internal membrane 28 and the upper cation exchange membrane 26 by the electric field generated by the cathode 12, while anionic species, including chlorine (Cl ), lactate (Lac), and negatively charged amino acids (AA ) are drawn through the bottom internal membrane 28 into the waste stream 31 flowing between the bottom internal membrane 28 and the lower cation exchange membrane 26. A voltage source (power supply) 27 provides the opposing charges to the anode 12 and cathode 14 to which it is electrically connected to provide the electric field across the device 10.

[0071] FIG. 7 shows that, for certain operating conditions, the concentration of sodium in the harvest stream decreases below the detection limit of the bioanalyzer. Plotted therein, as a function of electric current between the electrodes through the device, are the concentrations of sodium ions (Na+) in FIG. 7, potassium ions (K+) in FIG. 8, Attorney Docket No. mit-25891 pct ammonia ions (NH4+) in FIG. 9, glucose in FIG. 10, glutamate in FIG. 11, and glutamine in FIG. 12 at harvest-stream flow rates of 5 ml / min. 54, 10 ml / min. 56, and 15 ml / min. 58 and at waste-stream flow rates of 5 ml / min. 60, 10 ml / min. 62, and 15 ml / min. 64. The concentration of potassium was reduced by up to 84%, as seen in FIG. 8. FIG. 9 shows that ammonium was significantly removed, with up to 83% reduction at conditions of 15 ml / min and 1.5 A. Neutral molecules, such as glucose, are not significantly removed at the same conditions at which ammonium was most reduced, as seen in FIG. 10.

[0072] In these plots, open markers with dotted lines represent levels of sodium too low to be measured by the FLEX 2 Bioanalyzer from Nova Biomedical. For this experiment we took fresh cell-culture media (with no ammonium) and ’spiked’ it by adding ammonium to reach a concentration similar to spent cell-culture media from our perfusion bioreactor. FIGS. 7-12 show that, at many of the operating conditions tested, this device significantly decreases the concentration of ammonia in the harvest stream. Background electrolytes are also significantly removed but can be re-added to the harvest stream. Nutrients with neutral charge are not removed from the harvest stream unless the device is operated at a high electrical current that significantly changes the pH inside the device. Accordingly, FIGS. 7-12 show the removal of charged waste products (ammonium) while retaining neutral nutrients, such as glucose and most amino acid. In other exemplifications, this apparatus and methods maybe used to remove charged impurities such as DNA or host cell proteins (HCPs) in addition to or instead of removing ammonium (and, optionally, lactate).

[0073] Charged metabolites, such as glutamate, are also removed, though at a lower rate than ammonium due to their lower electrophoretic mobility, with FIG. 27, which is discussed below, showing a peak reduction of 68%.

[0074] Neutral amino acids, such as glutamine, are also removed at a lower rate than ammonium; however, as FIG. 25 shows, operating conditions with high current begin to significantly separate these molecules. It is likely that increases in pH near the CEM surface of the harvest stream are caused by selective transport of hydrogen ions through the CEM. A significant pH shift would cause typically neutral amino acids, such as glutamine, to acquire charge as the pH of the solution rises above their pl value (pl = 5.65), leading to their removal by the ICP process. Measurements of the waste stream showed comparable increases in the concentration of glutamate and other neutral metabolites (at normal pH), corroborating our hypothesis on the removal of glutamate.

[0075] Batch culture with regenerated and spent cell-culture media:

[0076] FIGS. 13-20 include plots for viable cell density (VCD) (FIG. 13), cell viability (FIG. 14), ammonium (FIG. 15), lactate (FIG. 16), glutamine (FIG. 17), glucose (FIG. 18), glutamate (FIG. 19), and monoclonal antibody (mAb) titer (20) over six days from Attorney Docket No. mit-25891 pct feeds of (a) 100% fresh culture media 66, (b) 75% fresh and 25% recycled culture media 68, (c) 50% fresh and 50% recycled culture media 70, (d) 25% fresh and 75% recycled culture media 72, (e) 75% fresh and 25% spent culture media 80, (f) 50% fresh and 50% spent culture media 82, (g) 25% fresh and 75% spent culture media 84.

[0077] In batch culture experiments, the regenerated cell-culture media outperformed every mixture of spent cell-culture media with respect to cell viability and peak viable cell density (VCD). FIG. 14 shows that cell viability for all cultures using regenerated cell-culture media stayed above 90%, while cultures with 75% spent cell-culture media declined to 38% viability. Additionally, when replacing 25% of fresh cell-culture media with regenerated cell-culture media, there is virtually no difference in the peak VCD reached (FIG. 13). Replacing 50% fresh cell-culture media with regenerated cell-culture media decreased peak VCD by only 24%. The fresh and regenerated media cultures showed similar metabolic flux for ammonium, lactate, glutamine, glucose, and glutamate (FIGS. 15-19), while some cultures with spent cell-culture media produced excess waste and reduced the rate of glucose consumption.

[0078] Antibody production showed similar advantages of regenerated cell-culture media over spent cell-culture media (FIG. 20), but the antibody titer was not directly correlated with VCD. For example, the culture with 25% spent cell-culture media and culture with 75% regenerated cell-culture media reached a similar peak VCD of ~4M / mL, but the antibody titer of the regenerated culture was significantly lower. This mismatch between VCD and productivity indicates a likelihood that regenerated cellculture media contains sufficient metabolites to allow cells to grow to high density, but there might be a reduction of one or multiple components that are critical for antibody production.

[0079] Mock-perfusion with regenerated and spent cell-culture media:

[0080] In mock-perfusion culture with 0.67 WD, the regenerated cell-culture media outperformed spent cell-culture media at all mixing ratios once the culture reached high density (~5 M / mL) on day 4 where accumulated waste in unprocessed spent cellculture media inhibited cell growth. Replacing 25% of fresh cell-culture media with regenerated reduced peak VCD by 12%, while replacing the same amount with spent cell-culture media reduced peak VCD by 35% (FIG. 21). Cell viability for all test conditions (both with regenerated and spent cell-culture media, at different mixing ratios) remained above 90%, except the mixture of 75% spent cell-culture media (FIG. 22). Replacing half of the fresh cell-culture media with regenerated cell-culture media reduced the peak VCD by 29% while replacing with spent cell-culture media reduced the peak VCD by 8%. In general, percentage reductions in peak VCD using regenerated cell-culture media are smaller than the percentage of fresh cell-culture media replaced. Attorney Docket No. mit-25891 pct

[0081] Metabolite profiles for ammonium, lactate, glutamine, glucose, and glutamate are shown in FIGS. 23-27.

[0082] In mock-perfusion, regenerated cell-culture media had little advantage over spent cell-culture media for antibody production (FIG. 28), with only the 50% mixture ratio showing a significant increase in antibody titer. The mock-perfusion setup already reduces waste accumulation, so antibody production in most of the culture conditions is not waste-inhibited but nutrient-constrained, leading to similar performance of regenerated and spent cell-culture media.

[0083] PMI Analysis and Projection:

[0084] To understand the impact of cell-culture media recycling on antibody production efficiency, we analyzed the water process mass intensity (PMI) of the cultures. This calculation was performed by dividing the mass of fresh cell-culture media used in each culture by the mass of antibody produced and then normalizing and comparing values to the 100% fresh cell-culture media culture. Recycling spent or regenerated cell-culture media in batch or mock-perfusion conditions decreases antibody production, as shown in FIGS. 29 and 30, respectively, which plot the normalized monoclonal antibody (mAb) harvest for culture media with 25% recycled media 68, with 50% recycled media 70, and with 75% recycled media for spent and regenerated media. Despite the decreased productivity, all culture conditions with regenerated cell-culture media led to a reduced water PMI in batch and mock-perfusion (FIGS. 31 and 32), with a PMI reduction of up to 33% achieved in batch culture. Spent cell-culture media, by comparison, produced higher PMI values for all mixture ratios except 75% recycled, compared with the case using 100% fresh cell-culture media.

[0085] To explore strategies for utilizing cell-culture media regeneration, we created PMI projections (Table 1, below) for three potential scenarios depicted in FIGS. 33-35, the details of which are described below. The first two scenarios involve running a primary perfusion culture and then using the spent cell-culture media in separate secondary batch and perfusion cultures, respectively. The first two scenarios require additional equipment and other raw materials to run the secondary culture, so a third scenario was considered extending a perfusion culture by self-recycling spent cellculture media.

[0086] In FIG. 33, fresh media 98 is fed through a conduit into a vessel for a primary perfusion culture 86. The spent media 95 from the primary perfusion culture 86 is withdrawn through a conduit to a chromatography column 90 that produces a downstream flow 92 that is removed via one conduit and an additional product that includes the culture media and that is directed through another conduit to an ICP waste separation device 10 that produces a waste stream 31 and regenerated culture media 94 Attorney Docket No. mit-25891 pct that is directed through a conduit into a vessel containing a secondary batch culture 88 to which fresh media 98 is also fed via another conduit.

[0087] The apparatus of FIG. 34 is similar to that of FIG. 33, except a vessel with a secondary perfusion culture 100 is substituted for the vessel for the secondary batch culture 88 of FIG. 33. The apparatus of FIG. 35, meanwhile, provides for self-recycling perfusion, utilizing a continuous chromatography column 90 and an ICP waste separation device 10 that releases regenerated media through a column directly back into a vessel containing a self-recycling perfusion culture 102.

[0088] Table 1: Projections of water PMI reduction for varying recycling strategies:

[0089] Ammonium and Lactate Reduction for Spent Media Processed by ICP Separation Device:

[0090] Measured levels of ammonium (FIG. 36) and lactate (FIG. 37) for spent media 95 and regenerated media 94 processed by ICP waste separation are plotted in FIGS. 36 and 37. Spent media was generated by the microfluidic cell retention perfusion culture described above.

[0091] Calculations for Projected PMI of Varying Recycling Strategies:

[0092] Scenario 1:

[0093] The antibody titer measured on the final day for each batch culture was used to estimate the total productivity per liter of a secondary batch culture done with each mixture ratio of regenerated and spent cell-culture media. The antibody production was calculated for a secondary batch culture with 100% fresh cell-culture media that used the same amount of fresh cell-culture media involved in each recycled media mixture ratio. The change in water PMI was calculated by comparing the PMI values of the equivalent fresh volume secondary batch culture for each mixture ratio of recycled secondary batch culture. Attorney Docket No. mit-25891 pct

[0094] Scenario 2:

[0095] The antibody titer measured on each day of mock-perfusion culture was multiplied by the cell-culture media replacement rate (0.667 WD) to estimate the total productivity per liter of a secondary perfusion culture done with each mixture ratio of regenerated and spent cell-culture media. The antibody production was calculated for a secondary perfusion culture with 100% fresh cell-culture media that used the same amount of fresh cell-culture media involved in each recycled media mixture ratio. The change in water PMI was calculated by comparing the PMI values of the equivalent fresh volume secondary perfusion culture for each mixture ratio of recycled secondary perfusion culture.

[0096] Scenario 3:

[0097] The antibody titer measured on the final day of mock-perfusion culture for each recycling mixture ratio was used to estimate the productivity of an existing perfusion culture run extended with recycled cell-culture media. The antibody production was calculated for a perfusion culture with 100% fresh cell-culture media that used the same amount of fresh cell-culture media involved in each recycled cell-culture media mixture ratio. The change in water PMI was calculated by comparing the PMI values of the equivalent fresh volume perfusion culture for each mixture ratio of recycled perfusion culture.

[0098] Ammonium and Lactate Reduction for Low-Ammonium Spent Media Processed by ICP Separation Device:

[0099] Measured levels of ammonium and lactate for spent media and regenerated media processed by ICP waste separation are respectively plotted in FIGS. 38 and 39. Low-ammonia spent media was generated by a 1L perfusion culture of NIH VRC01 CHO Ki cells in AMBIC 1.1 media that lasted 56 days.

[0100] Mock-Perfusion with Low-Ammonium Spent Media:

[0101] Results of mock-perfusion Chinese hamster ovary (CHO) culture use varying mixtures of fresh, regenerated, and spent media over four days are shown in FIGS. 40- 46 for 100% fresh media 66, 75% fresh media and 25% recycled media 68, 50% fresh media and 50% recycled media 70, 75% fresh media and 25% spent media 80, and 50% fresh media and 50% spent media 82. Viable Cell Density (VCD) is plotted in FIG. 40. Percent cell viability is plotted in FIG. 41. Ammonium concentration is plotted in FIG. 42. Lactate concentration is plotted in FIG. 43. Glutamine concentration is plotted in FIG. 44. Glucose concentration is plotted in FIG. 45. Glutamate concentration is plotted in FIG. 46. Attorney Docket No. mit-25891 pct

[0102] An embodiment of the device 10 without an endplate 29 that would generate a counterflow across the internal membrane 28 (a single internal membrane 28 on the cathode side is employed in this exemplification) such that the flow is linear through this device 10, where ammonia (NH4+) is extracted through the upper cation exchange membrane 26, while the remainder of the feed, including dissolved salts, is extracted in a singular output flow 106 to maximize the recovery of culture media.

[0103] Self-Recycling Perfusion Culture Process:

[0104] A system for conducting a self-recycling perfusion culture process is shown in FIG. 48. A container of feed composition 104 including cell-culture media is provided with a conduit, including a flow-control valve extending between the source of feed composition 104 and a self-recycling perfusion culture bioreactor 102 for growing mammalian cells. Another conduit extends between the self-recycling perfusion culture bioreactor 102 (here, a soomL stirred tank bioreactor from Applikon) and is configured to provide for the flow of spent cell-culture media with cells to a cell retention device no. The cell retention device no separates the cells from the culture medium and returns the cells to the self-recycling perfusion culture bioreactor 102 via another conduit. The separated spent culture, meanwhile, is extracted from the cell retention device 110 and passed via a conduit to a culture harvest vessel 112.

[0105] Meanwhile, a separate collection of culture medium and cells is removed from the self-recycling perfusion culture bioreactor 102 and fed via another conduit to a bleed pump 108 that monitors and controls the viable cell density in the self-recycling perfusion culture bioreactor 102 by measuring the cell density and removes old or excess cells to maintain the stability of viable cells in the culture media before sending the culture media to a culture harvest vessel 112 that separates remaining cells and debris from the culture broth to produce a harvest stream of culture media that substantially cell free, which is then passed via a conduit to a secondary filtration device 114 (here, a depth filter grade XoHC from Millipore Sigma) that removes any remaining cells or cell fragments, ensures sterility, and removes any other particles to a protein A chromatography device 116 (a HITRAP MABSELECT prismA protein A affinity column from Cytiva) that captures a therapeutic antibody that is released in an eluant 108 via one column, while a flow-through composition, including the processed culture media, is passed from the protein A chromatography device 116 via a conduit to the ICP waste separation device 10 to remove the ammonia (and, optionally, the lactate) therefrom, as is described above before being passed via another conduit to a vessel for regenerating the culture media 94 to restore its nutrients (including, e.g., glutamine, glucose) and to reestablish an optimal pH level and electrolyte content. Attorney Docket No. mit-25891 pct

[0106] Multi-Stage Single-Layer Waste Separation Increases the Yield of Regenerated Media:

[0107] For an initially high waste concentration, culture media separation can be practiced via a series of ICP waste separation devices 10, as shown in FIG. 49, wherein the waste stream 31 of the first stage is passed via a conduit as a feed through the inlet 18 of the second stage ICP waste separation device 10.

[0108] Full Recovery Regeneration Using Single-Layer ICP Device:

[0109] A single-layer ICP waste separation device 10 that lowers the harvest and waste streams (which can be recombined here and in the other embodiments) is shown in FIG. 50. Operation in the “limiting” regime can halve the ammonia concentration. Low initial waste concentration streams can be mixed after a single pass to recover 100% of spent media. FIG. 50 shows the flow of cations (C+) 30 into the device 10 through the cation-exchange membrane 26. Also shown are glucose (Glue) 112, chlorine ions (Cl ) 46, lactate (Lac ) 44, amino acid anions (AA ) 42 on the side of the bottom internal membrane 28 closest to the anode 14. On the upper side of the top internal membrane 28, sodium ions (Na+) 40, ammonium ions (NH4+) 38, and positively charged amino acids (AA+) 36 pass through the upper cation exchange membrane 26 to the cathode 12. Neutral amino acids (AA) 116 remain in the harvest stream 33 along with glutamine (Gin) 114 and glucose (Glue) 112. The upper cation exchange membrane 26 blocks the passage of anions (A ) into the system.

[0110] In FIGS. 51 and 52, the concentrations of ammonia (NH4+), lactate (Lac), glutamate (Glu), and sodium (Na+) are plotted for the harvest stream passed through a single stage 120, two stage 122, and three stages 124, and for the waste stream passed through a single stage 126, two stage 128, and three stages 130.

[0111] The separation of the harvest (diluate) stream via a harvest outlet 20 and the waste (concentrate) stream via a waste outlet 22 in the ICP waste separation device is shown schematically in FIG. 53, while the combined output without separation of (or with the recombination) of harvest and waste streams via a combined outlet 132 is shown in FIG. 54, as can be practiced with the methods and apparatus described herein.

[0112] Discussion:

[0113] We demonstrate that, by recycling spent cell-culture media after removing waste products selectively, we can reduce the water PMI of the process by up to -33%— a significant figure when compared to previous recycling efforts that reduced water PMI by 18% (Madabhushi, et al.). The benefit of regenerated cell-culture media over direct recycling of spent cell-culture media is dependent upon cell line, cell-culture media formulation, and culture conditions, with FIGS. 38 and 39 showing an example of low- ammonium spent cell-culture media where regeneration provides no benefit. Attorney Docket No. mit-25891 pct

[0114] Separating waste and reusing cell-culture media presents two potential use cases with the device either integrated for recycling during the perfusion run or setup for processing the flow-through of antibody harvest after the run. It is worth noting that the water PMI projections in scenarios 2 and 3 are based on data from mock-perfusion cultures that reach lower peak VCD than actual perfusion cultures, so it is possible that in the future, water PMI can be further reduced by gathering data from recycling with real perfusion bioreactors. Although the projected PMI reductions in self-recycling were less than in secondary batch culture, the self-recycling of perfusion cultures may ultimately be preferable for economic and environmental reasons.

[0115] We demonstrated the feasibility of a single-stage ICP-based electrokinetic separator capable of processing up to 0.9 L per hour (10 mL / min), but the layered design of the device allows for easy scaling down or up to accommodate diverse spent cell-culture media volumes produced by bioreactors in process development or manufacturing. The device returns 50% of the input cell-culture media as a harvest stream lower in waste products and other molecules with a net charge. This recovery rate, however, is another parameter that can be optimized. In our current device, increasing the recovery rate by increasing the flow rate of the harvest stream relative to the waste stream lowers the waste removal efficiency. Still, it may be possible to explore asymmetric channel geometries or multi-stage devices in the future that increase the recovery rate and, therefore, have an even larger impact on improving the sustainability of biomanufacturing. Electrokinetic regeneration of spent cell-culture media should theoretically be feasible with methods other than ICP, such as electrodialysis. However, it is likely that designs that incorporate anion exchange membranes may be more susceptible to biofouling and reduced efficiency. The cation exchange membranes utilized in our design can also experience fouling primarily due to the mineralization of divalent cations, such as calcium, typically found in culture media, especially after a long-term (days) operation for seawater desalination (with relatively high Ca concentration). We did not, however, observe increased membrane resistance in our experiments, likely because the concentration of divalent cations in the cell-culture media was relatively low.

[0116] We have studied the removal of key waste products, including ammonium and lactate, but there are other impurities present in spent cell-culture media, such as hostcell proteins and DNA. Given their generally charged characteristics, they may also be removed into the waste stream (enabling further harvesting / downstream processing). Additional iterations of the electrokinetic regeneration process may alter the device geometry for greater removal of these impurities with comparatively lower electrophoretic mobilities, or it may be beneficial to pre-filter cell-culture media through an adsorptive depth filter. Additionally, when characterizing the impact of Attorney Docket No. mit-25891 pct recycled cell-culture media upon antibody production, measuring critical quality attributes (CQAs), such as glycosylation profiles, (in addition to titer) will be advantageous for further development of the process. Adjusting operating parameters, such as the electrical current of the device, or adding specific nutrient supplements are refinements of the separation process that may optimize specific CQAs.

[0117] Conclusion:

[0118] We have demonstrated an ICP separation process that removes waste from spent cell-culture media and improves the efficiency of material usage when culturing with recycled cell-culture media. This work is an advancement towards decreasing the raw material cost associated with mAb production, which may allow increased affordability and access to medication. Additionally, these regeneration and recycling processes can decrease the water consumption associated with biomanufacturing, reducing the environmental impact and rendering biomanufacturing facilities more sustainable to be located in many communities experiencing persistent or temporary water stresses.

[0119] In describing embodiments, herein, specific terminology is used for the sake of clarity. For the purpose of description, specific terms are intended to at least include technical and functional equivalents that operate in a similar manner to accomplish a similar result. Additionally, in some instances where a particular embodiment includes a plurality of system elements or method steps, those elements or steps may be replaced with a single element or step. Likewise, a single element or step may be replaced with a plurality of elements or steps that serve the same purpose. Further, where parameters for various properties or other values are specified herein for embodiments, those parameters or values can be adjusted up or down by i / iooLh, i / soth, 1 / 20111, i / ioth, 1 / 5*, i / 3rd, 1 / 2, 2 / 3rd, 3 / 4*, 4 / 5*, 9 / 10*, 19 / 20*, 49 / 50*, 99 / 100*, etc. (or up by a factor of 1, 2, 3, 4, 5, 6, 8, 10, 20, 50, 100, etc.), or by rounded-off approximations thereof or within a range of the specified parameter up to or down to any of the variations specified above (e.g., for a specified parameter of 100 and a variation of 1 / 100*, the value of the parameter may be in a range from 0.99 to 1.01), unless otherwise specified. Further still, where methods are recited and where steps / stages are recited in a particular order— with or without sequenced prefacing characters added for ease of reference— the steps / stages are not to be interpreted as being temporally limited to the order in which they are recited unless otherwise specified or implied by the terms and phrasing.

[0120] While this invention has been shown and described with references to particular embodiments thereof, those skilled in the art will understand that various substitutions and alterations in form and details may be made therein without departing from the scope of the invention. Further still, other aspects, functions, and advantages are also Attorney Docket No. mit-25891 pct within the scope of the invention; and all embodiments of the invention need not necessarily achieve all of the advantages or possess all of the characteristics described above. Additionally, steps, elements and features discussed herein in connection with one embodiment can likewise be used in conjunction with other embodiments. The contents of references, including reference texts, journal articles, patents, patent applications, etc., cited throughout the text are hereby incorporated by reference in their entirety for all purposes; and all appropriate combinations of embodiments, features, characterizations, and methods from these references and the present disclosure may be included in embodiments of this invention. Still further, the components and steps identified in the Background section are integral to this disclosure and can be used in conjunction with or substituted for components and steps described elsewhere in the disclosure within the scope of the invention.

Claims

Attorney Docket No. mit-25891 pctCLAIMSWhat is claimed is:

1. A method for processing spent cell-culture media from a bioreactor using an ion- concentration-polarization device, the method comprising: providing an ion-concentration-polarization device comprising alternating layers of ion-selective membranes and spacer layers; applying an electric field to the ion-concentration-polarization device; introducing a feed, including spent cell-culture media and nutrients, from the bioreactor into the ion-concentration-polarization device; separating a charged waste product from the feed in the ion-concentration polarization device; and removing the remainder of the feed, including the spent cell-culture media, from the ion-concentration-polarization device in a combined flow that is not differentiated via separate diluate and concentrate streams.

2. The method of claim 1, wherein the ion-selective membranes are cation exchange membranes.

3. The method of claim 1, wherein the spacer layers are millifluidic spacer layers.

4. The method of claim 1, wherein the charged waste product comprises ammonia.

5. The method of claim 4, wherein the charged product further comprises lactate.

6. The method of claim 1, wherein the nutrients comprise amino acids.

7. The method of claim 1, further comprising transporting the spent cell-culture media into the ion-concentration-polarization device using a peristaltic pump.

8. The method of claim 1, further comprising generating the electric field using a power supply and monitoring the electric field using a conductivity meter.

9. The method of claim 8, wherein the power supply supplies and measures voltage and electrical current, and the conductivity meter measures the conductivity of the harvest stream.Attorney Docket No. mit-25891 pct10. The method of claim 1, further comprising controlling flow rates at harvest and waste outlets of the ion-concentration-polarization device using flowmeters and control valves to maintain equivalent flow rates.

11. The method of claim i, further recycling the spent cell-culture media and nutrients in the bioreactor to culture additional cells.

12. An ion-concentration-polarization system for processing spent cell-culture media from a bioreactor, the system comprising: an ion-concentration-polarization device comprising alternating layers of ion-selective membranes and spacer layers; and a power supply configured to apply an electric field to the ion- concentration-polarization device, wherein the ion-concentration-polarization device is configured to receive a feed, including spent cell-culture media and nutrients, from the bioreactor via a feed stream and to separate a charged waste product from the remainder of the feed, wherein the remainder of the feed is not differentiated via separate diluate and concentrate streams.

13. The ion-concentration-polarization system of claim 12, wherein the ion-selective membranes are cation exchange membranes.

14. The ion-concentration-polarization system of claim 12, wherein the spacer layers are millifluidic spacer layers.

15. The ion-concentration-polarization system of claim 12, wherein the charged waste product comprises ammonia, and wherein the nutrients comprise amino acids.

16. The ion-concentration-polarization system of claim 15, wherein the waste product further comprises lactate.

17. The ion-concentration-polarization system of claim 12, further comprising a peristaltic pump configured to transport the spent cell-culture media into the ion-concentration-polarization device.

18. The ion-concentration-polarization system of claim 12, further comprising a conductivity meter configured to monitor the electric field by measuring the conductivity of the harvest stream.Attorney Docket No. mitsgSgipct19. The ion-concentration-polarization system of claim 12, further comprising flowmeters and control valves configured to control flow rates at harvest and waste outlets of the ion-concentration-polarization device to maintain equivalent flow rates.

20. The ion-concentration-polarization system of claim 19, further comprising: measurement devices for measuring at least one of flow or composition in at least one of the streams; a controller in communication with the measurement devices, the flowmeters, and the control valves: a computer-readable medium in communication with the controller and nontransitorily storing a software program configured to instruct the controller to control the power supply and the measurement devices and to receive measurements from the measurement devices.

21. The ion-concentration-polarization system of claim 12, wherein the ion- concentration-polarization device further comprises electrode layers containing titanium plates coated with Ru-Ir and constantly rinsed with a sodium sulfate solution.

22. The ion-concentration-polarization system of claim 12, wherein the spacer layers are laser cut to have a three-layered channel, and plastic mesh is placed in intermediate, harvest, and waste channels of the spacer layers for mechanical stability and to stabilize electroosmotic flow generated near the ion-selective membrane surface.

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