CN117959819A

CN117959819A - System and method for reducing ammonia and lactic acid in cell culture media for recovery and reuse

Info

Publication number: CN117959819A
Application number: CN202311838251.2A
Authority: CN
Inventors: 黄迎庆
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-12-28
Filing date: 2023-12-28
Publication date: 2024-05-03

Abstract

The present invention discloses a novel integrated system that is capable of continuously processing and recovering media from large scale cell culture depleted cultures to reduce cost and waste. The method includes clarification, intermediate concentration, pH adjustment, electrodialysis separation and analysis of possible cells and cell debris. After the initial cell separation, the cells and cell debris are removed by a further filtration step to clarify the medium while recovering the amino acids and nutrients in the permeate. Optional ultrafiltration may be further enriched in protein-like components prior to electrodialysis. An applied electric field permeates lactate, salt and ammonia from the medium through the selective membrane into the concentrate flow path. The culture medium subjected to deamination and deflocculating acid enters a quality control flow to carry out component analysis, and then can be used as a raw material of the culture medium to enter a bioreactor for repeated use. The system can significantly reduce the environmental impact and expense of the large-scale biological production industry (e.g., cell culture meats, etc.) by recovering depleted media in cell culture, particularly in cell perfusion culture.

Description

System and method for reducing ammonia and lactic acid in cell culture media for recovery and reuse

Technical Field

The present invention relates to systems and methods for clarifying and recovering spent cell culture media by removal of cells, debris, lactic acid and ammonia to allow reuse or further processing. More specifically, a system solution is disclosed that utilizes electrodialysis using filtration, pH adjustment, and continuous or semi-continuous operation. The system can save culture nutrition and costs while reducing waste and waste liquid emissions by recovering valuable amino acids, growth factors and other components from the used culture medium.

Background

The artificially cultivated meat can supplement the traditional livestock meat as part of the protein source in an environment-friendly way. The artificial cultivation of meat can reduce the emission of greenhouse gases, the requirements of land and water, and is remarkably beneficial to animal welfare.

To produce cultured meat, animal cells having the desired characteristics are inoculated into a bioreactor with a properly designed medium and allowed to proliferate. The cell culture medium must contain essential nutrients such as amino acids, glucose, and other key components such as growth factors and albumin that are critical to mediating and supporting cell growth. During cell culture, living cells produce metabolic wastes such as lactic acid and ammonia, which need to be eliminated or controlled within a specific range to prevent cell growth from being inhibited and to obtain high cell yield.

Various cell perfusion techniques separate cells from a cell culture medium and extract a fraction of the nutrient-depleted medium from the cell culture system. Fresh medium is then added to the bioreactor system to supplement nutrients and other key components, such as growth factors.

Cell culture media is a key cost driver in the meat industry. Even spent media contain valuable components such as amino acids, growth factors, albumin, recoverable and recyclable buffer components. Without recycling, these spent media can present additional challenges to waste management because they carry large amounts of organic material. The culture medium may be derived from a cell perfusion culture process or from a fed-batch process, with the meat cells being finally harvested as the residual culture medium after production.

The biomedical industry has traditionally not recovered media due to lack of appropriate motivation measures and recycled media process regulatory challenges. There is no report that a large scale solution can effectively achieve these objectives.

In order to recover the cell culture medium, the key metabolic waste products such as lactic acid and ammonia must be reduced to a certain level and the residual cell debris needs to be removed.

The most efficient method of separating cell debris is by filtration. Filtration may be achieved by depth filtration or tangential filtration. Tangential Flow Filtration (TFF) for cell perfusion or cell separation provides a good choice for clarification of cell culture fluids.

TFF can be performed in two ways: single Pass Tangential Flow Filtration (SPTFF) and cyclic tangential flow filtration (RTFF).

In single pass tangential flow filtration, the feed solution passes through the membrane once and the purified product is collected as permeate on the other side of the membrane. The feed solution that does not pass through the membrane is enriched in cell debris, known as reflux, or retentate, which may be a by-product. The method is simple and efficient, but may be limited by membrane pore blockage, and membrane throughput.

In circulating tangential flow filtration, the feed solution is continuously circulated through the filter by a pump. The clarified medium is collected as permeate on the other side of the membrane and the feed solution is recycled until the desired product yield is reached. This method requires additional equipment such as pumps and processing vessels, and may be more complicated to set up and operate.

SPTFF is generally simpler and more efficient, but RTFF can achieve higher tangential flow rates and achieve recovery of higher concentrations of purified product.

The TFF step also allows recovery of media components, such as macromolecules, albumin, growth factors, exosomes or other cellular products, to harvest their value and reduce wastewater.

Filtration can be used to clarify cell debris and to separate macromolecules, but it is not an efficient method for removing ammonia and lactic acid from spent media. The removal of gases such as ammonia is industrially achievable by degassing membranes, although the process is slow and requires a large amount of membrane surface area.

Purification of small molecules can be accomplished by ion exchange chromatography. However, the chromatography step consumes a large amount of water, and the chromatography resin is expensive, not easily recovered, and not naturally degradable, making it cost-effective and not environmentally friendly.

Dialysis has been tested in small batches of recovery medium. However, dialysis is slow, inefficient, and can be expensive because of its low throughput, requiring a large membrane surface.

The electrodialysis technology improves the mass transfer rate in the dialysis through an electric field, and can realize the selectivity of ion transmission in the ion exchange membrane by adjusting the pH value of the electrodialysis feed stream and adjusting the technological parameters such as voltage, current, feed flow and the like. Electrodialysis is a process that uses electricity to separate ions from a solution through ion-selective membranes, and can be used for desalination of sea water to remove salt ions, leaving fresh water behind.

In Electrodialysis (ED), the solution to be treated (e.g. seawater or wastewater) is fed into a series of flow cells, each formed of two different types of ion selective membranes, separated by spacers to prevent direct contact. A direct current is applied across the cell causing ions to migrate toward and through the oppositely charged membrane. Positive ions (e.g., sodium) migrate toward and through the negatively charged membrane, while negative ions (e.g., chloride) migrate toward the positively charged membrane.

The system consists of multiple layers of alternating cation exchange membranes (negatively charged) and anion exchange membranes (positively charged) that selectively allow positive and negative ions, respectively, to pass through.

As ions pass through the membrane they will concentrate on the other side, thereby reducing the salt concentration in the solution. Electrodialysis is an efficient method of producing desalinated water from solution.

Electrodialysis is particularly important in areas where fresh water resources are scarce. In addition to desalination of sea water, electrodialysis is also used for wastewater treatment, helping to remove ionic contaminants in industrial wastewater. It is also of great use in the food industry, including the desalination of whey in cheese production and the purification and concentration of various foods.

Electrodialysis is very energy efficient, especially when treating low to medium salinity water, and can selectively remove ions.

In order to protect the electrodialysis membranes, prefiltering is required to remove cell debris and excess organic components from the culture medium, and pH adjustment to a suitable range is required.

The byproducts of electrodialysis are acids and bases in the electron stream near the electrodes, which byproducts may be recovered for recovery for pH adjustment and other applications.

The invention discloses a novel economical and efficient process combining TFF and electrodialysis technologies, which is used for removing lactic acid and ammonia from a clarified and exhausted culture medium for recovery.

The invention discloses an economical and efficient method for treating and recycling components of a spent culture medium, which combines an efficient filtration technology with electrodialysis to recycle the culture medium.

Filtration and electrodialysis are generally batch operations, and large-scale biological manufacturing including meat analogue, synthetic biological products, if produced on a large scale, require continuous production processes to achieve process enhancement, investment reduction, and efficiency improvement.

These techniques enable continuous or semi-continuous operation of medium recirculation, helping to achieve industrial mass production of cell culture meats, synthetic biology.

Disclosure of Invention

The following summary is provided to illustrate some general inventive steps of systems, methods, devices, and apparatus in the specification. This summary is not an extensive overview of the invention, and is not intended to limit the scope of the application claims thereto.

In some embodiments thereof, the present invention discloses a novel system for large scale recovery of used cell culture media from large scale cell culture, cell differentiation, cell washing, or other types of biological production to achieve cost-effective reuse. The system utilizes filtration, pH adjustment and electrodialysis to reduce lactic acid and ammonia while removing cells and impurities. Continuous operation minimizes waste and restores the medium for further biological treatment.

In some aspects, microfiltration, including direct flow filtration and tangential flow filtration, may be used for clarification filtration of the media. Tangential flow filtration can achieve a retention of more than 90% of proteins such as growth factors or recovery and shut-off of 90% depending on the membrane pore size. Tangential flow filtration may use single pass and recycle modes. The preferred range of tangential flow filtration membrane pore size for clarification is 0.1-0.45 μm.

In some aspects, the TFF step is used to clarify cell debris, while in some other aspects TFF may also be used to concentrate media components or cell products, e.g., macromolecules, exosomes, etc., using a suitable membrane of a cutoff molecular weight, such as less than 0.1 microns, etc.

In other preferred aspects, the pH is adjusted to produce a clarified medium for subsequent electrodialysis. The pH adjustment of the clarified medium may be performed in a batch process or in a continuous mode. The invention discloses application of the two methods.

The isoelectric point of most amino acids and many cellular proteins is between pH 5-6.5. The pH of the medium is set to 4.5-6.5 to ensure solubility while converting lactic acid and ammonia to charged ions for removal.

In yet another aspect, electrodialysis utilizes an electric field to extract ionic lactic acid ions and ammonium ions from a pH-adjusted medium through a selective membrane, enriching them in a concentrate stream. Electrodialysis under suitable conditions can achieve lactic acid removal efficiencies of up to 90%.

In various aspects, the integrated system can concentrate valuable components such as growth factors while achieving desired levels of lactic acid and ammonia to prevent culture inhibition. The continuity and compactness of the system of the present invention provides a sustainability advantage over other techniques. By adopting the recovered cell culture medium, the wastewater treatment and production costs are reduced, and the component requirements of the fresh culture medium are satisfied. Meanwhile, the extracellular products such as organic acid and the like can be recovered from the electrodialysis concentrated solution by further treatment.

Drawings

The novel features believed characteristic of the illustrative embodiments are set forth in the appended claims. However, the exemplary embodiments as well as a preferred mode of use, further objectives, and descriptions thereof, will best be understood by reference to the following detailed description of one or more exemplary embodiments of the disclosure when read in conjunction with the accompanying specification. The accompanying drawings, wherein:

FIG. 1 shows a flow diagram of a media recovery system comprising three modules, clarification, pH adjustment and electrodialysis.

FIG. 2 shows a flow diagram of a media recovery system in which an initial clarification and component enrichment module includes DFF and TFF sections.

FIG. 3 shows a flow diagram of a media recovery system, wherein the initial clarification includes only TFF.

Fig. 4 shows a flow diagram of a media recovery system for a perfusion system, wherein a cell perfusion filter may simultaneously achieve cell separation, and the filtration system may also include the following direct current filtration (DFF) for additional clarification or bioburden reduction.

Figure 5 shows a flow diagram of a modular TFF system for cell separation or clarification, wherein multiple modular TFFs in series may achieve greater processing capacity.

FIG. 6 shows a flow diagram of a TFF system using multiple filters for cell separation or clarification, where the filters can be cleaned and regenerated on-line.

FIG. 7 shows a flow diagram for a single pass TFF system including multiple filters for cell separation or clarification, where the filters can be cleaned and regenerated on-line.

Fig. 8 shows a flow diagram comprising a plurality of filter direct current filtration systems for prefiltering and clarification to protect the electrodialysis device, wherein the filters can be cleaned and regenerated on-line.

Fig. 9 shows a flow chart of the pH adjustment module, where 9A is a batch process using a mixing tank, and 9B shows a continuous operation using an in-line mixer.

Fig. 10A illustrates the principle of operation of electrodialysis, with most of the amino acids remaining in the thin solution and lactic acid and ammonium ions migrating into the concentrate stream, while fig. 10B is a simplified diagram of the electrodialysis system, where other associated piping and wiring is omitted.

Fig. 11 shows the principle of operation for separating ammonia and lactic acid using a multi-stage electrodialysis series.

Fig. 12 shows the working principle of a plurality of parallel electrodialysis with on-line membrane regeneration capability and capable of continuous operation.

Detailed Description

The following description details a preferred embodiment of an automated media recovery system. However, the specific examples provided constitute exemplary embodiments only. The terms and expressions throughout this document should not be construed solely in terms of literal or dictionary definitions. Rather, the text and description is intended to embody the inventive concepts and innovations to explain the recycling process and its advantages. Thus, variations in the arrangement of system components, workflow sequences, instrumentation, or proportions are possible within the scope of the invention. Additional embodiments may utilize alternative configurations or modified elements to achieve medium recovery. The specific embodiments disclosed are merely representative of functional exemplary applications and do not limit the scope of the possible manifestations covered in the claims.

Reasonable modifications, integration and replacement of the components that maintain the depleted media processing recovery function should also be considered within the scope of protection. These declarative statements are intended to enumerate the innovative conceptual technical ideas to create a solution with similar functionality.

Accordingly, the summarized media recovery system and technical concepts should be construed based on beneficial industrial applicability and are not strictly limited to the exemplary specification, drawings, and description selected for illustration purposes. Systems and system extensions consistent with or similar to the principles of the present invention are intended to be within the scope of the claims of the present invention.

Figure 1 illustrates an integrated continuous system for recovering the components comprised by spent media 1 from a cell culture bioreactor to reduce overall production costs and environmental impact. By separating and recovering valuable media components such as growth factors, proteins and micronutrients, the system significantly reduces the raw material costs of ongoing mass production.

As shown in fig. 1, the automated process includes three primary recovery and characterization subsystems/modules, including: clarifying and separating macromolecules; regulating pH and electrodialysis; the control and quality control parts have not been shown in the figures.

First, the cell culture produced by the bioreactor or fermenter is first passed to a primary cell separation apparatus, preferably comprising filtration and/or centrifuges to effect primary cell separation. Spent medium 1 after primary cell separation passes through clarification (filtration) module 2. Module 2 may include additional filtration to separate larger particles and cellular products or macromolecules in the medium. Alternatively, if a TFF device is used in module 2, the cell suspension 5 may be clarified in one step directly in clarification module 2. These processes are used to clarify the medium to remove components such as cell debris that affect electrodialysis function, as shown in fig. 1. The clarified medium is pH adjusted in pH adjustment module 3 before further treatment in electrodialysis module 4. The discharge of the electrodialysis module 4 comprises a diluent flow 8 and a concentrate flow 7.

In some embodiments thereof, and depending on the cell culture process and perfusion technique, clarification module 2 may include a Direct Flow Filtration (DFF) assembly and/or a Tangential Flow Filtration (TFF) assembly. In one embodiment, the medium used to effect primary cell separation using centrifugation in a centrifuge, small amounts of cell debris are still present. In one mode, a prefilter may be used in a direct filtration mode to clarify cell debris. The TFF step may then be used to enrich the medium components, such as albumin growth factors, or cell products, such as fat particles, exosomes, milk proteins, egg proteins, etc., in a precision fermentation. The embodiment of fig. 2 shows a flow diagram of a medium recovery system, wherein the initial clarification includes DFF system 6 and TFF system 9. The permeate may contain lower molecular weight components such as glucose, amino acids, and media buffer components, among others. In some embodiments, since TFF may clarify media including higher levels of cell debris, it may not be necessary to further use a direct flow filter after TFF permeate, but may include providing additional protective filters for subsequent steps. In the figure, retentate 11 may be passed to the next operation for further processing.

In some illustrative other embodiments, a macroporous membrane having a pore size of 1 μm or greater is used to intercept cells in TFF or ATF cell separation, rather than a centrifugation step. Macroporous TFF or ATF may also be used as a process-enhanced cell perfusion device. The medium harvested after separation of the cells by the perfusion device still has a certain level of cell debris and may be further clarified using a further filtration step prior to pH adjustment of the electrodialysis operation. FIG. 3 illustrates a media recovery system wherein the initial clarification includes only a tangential flow filtration system.

In some embodiments, if a smaller pore size membrane (0.45 um or less) is used in perfusion or cell separation, the dc clarification step shown in the figure is not necessary. In particular, fig. 4 shows a medium recirculation system for a cell perfusion system, wherein perfusion filter service can perform primary cell separation, and at the same time can optionally include the following direct current filtration (DFF) for additional clarification. The perfusion bioreactor 12 is included in this embodiment and the cell suspension may be fed to the TFF module 9 by a perfusion pump 14 through a feed line. One or more valves 13 may be used to control and regulate the flow of cell culture medium circulation and to control/regulate the flow of culture medium to the next module.

In other embodiments, the TFF system in the clarification and macromolecule separation steps may include multiple modular systems, which may be connected in parallel or in series, to achieve greater capacity. Figure 5 shows a flow diagram of a modular TFF system for cell separation or clarification, wherein the modular TFFs are connected in series to achieve greater capacity. Permeate from a previous stage TFF module is coupled by feed line 15 and may be fed to the feed loop of the next stage reflux loop as shown. Each modular system may have multiple TFF filters to increase capacity. It should be noted that these modular systems may also be arranged in parallel to achieve higher capacities. One or more pressure monitors 16 may be employed to monitor the pressure of the medium in the flow path. The TFF system can be used for cell perfusion, for example, by matching with proper filters, flow rates and pumps.

Still further, some embodiments may include a regeneration device that may clean and regenerate filter membranes on-line, as shown in FIG. 6. In particular, a number of modular filter TFF systems for cell separation or clarification are depicted in fig. 6, wherein the filters can be cleaned and regenerated on-line. The system diagram configuration in this illustration allows for regeneration of the filter by isolating the filter from the material to be treated and by flushing and cleaning by injection of appropriate buffers and cleaning reagents. In particular, one or more containers 17 are provided for temporary storage of the rinse and cleaning solution, or for providing a conduit for external introduction of the rinse solution or cleaning solution. The cleaning solution and the rinsing solution are either delivered to the filter and contacted with the membrane filter to regenerate the filtration membrane or rinse the filter of residual cleaning agent. During the cleaning step, the sample or material being processed may bypass these filters and be processed by other filters, so the process may be continued. After the cleaning procedure is completed, the filter can be reconnected and used for production. A retentate or retentate reservoir 19 is shown for temporary storage of enriched cells or cell debris etc. In some embodiments, the described filtration system may be used for cell perfusion and tank 12 is a perfusion bioreactor.

In some embodiments, the TFF system used in the filtration module is a system capable of fully or partially achieving single pass tangential flow filtration (SpTFF), as shown in FIG. 7. In particular, the figure shows a multi-filter single pass TFF system for cell separation or clarification. A plurality SpTFF of filters (30). In turn, the depleted medium continues to flow through the retentate (retentate or reflux) path of each filter, concentrating the cell debris or macromolecules 11 in the retentate, and the permeate 10 is available for the next operation. Preferably, in-line filter regeneration may be performed on each filter by isolating the filter to be cleaned and allowing the material being treated to bypass the filter being cleaned. The isolated filter is rinsed, cleaned, flushed, and may be redeployed for material processing after the cleaning step is completed. The cleaning/rinsing solution may be recirculated through the filter or discharged through the rinsing line 20.

In yet another embodiment, the filtration module employs a plurality of straight flow filtration filters, which may be connected in parallel or in series. This is illustrated in the following example by the embodiment of figure 8 which illustrates a multiple filter direct current filtration system for prefiltering and clarification of cell depletion media (1) to protect electrodialysis devices. In this respect, the filtration system comprises a parallel connected filter 9 capable of in-line filter regeneration in a similar manner as described in fig. 7. The filter can be isolated from the feed liquid by closing the valve and cleaned, and can be redeployed after the cleaning step is completed. In a preferred implementation, the pressure sensor 16 enables real-time monitoring and automatic control of the transmembrane pressure and system.

Hollow fiber membranes, spiral membrane filters, flat cassette filters or ceramic membranes, 0.1 μm to 0.45 μm filters can be used to clarify cells, cell debris, particles and aggregates, while membrane filters of 0.1 μm or smaller pore size can be used for concentrating and recovering valuable media proteins and supplements for reuse.

Furthermore, in some preferred implementations, the TFF permeate flows into temporary storage tanks, allowing continuous adjustment of pH to an optimal window of pH 5.5-6.0 for subsequent electrodialysis separation.

Operations for performing the filtration module for cell separation and cell debris clarification and enrichment of the medium components are only selectively provided herein. The present disclosure does not set forth all potential options in detail. And many other variations exist that can be achieved without the need for inventive work and are within the scope of the present invention.

PH regulating module

In some aspects thereof, the pH adjustment subsystem may be operated in batch mode using a vessel with mixing functionality, such as mixing tank 23 shown in fig. 9A, wherein pH adjusting reagents may be contained in tank 21, and clarified media is contacted and mixed with the pH adjusting reagents in mixing tank 23, adjusted to the appropriate pH, in preparation for a subsequent electrodialysis step. In other aspects, it may be preferable to employ an in-line mixer, such as the static in-line mixer 24 shown in fig. 9B. In a preferred aspect, the pH adjustment system minimally includes a pH meter, a mixing device, and a pH adjusting agent source such as an acid and a base solution.

The use of an in-line mixer, optionally preferably includes a computer control system 90 with a feedback loop, an upstream amino acid analysis instrument, a conductivity meter before the addition of the pH adjusting reagent, and a pH measuring device after the mixer is shown in fig. 9A and 9B. For process control and to provide feedback to a computer. These devices are not fully shown in the figures.

Electrodialysis module

In an exemplary aspect, electrodialysis uses an electric field and ion exchange membranes to remove salts and other ions from a sample. It involves alternating cation and anion exchange membranes which migrate toward oppositely charged electrodes when a current is applied between the membranes, and which selectively allow ions to pass through. The electrodialysis sectional view module shows the module with anode 27 and cathode 26 with the input material entering the dilute flow path, ions removed from the dilute flow being carried away by the concentrate flow (C-flow), and electrolyte flow 28 flowing through the chamber adjacent the electrodes. The process is widely used for desalination of sea water, in particular in the treatment of low to medium salinity water. Here, the concentrated stream may be depleted medium, buffer, or pure water.

For ion exchange membranes, particularly for anion exchange applications, functional groups with the lowest pKa, such as sulfonic acid (-SO 3H) groups, are typically used, particularly in cation exchange membranes, where their pKa value is very low, below 1.

The functional groups used for the anion exchange membrane generally include quaternary ammonium salts and the like. These groups are basic in nature (as opposed to acidic) and can attract and permeate anions. The principle of electrodialysis is shown in figure 10A. For simplicity, the present invention shows only that the main flow comprises the medium-carrying components, the diluted flow 8 represents the salt-reduced medium, and the concentrate flow 7 is enriched with ions from the diluted flow, as in fig. 10B.

Most of the amino acids used in the medium have pKi between 5 and 6.5 as shown in table 1 below.

Table 1: isoelectric point of commonly used amino acids

The depleted medium entering the diluent stream 8 is pH adjusted for dialysis. The majority of amino acids used in the medium have pKi between 5 and 6.5. The pH was adjusted to between 5 and 6.5 at the first dialysis.

Illustratively, an Electrodialysis (ED) module may include one or more pH adjustment and electrodialysis steps. In an exemplary first step, the pH of the clarified medium may be adjusted to between 5 and 6.5 for a first dialysis. At a pH between 5 and 6.5, the ammonium ion pKa of ammonia is around 9.25, mainly in the form of NH ₄ ⁺ ions, which can pass through a cation exchange membrane, which allows only positively charged ions (cations) to pass through, while blocking negatively charged ions (anions). Cation exchange membranes are typically made of sulfonated polystyrene or the like, wherein the sulfonic acid groups provide cation exchange sites.

At pH 5-6.5, lactic acid has a pKa of 9.25 and exists in anionic form (LT-). The lactate ions may pass through a positively charged anion exchange membrane. The anion exchange membrane can block positively charged ions (cations). Preferably, the material for the AEM generally comprises quaternary ammonium groups, which promote anion exchange.

Depending on the amino acid profile of the clarified media, the pH of the feed may be adjusted to a value between pH 5 and 6.5 and fed into the diluent channel. Most of the amino acids and glucose are neutrally charged and pass through the diluent channel, while NH ₄ ⁺ and LT ^- pass through the semipermeable membrane into the concentrate stream, as shown in fig. 10.

In some non-limiting aspects, the automatic sensor and pH condition feeding apparatus can maintain the medium pH. The proper pH ensures that most proteins remain solubilized while converting ammonia to charged ammonium ions that migrate in the electric field. The electrodialysis stack then concentrates these ions into a concentrate through ion selective membranes, reducing the lactic acid and ammonia concentration by 50% or more in 5 hours. The desalted solution exits the electrodialyser in a dilute liquid stream for reuse.

In some alternative embodiments, the on-line sensor and analysis continuously quantifies amino acid profile, glucose, vitamins, growth hormone levels, and other markers of interest to monitor recovery efficiency and characterize the media composition after electrodialysis. The feedback control logic may automatically adjust upstream process parameters to optimize the solution for the restoration of the culture re-entry. A multi-step Electrodialysis (ED) process can be used to further reduce ammonia and lactic acid levels and recover amino acids by readjusting the pH of the diluent or concentrate and performing another step ED again, as shown in fig. 11. More specifically, fig. 11 shows the principle of operation with a plurality of electrodialysis steps in series, wherein the concentrate or dilute stream of the first step can also be treated by another ED step to further separate lactic acid or ammonia from the amino acids.

For example, the second Electrodialysis (ED) may involve adjusting the pH to approximately pKa 3.85 of lactic acid, where lactic acid is mostly neutral and most amino acids (other than aspartic acid and glutamic acid) are positively charged, which amino acids may pass through an anion exchange membrane (positively charged). Other positively charged amino acids, residual ammonia ions are positively charged, and can pass through an anion exchange membrane with acidic functional groups such as sulfonic acid. In this step, amino acids may be removed from the concentrated stream of the first step, and a majority of neutral lactic acid may be enriched in the diluent stream for further processing.

The side chain amino acids are shown in Table 2.

TABLE 1 pI of amino acids with ionizable side chains

Another alternative electrodialysis step is to adjust the concentrate pH of the first electrodialysis step (feed pH 5-6.5) or the second electrodialysis step (feed pH 3.85) to approach the pKa pH 9.25 of ammonia, except that lysine and arginine are positively charged, while the other amino acids are negatively charged, and pass through a cationic semipermeable membrane. Lysine and arginine are mostly still negatively charged and pass through the anion exchange membrane. It should be noted that if a quaternary ammonium based film is used, the number of times the positively charged ions are repelled relatively weakly. However, if the anion exchange membrane uses groups with higher pKa, the rejection and blocking of positively charged ions can be improved. The main purpose here is to recover the amino acids in the previously concentrated stream.

The present invention discloses only exemplary embodiments of a multi-stage ED process, with other similar process steps. For example, the first dialysis is performed by first removing lactic acid by adjusting the pH of the lactic acid close to pI, so that the amino acids will be enriched in the concentrate stream.

In some embodiments, to achieve continuous treatment, multiple electrodialysis stacks may be arranged in parallel and in-line membrane cleaning and regeneration may be performed. A plurality of electrodialysis units 4 are connected in parallel as shown in fig. 12, which allows individual electrodialysis units to be isolated from the feed solution and rinsed and cleaned with appropriate buffers and cleaning reagents. In particular, one or more containers 17 are used for temporary storage of the rinsing and cleaning solution or for providing an external supply of the rinsing solution or cleaning solution, which is fed to the electrodialyser via a supply line 20 and is brought into contact with the electrodialysis membranes for cleaning the filtration membranes and for rinsing the cleaning agent. In the cleaning step, the sample or material being treated may bypass these electrodialysis units undergoing membrane regeneration and be treated by other electrodialysis units, so the process may be carried out continuously. After the cleaning procedure is completed, the electrodialyser can be re-connected and used for production. In general, the present invention, automated processes can continuously regenerate spent media to support long-term continuous production of bioreactors while recovering valuable components of the spent media that were found in existing perfusion processes. The system can be easily expanded by combining large processing units with higher capacity or expanding more units in parallel to meet the needs of an industrial scale process.

In some embodiments, the medium recovery of perfusion bioreactors using 2um tangential flow filtration membranes for cell interception is exemplified (as shown in figure 3). The spent media from perfusion is passed through a single pass Tangential Flow Filtration (TFF) system (SpTFF system as shown in fig. 7, with filters having pore size grades of 0.2-0.45 μm) to remove residual cell debris and other particulate matter, providing clarified filtration prior to electrodialysis. In some embodiments, 4 SpTFF filter units are connected in series, with on-line filter regeneration capability. This single pass operation can concentrate cell debris and impurities up to 10 times while collecting more than 90% of the feed to restore the media quality. In one embodiment of a 2,000l batch perfusion bioreactor, TFF perfusion may incorporate a tubular TFF filter having a surface area of 2m ² and a transmembrane flux of between 10-100LMH may be achieved. The filter may be capable of being regenerated in combination on-line. After perfusion and collection of spent cell culture medium, the 0.2 μm tangential flow filtration clarified and 90% of the medium volume in the permeate was collected within 5 hours. If the continuous process is not performed, the permeate buffer may be collected in a holding vessel for pH adjustment for electrodialysis operation.

The permeate stream is mixed with a pH adjusting reagent in pH adjusting tank 23 to stabilize the pH between 5.6-5.8 prior to electrodialysis. The reagent may be an acidic titrant solution introduced by a metering pump. Optimizing the pH in this range ensures that most amino acids are electrically neutral and that the cellular proteins remain soluble while also converting ammonia to charged ammonium ions for subsequent removal.

The pH-adjusted, TFF clarified medium is then fed to an electrodialysis unit. An electric field is applied causing negative chloride ions and lactate ions to migrate toward the anode into the concentrate stream and store in the concentrate stream container. Positively charged ammonium cations flow in the opposite direction to the cathode and also into the concentrate stream. The culture medium now partly freed of lactic acid and ammonia leaves the electrodialyser through a dilution flow outlet for storage or reuse. For a treatment capacity of 2,000L, a single stage electrodialysis can use a surface area of optimally 50m ². Electrodialysis can reduce lactic acid and ammonia by 50% or more. Recovery of 70% or more of amino acids can be achieved.

The electrodialysis concentrate stream may be subjected to further electrodialysis to recover valuable components, such as organic acids, including lactic acid. At the same time, the dilutions were analyzed by the QC module to confirm the quality of the dilutions were returned to the bioreactor or to the process for the medium configuration.

It is contemplated that the described media recovery system may be implemented as an integrated stand-alone device or in a distributed model. In one embodiment, tangential flow filtration, electrodialysis, concentration, quality control, and control system components are implemented on a single compact hardware device, independent of external communication. Reasonable modifications and adaptations of these embodiments are within the scope of this invention. Accordingly, applicant intends to cover closely related configurations consistent with the inventive concepts.

The above described medium recovery module may be used for additional processing as part of a large scale plant system. It is also within the scope of the invention to add additional processing unit operations such as chromatography, heat exchange or temperature control, freeze drying, evaporation.

Industrial application

The disclosed cell culture medium recovery system has significant utility in industrial scale culture meat production facilities or other large scale biological production. As this emerging industry evolves from small volume processes to large scale production chains meeting global protein requirements, efficient recovery and reuse of depleted growth media can provide tremendous value. The integrated, continuous recovery method enables the large bioreactor or cell perfusion culture process to run continuously for a long period of time, thereby minimizing emissions. The operating cost is reduced by recovering the medium components, and the investment of the medium recovery apparatus can be rationalized. Furthermore, the compact modular design enables the recovery device to be supported in multiple bioreactors. By recovering the active components of the culture medium, the system can reduce both cost and environmental waste. Therefore, the innovation can effectively support the cost reduction and synergy of biological processes such as large-scale cell culture meat and the like.

Reference is made to:

1. Su Man Qian Dela NAS, ind Nagamori, masanobu Horie, masahiro Kino-Oka, engineering Bioprocess Biosyst, month 1 of 2017 by dialyzing the medium to expand human-induced pluripotent stem cells in suspension culture; 40 (1):123-131.

Carey and Giuliano (2011) amino acids, peptides and proteins. Organic chemistry 8 th edition, 25,1126McGraw Hill,ISBN-13:978-0077354770.

Claims

1. A system for recovering a cell culture medium, comprising:

A filtration system for clarifying the used medium and/or enriching some of the medium components;

A pH adjusting module for the clarified feed liquid; and

An electrodialysis module for removing lactic acid and ammonia ions from the clarification media;

preferably, the TFF system is part of a cell perfusion culture system operating in tangential flow mode or alternating flow tangential flow filtration mode.

2. The system of claim 1, wherein the filtration system comprises one of:

Tangential flow filtration systems;

a direct current filtration system; or alternatively

A combined system comprising tangential flow and straight flow filtration sections.

3. The system of claim 1, wherein the filtration system comprises a tangential flow filtration system having at least some filters connected in series and capable of operating in a full or partial single pass mode.

4. The system of claim 1, wherein the filtration system comprises a plurality of modular tangential flow filtration subsystems arranged in parallel or in series; wherein in the case of tandem, the retentate of the preceding modular subsystem is connected to the retentate circulation flow path loop of the following subsystem and the permeate is used for electrodialysis.

5. The system of claim 1, wherein the filtration system consists of a plurality of filters in one or more modular systems that can be filtered in a cyclic tangential flow mode, a single pass tangential flow mode, a straight flow mode, or a mixed mode, the filters can be connected in parallel or in series, and the filtration system comprises an on-line regeneration system that allows the cleaning and flushing of filter membranes, the filter regeneration system comprising:

a. One or more containers for rinsing and cleaning solutions or an external input line for supplying rinsing or cleaning solutions to the filter and in contact with the filter membrane;

b. The flushing and cleaning solution inlet line can deliver the flushing and cleaning solution to the filter through the feed side or permeate side, as well as an applicable control valve;

c. the cleaning solution outlet pipe can be connected to a filter feed side or permeate side line, to carry the rinse and cleaning solution to one or more cleaning solution containers for recirculation or discharge, and to a suitable control valve;

d. Pipes and valves that can isolate one or more filters for cleaning and regeneration allow samples or materials being processed to bypass these filters being cleaned and be processed by other filters.

6. The system of claim 1, wherein the electrodialysis module for reducing lactic acid and ammonia in a cell culture medium comprises:

a. a plurality of electrodialysis devices connected in series or parallel;

b. one or more vessels for storing or externally-connected pipes for cleaning and rinsing fluids

C. The matched pipeline can convey the cleaning liquid and the flushing liquid to the electrodialysis unit, enter electrolyte flow, dilution flow and concentration flow channels and contact the ion exchange membrane;

d. a washing and rinsing liquid outlet pipe, a pipe capable of transferring washing and rinsing liquid from the electrodialysis unit to one or more cleaning solution buffer containers for circulation washing mode or draining, and a suitable control valve; and

E. Piping and valves capable of isolating one or more electrodialysis columns for cleaning and regeneration allow feed to be treated to bypass the electrodialysis unit undergoing membrane regeneration and be treated by other units.

7. The system of claim 1, wherein the pH adjustment module comprises:

a. at least one container containing a pH adjusting reagent or a pipe externally connected with the pH adjusting reagent;

b. At least a pump or valve capable of controlling the addition of the pH adjuster;

c. the mixing device mixes the pH regulator with the culture medium; and

D, the pH value after mixing is measured by a pH measuring device;

Preferably, the mixer used in the pH adjustment module is an in-line mixer, and has the capability of continuously adjusting the pH of the feed liquid.

8. The system of claim 1, wherein the electrodialysis module comprises a plurality of electrodialysis cells connected in series or parallel; these units may use the same or different feed pH ranges; the subsequent stages in series may be fed with the dilute or concentrate streams of the previous stage adjusted to the specified pH.

9. A method of recovering a cell culture medium comprising:

a. clarifying the medium or concentrating the components of the depleted medium using a filtration operation;

b. Regulating the pH value of the clarified culture medium; and

C. Removing lactic acid and dissolved ammonia from the pH adjusted medium using one or more electrodialysis steps;

Preferably, wherein the first step electrodialysis feed has a pH of between 5 and 6.5.

10. The process of claim 9 wherein the inlet pH of the first electrodialysis step is between 5 and 6.5 and the second step is carried out using the lactic acid rich feed of the previous step at a pH of 3.5-4.2 to the second step to further enrich the lactic acid and recover the cell culture medium components.

11. A process according to claim 9 wherein the inlet pH of the first electrodialysis step is between 5 and 6.5 and the ammonia-rich sample in the second or third step is reused in the feed pH 8.7-10 to separate ammonia from other amino acids by electrodialysis, where ammonia is enriched in the dilution stream.

12. The method of claim 9, further involving one or more additional unit operations from: performing chromatography; concentrating; drying; crystallizing; filtering by a terminal; or evaporation, etc.

13. The method of claim 9, wherein the recovery and regeneration system is on a culture medium used in a culture process for mammalian cells, yeast cells, bacterial cells for the production of food, pharmaceutical or cosmetic ingredients (including exosomes) for cell culture.