Device for cell maintenance and method thereof

Abstract

A device for maintaining cells comprises a pair of hollow shafts (107) having a first hollow shaft (107A) and a second hollow shaft (107B) that are suspended within a container (101). A bidirectional pump (109) is configured to circulate the medium within the container by the rotation of the bidirectional pump (109) together with the hollow shafts (107) through an external circulation tube (113) and to generate pressure within the cell compartment (105) associated with the second hollow shaft (107B), and simultaneously, the cell compartment associated with the first hollow shaft (107A) is configured to accumulate cells on the surface of a flexible filter (203) by creating a vacuum through the bidirectional pump. The device (100) is a closed-loop system that controls the pumps and inlets / outlets according to the required time and amount. The present invention helps to maintain cells and separates cells from the circulating culture medium within the container.

Description

Technology Field

[0001] The present invention relates to an apparatus and method for maintaining cells. More specifically, it relates to an apparatus for separating cells from a medium during a cell culture process and improving batch life to increase batch yield and overall process output. Background Technology

[0002] Cell separation is a required operational step for the perfusion process. The perfusion process is an important technique used in the biopharmaceutical industry for various purposes, primarily related to the production of biopharmaceuticals such as monoclonal antibodies, vaccines, and other therapeutic proteins. There are several reasons suggesting the necessity of cell separation and perfusion during the upstream batch execution of biomolecules.

[0003] Perfusion culture is a continuous cell culture system that maintains optimal growth conditions by continuously supplying fresh media and removing consumed media. This dynamic approach enables the achievement of high cell density and consistent product quality, reduced production time, reduced facility footprint and operation, and improved product consistency. A key component of this system is cell retention, which involves physically separating cells from the culture medium through techniques such as tangential flow filtration or membrane filtration. By retaining cells within the bioreactor, continuous culture becomes possible, leading to increased productivity and consistent product quality.

[0004] Cell maintenance devices are central to the perfusion process because their function is to maintain cells within the vessel / bioreactor. Other mechanisms, such as the addition of fresh media and the removal of used media, constitute the basic operation of pumps (i.e., harvest pumps, media addition pumps) that manage fresh media addition and used media removal through predefined logic and recipes for perfusion.

[0005] Apoptosis, a spontaneous process during cell culture, leads to cell lysis, which releases cell debris into the culture medium. This debris, composed of cellular components, can negatively impact the culture by hindering cell growth, reducing product yield, and impairing product quality. Cells also generate waste byproducts from their metabolism, such as lactate and ammonia, which are detrimental to the cell growth and expression of the required product and to the quality of the product. Cell separation techniques help remove these cell culture byproducts, such as cell debris, lactate, and ammonia, through the harvesting line, leading to a more efficient process that delivers higher-quality products.

[0006] The retained cells are separated and maintained within the same environment, such as a bioreactor, where fresh media is supplied to volume-compensate the harvested used media. The continuous removal of these byproducts and the addition of fresh media lead to an increase in cell number (growth) and batch life compared to the batch culture process into the bioreactor. The concept of more cells leads to the production of more product, which in turn leads to the production of a larger volume of product into the bioreactor.

[0007] In traditional batch processes, cells may experience stress due to fluctuations in nutrient availability and waste accumulation. Perfusion minimizes these fluctuations to reduce cellular stress and improve overall culture life and productivity.

[0008] Therefore, cell separation and perfusion are essential technologies in the biopharmaceutical industry because they enable cell isolation and maintenance, ensure the availability of fresh culture media for separated and progressively concentrated cells, facilitate continuous production, and improve operational productivity and product quality. These cell separation techniques are crucial for meeting the high-quality standards and production demands of the biopharmaceutical industry.

[0009] There are currently various tools used as cell maintenance devices. Current cell separation devices for biopharmaceuticals, along with their bottlenecks, are described in the table below:

[0010]

[0011]

[0012]

[0013]

[0014] Therefore, it is necessary to invent a device for cell maintenance that overcomes the difficulties described above. The problem to be solved

[0015] The main objective of the present invention is to provide an apparatus and a method for maintaining cells.

[0016] Another objective of the present invention is to provide an apparatus and method for cell maintenance that enables the isolation and maintenance of cells.

[0017] Another objective of the present invention is to provide an apparatus and method for maintaining cells to improve product purity.

[0018] Another objective of the present invention is to provide an apparatus and method for cell maintenance to enable continuous production and to increase the yield efficiency of the current production process.

[0019] Additionally, the object of the present invention is to provide an apparatus and method for cell maintenance that provide a simple structure, total containment, and cost efficiency.

[0020] Another objective of the present invention is to provide an apparatus and method for cell maintenance that does not involve manual intervention and connection and provides an error-free setup.

[0021] Another objective of the present invention is to provide an apparatus and method for cell maintenance that increases the efficiency of bioreactor cell culture operations.

[0022] Another objective of the present invention is to provide an apparatus and method for maintaining cells for small-scale or pilot-scale application or for use on a commercial scale.

[0023] Additionally, the object of the present invention is to provide an apparatus and method for cell maintenance to eliminate shear stress and negative effects on cell health and viability.

[0024] Additionally, the object of the present invention is to provide an apparatus and method for cell maintenance to eliminate problems associated with conventional techniques and prior art. means of solving the problem

[0025] The present invention relates to an apparatus and method for cell maintenance. The apparatus comprises at least one pair of vertically extended hollow shafts suspended above a vessel of a bioreactor, each hollow shaft being connected to at least one cell compartment at its lower end, each said cell compartment being fluidly connected at the lower end of an individual hollow shaft, each hollow shaft being vertically extended, the hollow shafts being fluidly connected to one another at their upper ends via an external circulation tube, and a drainage tube being fluidly connected to the external circulation tube. A bidirectional pump is positioned above the external circulation tube or in line with the external recirculation tube, and a drainage pump is positioned above the drainage tube or in line with the drainage tube. This configuration is a closed-loop system controlled by pumps and inlets / outlets according to the required time and volume. The present invention enables the automation of the process and a non-human intervention process. Brief explanation of the drawing

[0026] FIG. 1 illustrates a schematic diagram of a device for maintaining cells according to an embodiment of the present invention. FIG. 2 illustrates a schematic diagram of cell compartments of a cell maintenance device according to an embodiment of the present invention. FIG. 3 illustrates a schematic diagram of cell compartments of a cell maintenance device during increased pressure within the cell compartment, according to an embodiment of the present invention. FIG. 2 illustrates a schematic diagram of cell compartments of a cell maintenance device during increased vacuum within the cell compartment, according to an embodiment of the present invention. FIG. 5a illustrates a side view of a flexible filter of cell compartments of a cell maintenance device according to an embodiment of the present invention. FIG. 5b illustrates a side view of a support mesh of cell compartments of a cell maintenance device according to an embodiment of the present invention. FIG. 6 illustrates viable cell density and survival percentage in different variations of the device according to an embodiment of the present invention. Specific details for implementing the invention

[0027] Before describing the invention in detail, it should be understood that the application of the invention is not limited to the details of the configuration and arrangement of the parts illustrated in the accompanying drawings. The invention enables other embodiments as described above and as illustrated in different drawings, and can be practiced or performed in various ways. It should be understood that the language and terminology used herein are for illustrative purposes only and not for limiting purposes.

[0028] In this document, the term "exemplary" is used herein to mean "serving as an example, case, or illustration." Any embodiment or implementation of the presented subject described herein as an "exemplary" is not to be interpreted as being more preferred or advantageous than other embodiments.

[0029] Although the present disclosure allows for various modifications and alternative forms, specific embodiments of the present disclosure are illustrated by way of example in the drawings and will be described in detail below. However, it should be understood that the present disclosure is not intended to be limited to the specific forms disclosed, and on the contrary, the present disclosure covers all modifications, equivalents, and alternatives that fall within the scope of the present disclosure.

[0030] The terms “include,” “including,” or any other variations thereof are intended to cover a non-exclusive inclusion such that a method of including a list of acts may include not only such acts but also other acts that are not explicitly enumerated or are inherent in such a method. In other words, one or more acts in a method carried out by “include” do not exclude the existence of other acts or additional acts in the method without entailing further restrictions.

[0031] The terms “flexible filter,” “membrane,” “flexible membrane,” or “cell maintenance membrane” may be used interchangeably and may be understood as structures for filtration or to serve as selective barriers, regardless of material composition, configuration, or specifications. Technical terms should not be interpreted as imposing any restrictions on the scope of the invention.

[0032] The terms “hollow shaft,” “tubular shaft,” or “tubular element(s)” may be used interchangeably and understood as structures configured to allow the flow of materials, media, or fluids. Technical terms should not be interpreted as imposing any restrictions on the scope of the invention.

[0033] The term "batch" or "perfusion culture batch" refers to a single-cell lot cultured in a bioreactor from inoculation to harvest.

[0034] The present invention discloses an apparatus (100) for cell maintenance and a method thereof. FIG. 1 illustrates a schematic diagram of an apparatus (100) for cell maintenance according to the present invention. The apparatus (100) for cell maintenance may be configured to have a bioreactor having a vessel (101). The vessel (101) has a head plate that accommodates a flexible adapter that accommodates at least one shaft, on which at least one impeller (103) is mounted. It should be understood that different combinations may exist, such as the number of shafts, the number of impellers (103) mounted on the shafts, the arrangement of shafts such as vertical, horizontal, and size, the shape and number of flexible adapters through which the shafts enter the vessel (101) or bioreactor, a mechanism for transmitting mechanical action to the shafts, and the type, shape, location, and number of impellers (103).

[0035] In one embodiment, the impeller (103) is driven by a motor and provides a liquid flow of fluid contents within the chamber and provides sufficient mixing of the contents within the bioreactor vessel (101). The required gas enters the bioreactor through a sparger (any type: micro, open pipe, ring, etc.) positioned below the impeller (103). Upon introduction of the gas, the movement of the impeller (103) disperses gas bubbles and thoroughly mixes the contents of the vessel (101).

[0036] It should be understood that the bioreactor may be a different type of vessel (101) or a vessel (101) capable of performing an equivalent function, and that the invention is not specifically limited to the use of a bioreactor.

[0037] In one embodiment, the device (100) may include at least one pair of vertically extended hollow shafts (107). Each hollow shaft (107A, 107B) of the pair of hollow shafts (107) may be made of various materials, such as metal, or plastic composition, or alloy, or any other material suitable for application in a bioreactor.

[0038] In one embodiment, the pair of hollow shafts (107) comprises a first hollow shaft (107A) and a second hollow shaft (107B). Each hollow shaft (107A, 107B) is connected to at least one cell compartment (105) at its lower end. Each of the cell compartments (105) is flexibly connected at the lower end of each individual hollow shaft (107A, 107B), and each hollow shaft (107A, 107B) extends vertically. The pair of hollow shafts (107) is suspended within the container (101) and mounted through the head plate of the container (101) such that the upper end of the hollow shafts (107A, 107B) extends over the head plate of the container and the lower end of the pair of hollow shafts (107A, 107B) is maintained within the container (101).

[0039] The pair of hollow shafts (107) are preferably arranged in an orientation parallel to the impeller (103) of the vessel (101) of the bioreactor. Each of the hollow shafts (107A, 107B) is connected to an external circulation tube (113) at an upper end, and each of the external circulation tubes (113) is configured to have sensors (117) for detecting and measuring pressure and vacuum within the hollow shafts (107), the external circulation tubes (113), and the cell compartments (105). A bidirectional pump, as illustrated in FIG. 1, is configured to have an external circulation tube according to the specifications of the bidirectional pump. The function of the bidirectional pump (109) is to generate vacuum and pressure within the cell compartments (105). The bidirectional pump (109) recirculates the medium in both directions and passes the medium alternately through the hollow shafts (107). In one embodiment, the bidirectional pump (109) may include, but is not limited to, a peristaltic pump, a magnetic pump, and similar ones. The bidirectional pump (109) may be able to generate a pressure and vacuum within the range of 0.1 bar to 3 bar, but is not limited to. The pressure and vacuum may vary depending on the size and capacity of the device (100) associated with cell maintenance.

[0040] In some embodiments, a single bidirectional pump (109) may be configured with a plurality of pairs of hollow shafts (107) each having a cell compartment (105) attached to the lower end of each hollow shaft (107A, 107B). In some embodiments, the plurality of cell compartments (105) may be connected in series or sequentially along a single hollow shaft (107).

[0041] In one embodiment, a drainage pump (111) may be flexibly connected to an external circulation tube (113) through a drainage tube (115) and across a bidirectional pump (109), as shown in FIG. 1. The drainage pump (111) may be configured to drain the medium from the vessel (101) of the bioreactor.

[0042] FIG. 2 illustrates a schematic diagram of a cell compartment (105) of a cell maintenance device (100) according to the present invention.

[0043] A device (100) for cell maintenance may include a cell compartment (105). The cell compartment (105) may include a support mesh (201). A flexible filter (203) is mounted on the support mesh (201). The support mesh (201) acts as a seating platform for the flexible filter (203) during the creation of a vacuum within such cell compartment (105). The support mesh (201) also provides rigidity to the cell compartment (105). The flexible filter (203) is mounted together with the cell compartment (105) or the hollow shaft (107) through a process of sealing, welding, merging, or any other conventional method. The flexible filter (203) has a porous structural configuration that allows a medium to flow through to cause cell maintenance at the outer ends of the pores and hinders cell migration. The thickness of the flexible filter (203) is between 50 and 200 microns, and the size of the pores present in the flexible filter (203) is between 0.2 and 10 microns. The size of the mesh (pores) of the support mesh (201) is larger than the size of the pores in the flexible filter (203).

[0044] In some embodiments, the cell maintenance device (100) may include a rigid filter (not shown in the drawing) having a porous structural configuration that allows a medium to pass through and flow to cause cell maintenance in the pores and hinders cell migration. In one embodiment, as shown in FIG. 2, a cell compartment (105) is connected to the lower end of each hollow shaft (107A, 107B) of a pair of hollow shafts (107) suspended within a container (101). The plurality of cell compartments (105) may be configured in a series of sequences and may be mounted in line with each of the hollow shafts (107A, 107B) of the pair of hollow shafts (107), and the number of cell compartments (105) is not limited thereto.

[0045] FIG. 3 illustrates a schematic diagram of the cell compartments (105) of a cell maintenance device (100) while the pressure within the cell compartment (105) is increased according to the present invention. In a similar manner, FIG. 4 illustrates a schematic diagram of the cell compartments (105) of a cell maintenance device (100) while the vacuum within the cell compartment (105) is increased according to the present invention.

[0046] In one embodiment, the flexible filter (203) is expandable and contractible while pressure and vacuum are individually created within the cell compartment (105) as illustrated in FIGS. 3 and 4. As illustrated in FIGS. 1 and 3, when the bidirectional pump (109) is rotated clockwise, the culture medium in the vessel (101) flows from the first hollow shaft (107A) to the second hollow shaft (107B) through the cell compartments (105) and the connected external circulation tube (113) as illustrated in FIG. 1. The flow of the culture medium creates pressure within the cell compartment (105) associated with the second hollow shaft (107B) and creates a vacuum within the cell compartment (105) associated with the first hollow shaft (107A). As a vacuum is created within the first hollow shaft (107A) of the pair of hollow shafts (107) and simultaneously a pressure is created within the second hollow shaft (107B) of the pair of hollow shafts (107), as illustrated in FIGS. 3 and FIGS. 4, the flexible filter (203) of the cell compartment (105) associated with the first hollow shaft (107A) may contract, and simultaneously the flexible filter (203) of the cell compartment (105) associated with the second hollow shaft (107B) may expand. The flexible filter (203) of the cell compartment (105) associated with the first hollow shaft (107A) may contract due to the suction of a medium from the container (101) through the pores of the flexible filter (203) of the cell compartment (105).The size of the pores of the flexible filter (203) is smaller than the size of the cells, so that the cells are prevented from passing through the flexible filter (203) and accumulate on the outer surface of the flexible filter (203), and only the culture medium that does not contain cells flows into the cell compartment (105) associated with the first hollow shaft (107A) and, due to the function of the bidirectional pump configured with the external circulation tube (113), flows into the cell compartment (105) associated with the second hollow shaft (107B).

[0047] When a predetermined pressure or vacuum is reached within the cell compartment (105) or after a specific duration, the bidirectional pump may be operated in reverse mode and may rotate counter-clockwise.

[0048] When the bidirectional pump (109) rotates counter-clockwise, the culture medium in the vessel (101) flows from the second hollow shaft (107B) to the first hollow shaft (107A) through the cell compartments (105) and the connected external circulation tube (113), as illustrated in FIG. 1. The flow of the culture medium can create pressure within the cell compartment (105) associated with the first hollow shaft (107A) and can create a vacuum within the cell compartment (105) associated with the second hollow shaft (107B). As a vacuum is created within the second hollow shaft (107B) and pressure is simultaneously created within the first hollow shaft (107A), as illustrated in FIGS. 3 and FIGS. 4, the flexible filter (203) of the cell compartment (105) associated with the second hollow shaft (107B) may contract, and at the same time, the flexible filter (203) of the cell compartment (105) associated with the first hollow shaft (107A) may expand. The flexible filter (203) of the cell compartment (105) associated with the second hollow shaft (107B) contracts due to the aspiration of culture medium from the container (101). The size of the pores of the flexible filter (203) is smaller than the size of the cells, so that the cells are prevented from passing through the flexible filter (203) and accumulate on the outer surface of the flexible filter (203), and only the culture medium that does not contain cells flows into the cell compartment (105) associated with the second hollow shaft (107B) and flows into the cell compartment (105) associated with the first hollow shaft (107A) through the external circulation tube (113).At the same moment, pressure is generated in the cell compartment (105) associated with the first hollow shaft (107A), and as the culture medium flows through the cell compartment (105) associated with the first hollow shaft (107A), the cells that were externally adhered and accumulated on the pores of the flexible filter (203) of the cell compartment (105) associated with the first hollow shaft (107A) during the first cycle are released into the culture medium of the container (101).

[0049] The rotation of the bidirectional pump (109) in both directions will continuously generate and release pressure and vacuum into each cell compartment (105), where cell maintenance takes place in the bioreactor. The continuous expansion and contraction and the direction of medium flow in both the outgoing and incoming paths through the flexible filter (203) will not allow the filter to be blocked.

[0050] It should be understood that the number, size, and shape of the cell compartments (105) depend on the scale of operation and the type of application. The invention can be implemented in bioreactors of a minimum (i.e., 2 mL) to a maximum (i.e., 10,000 L) scale by linear scalability.

[0051] In one embodiment, a drainage pump (111) may be able to remove used medium that does not contain cells from the bioreactor according to a set perfusion rate. A level sensor may be installed within the vessel (101) of the bioreactor. The sensor may be a non-contact sensor installed at a mounting station, the function of which is to detect the level of the liquid. The level sensor may be able to detect the level of the culture medium within the vessel (101) of the bioreactor. A perfusion pump (not shown) may be configured to add fresh culture medium in consideration of the low liquid level of the culture medium within the vessel (101) of the bioreactor and to stop adding culture medium when a set level is achieved. The operation may also be performed by other means, such as flow rate and time-dependent pump rotation, to achieve a predetermined perfusion rate.

[0052] FIGS. 5A and 5B illustrate side views of a flexible filter (203) and a support mesh (201) of cell compartments (105) of a cell maintenance device (100) according to the present invention.

[0053] In some embodiments, the cell compartments (105) may be configured to rotate relative to the hollow shafts (107A, 107B) during cell maintenance. The cell compartments (105) have a spear shape and are mounted with each hollow shaft (107A, 107B) via bearings or associated means. During the rotation of the impeller (103) of the bioreactor, the angular momentum of the culture medium, and the aspiration of the culture medium, the cells may be evenly distributed on the outer surface of the flexible filter (203). In a similar manner, during the rotation of the impeller (103) of the bioreactor, during the angular momentum of the culture medium, and during the release of the flow of the culture medium, the cell compartment (105) can rotate and transmit centrifugal force that provides easy removal of cells from the outer surface of the flexible filter (203) of the cell compartment (105) and cells that are adhered to or accumulated on the outer surface of the flexible filter (203) of the cell compartment (105).

[0054] In addition, the perfusion and cell maintenance processes may be automatically driven by the rotation of a bidirectional pump (109), a drainage pump (111), and a perfusion pump (not shown). For the production concentration mechanism, an ultrafiltration technique is employed. An ultrafilter (119) may be placed in line with a drainage tube (115). The size of the filter should be selected based on the size of the molecules to be concentrated. For example, if molecules having a size of less than 50 kDa (kilodaltons) are to be concentrated in the bioreactor, an ultrafilter having a pore cut-off size of 50 kDa is used to allow all molecules exceeding 50 kDa to pass through the harvest / drainage line, and the size of the product to be concentrated to be maintained in the loop of the external circulation tube (113) and ultimately in the bioreactor vessel (101) is less than 50 kDa.

[0055] A method for maintaining a cell using a cell maintenance device (100) according to the present invention comprises the following steps:

[0056] a) a step of suspending at least one pair of hollow shafts (107) having a first hollow shaft (107A) and a second hollow shaft (107B) within a container (101);

[0057] b) connecting at least one cell compartment (105) to the lower end of each hollow shaft (107);

[0058] c) connecting the first hollow shaft (107A) and the second hollow shaft (107B) at their upper ends through an external circulation tube (113) and configuring a bidirectional pump (109) together with the external circulation tube;

[0059] d) a step of creating a vacuum within a cell compartment (105) connected to the lower end of the first hollow shaft (107A) by operating the bidirectional pump (109) in a clockwise direction;

[0060] e) a step of shrinking the flexible filter (203) of the cell compartment (105) connected to the lower end of the first hollow shaft (107A);

[0061] f) a step of aspirating culture medium from the container (101) through a flexible filter (203) into a cell compartment (105) connected to the lower end of the first hollow shaft (107A), and fixing cells on the outer surface of the flexible filter (203) of the cell compartment (105) connected to the lower end of the first hollow shaft (107A);

[0062] g) A step of flowing the aspirated culture medium from a cell compartment (105) connected to the lower end of a first hollow shaft (107A) into a cell compartment (105) connected to the lower end of a second hollow shaft (107B) until a predetermined pressure is created in the subsequent cell compartment (105) or until a predetermined vacuum is created in the preceding cell compartment;

[0063] h) A step of releasing culture medium from a cell compartment (105) connected to the lower end of a second hollow shaft (107B) by passing it through a flexible filter (203);

[0064] i) a step of creating a vacuum within a cell compartment (105) connected to the lower end of the second hollow shaft (107B) by operating the bidirectional pump (109) in a counter-clockwise direction;

[0065] j) A step of contracting the flexible filter (203) of the cell compartment (105) connected to the lower end of the second hollow shaft (107B);

[0066] k) a step of aspirating culture medium from the container (101) through a flexible filter (203) into a cell compartment (105) connected to the lower end of the second hollow shaft (107B), and fixing cells on the outer surface of the flexible filter (203) of the cell compartment (105) connected to the lower end of the second hollow shaft (107B);

[0067] l) A step of flowing the aspirated culture medium from a cell compartment (105) connected to the lower end of the second hollow shaft (107B) into a cell compartment (105) connected to the lower end of the first hollow shaft (107A) until a predetermined pressure is created in the subsequent cell compartment (105) or until a predetermined vacuum is created in the preceding cell compartment;

[0068] m) A step of releasing culture medium from a cell compartment (105) connected to the lower end of the first hollow shaft (107A) by passing it through a flexible filter (203);

[0069] n) a step of releasing cells from the outer surface of the flexible filter (203) of the cell compartment (105) connected to the lower end of the first hollow shaft (107A);

[0070] o) a step of repeating steps (d) to (n) until an optimal cell density is reached or until batch termination; and

[0071] p) A step of withdrawing a used medium that does not contain cells from a container (101) through at least one hollow shaft (107) by means of a drainage pump (111) during or after performing steps (a) to (o) of the above method.

[0072] In one embodiment, during the execution of a method for cell maintenance and perfusion, a level sensor simultaneously detects the level of culture medium in the vessel (101) and, upon detection of a low level, adds fresh culture medium from the culture medium feed line by a perfusion pump until a set level is achieved. The used medium is removed through the drainage tube (115) at a controlled flow rate controlled by the drainage pump (111). At the same time, fresh medium is added through the feed line (not shown) at a controlled flow rate controlled by a medium addition pump (not shown). The perfusion rate determines the relative flow rates of these pumps, which is the medium exchange rate within the vessel (101) of the bioreactor.

[0073] In one embodiment, the predetermined pressure or vacuum of the cell compartment (105) is between 0.1 bar and 3 bar, and when such pressure or vacuum is reached, the bidirectional pump (109) rotates to the opposite side.

[0074] From now on, the present invention will be illustrated by the following examples. However, while the following examples are merely for the purpose of further explaining the present invention, the scope of the present invention is not limited to specific embodiments.

[0075] yes

[0076] ㆍ Control cell maintenance system using alternating tangential flow filtration (ATF) (control variant):

[0077] In the experiment, the process for the control batch was executed by the following steps. Commercial cell culture medium (Name: ActiviPro, Manufacturer: Cytiva) was used in this study. The culture medium was prepared according to the hydration protocol recommended by the seller. Shake flasks of different sizes were utilized for the initial stages of cell proliferation as described in the flowchart below. Cell bank vials were removed from liquid nitrogen (LN2) vessels and thawed according to standard procedures. The cell bank vials were rehydrated in 125 mL shake flasks, and the shake flasks were incubated in a shaker at 37°C, 5% CO2, and 160 RPM for 3 to 4 days. Cell proliferation culture (seed culture) was performed according to standard procedures based on the number of cells required to inoculate a 5 L bioreactor scale. A standard 5L disposable bioreactor (SUB) of the STR (stirred tank reactor) was installed, followed by culture medium filling within the same SUB according to standard procedures. The bioreactor was inoculated with n-1 seed cultures. The inoculation criteria for the study were 0.4 to 0.6 million cells / mL. Process parameters were monitored and maintained according to standard process specifications. The experimental period for both the control and test groups was 20 days. The temperature was maintained at 37 ± 1.0 °C throughout the batch run. The pH was maintained between 7.1 ± 0.3 using 7% filter-sterilized bicarbonate or CO2 gas. The dissolved oxygen concentration was maintained at a setpoint of 40% by a combination of aeration and stirring strategies. Sampling of the culture medium was performed according to standard procedures. Cell growth was measured by the dye exclusion method to derive cell density (million cells / mL) and viability.After use, media samples were used to analyze offline pH, glucose, lactate, and other metabolite concentrations. Residual glucose concentrations of 2.0 gm / L or higher were maintained by the addition of a 20% w / v filter-sterilized glucose solution.

[0078]

[0079] In the control variant, ATF-2 (alternating tangential flow) was used for the perfusion process, and the culture medium perfusion strategy (RV / day) and other process parameters were followed as described in Table 1 below.

[0080]

[0081] The results achieved in the control cell maintenance system (ATF) through these arrangements and processes are shown in Table 2 and Figure 6 below.

[0082]

[0083] ㆍ Comparative Case Study on Novel Cell Maintenance Devices:

[0084] A control batch was executed using the strategy discussed in the above sections. The cell maintenance device (100) was tested for its functional operation in comparison to the control process. Different variables of the present invention mentioned in Table 3 were present. All other aspects of the bioprocess, such as seed proliferation culture, type of culture medium, seed density, process analysis (cell number, glucose, lactate), bioreactor parameters (pH, temperature, DO, RPM, etc.), and perfusion rate, were kept constant for the control batch as mentioned in the above sections.

[0085]

[0086] ㆍ Variation Example 1:

[0087] A single-impeller bioreactor was employed for this example. A single cell compartment (105) connected to each of the hollow shafts (107A, 107B) of the pair of hollow shafts (107), namely the first hollow shaft (107A) and the second hollow shaft (107B), was suspended in parallel with the single-impeller bioreactor. The cell compartment (105) associated with the first hollow shaft (107A) and the second hollow shaft (107B) can be considered individually as the first cell compartment (105) and the second cell compartment (105). Both cell compartments (105) associated with each of the hollow shafts (107A, 107B) of the pair of hollow shafts (107) were stationary and had a square shape. Recirculation was enabled by a bidirectional peristaltic pump accompanied by a vacuum threshold of 1 bar that triggers flow reversal. A flexible filter (203) of a cell compartment (105) having a thickness of 200 microns with a pore size of 5 microns separated the cell compartments (105) from the bulk culture medium.

[0088] A bidirectional pump (109) is employed to induce pressure and vacuum inside the cell compartments (105). During the clockwise operation of the bidirectional pump (109), the culture medium is propelled from the first cell compartment (105) to the second cell compartment. This flow creates pressure inside the second cell compartment (105) and a corresponding vacuum inside the first cell compartment (105). When a pressure or vacuum threshold is reached, the bidirectional pump (109) operates counter-clockwise and reverses the direction of flow. During the counter-clockwise operation of the bidirectional pump (109), the culture medium is propelled from the second cell compartment (105) to the first cell compartment (105). This flow creates pressure inside the first cell compartment (105) and a corresponding vacuum inside the second cell compartment (105). A device (100) comprising hollow shafts and an external circulation tube (113) enabled the transfer of culture medium between the first cell compartment (105) and the second cell compartment (105) and vice versa.

[0089] The second cell compartment (105), which is provided to have a flexible filter (203), was expanded when pressure was generated by operating the bidirectional pump (109) clockwise. At the same time, the first cell compartment experienced a vacuum due to the rheological properties of the recirculating culture medium. This vacuum enabled the accumulation of cells on the pores of the flexible filter (203) of the first cell compartment (105). As the culture medium continued to circulate, the vacuum on the first cell compartment side was strengthened to trigger a pressure or vacuum threshold and the bidirectional pump (109) to ultimately reverse the flow direction. The reversal of the flow direction was triggered when a preset vacuum in the first cell compartment (105) was reached or when a preset pressure in the second cell compartment (105) was reached. This flow reversal separated the cells from the pores of the flexible filter (203) of the first cell compartment (105) and propelled the cells into the bulk culture medium. Subsequently, the first cell compartment (105) was pressurized while the second cell compartment (105) developed a vacuum. This continuous process was continued throughout the entire batch duration to ensure efficient cell separation and resuspension.

[0090] The perfusion rate, an important process parameter, was controlled by the user to optimize nutrient supply and waste removal. The cell-free culture medium was continuously drained from the bioreactor and transferred to a collection vessel (not shown in the drawing).

[0091] Through the above process, the cell density inside the bioreactor was increased. This increase in cell density leads to a corresponding increase in membrane penetration vacuum (TMV) and membrane penetration pressure (TMP) across the flexible membranes (203). Consequently, the increased cell density reflects a reduction in the time interval between two pre-defined vacuum thresholds, which causes a higher frequency of reversal of the culture flow direction in subsequent stages of culture. The results achieved in Variant Example 1 have viable cell density and viability as shown in Table 4 and Fig. 6 below.

[0092]

[0093] ㆍ Variation Example 2:

[0094] The experiment was carried out in a 5L bioreactor equipped with a CHO cell line. The process steps and procedures following for the experiment were identical to the processes following for Variant Example 1. Variant Example 2 represents a modification of Variant Example 1, namely, cell compartments (105) were placed in the middle region of the bioreactor.

[0095] The results achieved in Variation Example 2 through this arrangement and process are shown in Table 5 and Figure 6 below.

[0096]

[0097] ㆍ Variation Example 3:

[0098] The experiment was carried out in a 5L bioreactor equipped with a CHO cell line. The process steps and procedures following for the experiment were identical to the processes following for Variant Example 1. Variant Example 3 represents a modification of Variant Example 1, namely, a bioreactor configured to have two impellers, a lower impeller mounted on a central shaft and an upper impeller, was utilized. In addition to two pairs of hollow shafts (107) having two cell compartments (105) arranged sequentially on each hollow shaft (107A, 107B), four cell compartments (105) attached to the lower ends of each hollow shaft (107A, 107B) were arranged parallel to the lower impeller, and the remaining four cell compartments (105) were arranged parallel to the upper impeller. The pairs were arranged to be spaced apart at a 90-degree angle.

[0099] The results achieved in Variation Example 3 through this arrangement and process are shown in Table 6 and Figure 6 below.

[0100]

[0101] ㆍ Variant Example 4:

[0102] The experiment was carried out in a 5L bioreactor equipped with a CHO cell line. The process steps and procedures following for the experiment were identical to the processes following for Variant Example 1. Variant Example 4 represents a modification to Variant Example 1, namely regarding the type of bidirectional pump (109). A magnetically driven disposable pump was placed within the device (100) as the bidirectional pump (109).

[0103] The results achieved in Variation Example 4 through this arrangement and process are shown in Table 7 and Figure 6 below.

[0104]

[0105] Variation Example 5:

[0106] The experiment was carried out in a 5L bioreactor equipped with a CHO cell line. The process steps and procedures following for the experiment were identical to the processes following for Variation Example 1. Variation Example 5 represents a modification of Variation Example 1, namely regarding the shape of the cell compartment (105). Instead of a square shape, round-shaped cell compartments (105) were adopted during the experiment.

[0107] The results achieved in Variation Example 5 through this arrangement and process are shown in Table 8 and Figure 6 below.

[0108]

[0109] Variation Example 6:

[0110] The experiment was carried out in a 5L bioreactor equipped with a CHO cell line. The process steps and procedures following for the experiment were identical to the processes following for Variant Example 5. Variant Example 6 represents a modification of Variant Example 5, namely regarding the dynamic nature of the cell compartment (105). While Variant Example 5 employed a statically fixed cell compartment (105), Variant Example 6 included a rotating cell compartment (105) mounted on a bearing. This design enabled the compartment to rotate about its own axis in response to changes in the direction of liquid flow. Specifically, at the initiation of liquid flow into the compartment, the combined force of the inflow flow induced by the impeller and the inertial flow triggers rotational motion. This rotational motion potentially improves cell detachment and reduces the frequency of cell accumulation on the pores of the cell compartment (105).

[0111] The results achieved in Variation Example 6 through this arrangement and process are shown in Table 9 and Figure 6 below.

[0112]

[0113] ㆍ Variant Example 7:

[0114] The experiment was performed in a 5L bioreactor equipped with a CHO cell line. The process steps and procedures following the experiment were identical to the processes following for Variant Example 1. Variant Example 7 represents a modification to Variant Example 1, specifically regarding the threshold value for flow direction reversal. The device was enabled by a peristaltic pump accompanied by a vacuum threshold of 3 bar that triggers flow reversal. Compared to Variant Example 1, Variant Example 7 extends the time interval between vacuum-triggered flow reversals. In this configuration, the system requires a 3-bar vacuum inside the cell compartment (105) to initiate flow reversal, thereby causing a longer duration between flow reversal cycles.

[0115] The results achieved in Variation Example 7 through this arrangement and process are shown in Table 10 and Figure 6 below.

[0116]

[0117] ㆍ Variant Example 8:

[0118] The experiment was carried out in a 5L bioreactor equipped with a CHO cell line. The process steps and procedures following the experiment were identical to the process following for Variant 7. Variant 8 represents a modification to Variant 7, specifically regarding the pore size of the flexible membrane. In this experiment, a flexible membrane having a pore size of 10 microns was employed. Variant 8 extends the time interval between vacuum-triggered flow inversions compared to Variant 7. In this configuration, the system requires a 3-bar vacuum inside the cell compartment (105) to initiate flow inversion, as the relatively larger pore size of the flexible membrane allows small fragments to pass through the membrane exiting the bioreactor through the harvest line, thereby causing a longer duration between cycles compared to Variant 7.

[0119] The results achieved in Variation Example 8 through this arrangement and process are shown in Table 11 and Figure 6 below.

[0120]

[0121] conclusion:

[0122] The key process output parameters for these experiments were cell density and cell viability. These indicators were used to evaluate the overall performance of the bioreactor system. To isolate the effects of experimental variables, all input parameters were kept constant across both the control and test groups. These controlled parameters included seed proliferation culture methodology, culture medium composition, initial cell culture density, process analysis (cell number, glucose, lactate), bioreactor operating conditions (pH, temperature, dissolved oxygen, etc.), and perfusion rate.

[0123] The experimental data presented in the following section exemplifies cell density and viability profiles for both the cell maintenance device (100) and the control process. In particular, test experiments employing the device (100) demonstrated superior performance in terms of cell density and viability compared to a conventional control process. These results suggest that further optimization and enhancement of the process could lead to much greater improvements in cell culture performance.

[0124] The cell maintenance device (100) has demonstrated superior performance in improving both cell density and viability. This increased cell proliferation directly leads to a significant increase in the production of target proteins synthesized by these cells.

[0125] The entire device (100) may be configured to have a closed-loop control system and an automation system through which all pumps of the system can be operated. The pumps, through automation, deliver / drain culture medium from the container (101) according to the required amount and time.

[0126] The advantages of embodiments of the present invention are illustrated in this specification:

[0127] In one embodiment, using the rotation of a bidirectional pump (109) of alternating repetitive rotation and using cell compartments (105) of a pair of hollow shafts (107) to aspirate and discharge culture medium helps to maintain cells and separate cells from the circulating culture medium. This further helps the cell perfusion process and improves the density of the culture.

[0128] In one embodiment, the device (100) suspended within the vessel (101) of the bioreactor enables continuous production and increases the efficiency of the current production process. Additionally, there is no involvement of manual intervention or connection, and it provides an error-free configuration.

[0129] All devices and methods disclosed and claimed can be made and practiced without excessive experimentation in the context of this disclosure. Although the systems, devices, and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the methods, systems, devices, and steps or sequences of steps described herein without departing from the concept, spirit, and scope of the present invention. Explanation of the symbols

[0130] 100: Device for cell maintenance 101: Bioreactor Vessel 103: Impeller 105: Cell compartment 107: Hollow shaft pair 107A: First hollow shaft 107B: Second hollow shaft 109: Bidirectional pump 111: Drainage pump 113: External circulation tube 115: Drainage tube 117 sensor 119 : Ultrafilter 201: Gigi Messi 203: Musical Filter

Claims

Claim 1 A cell maintenance device comprising at least one pair of vertically extending hollow shafts (107); each hollow shaft (107A, 107B) is connected to at least one cell compartment (105) at its lower end; each cell compartment (105) is flexibly connected at the lower end of each individual hollow shaft, each hollow shaft (107A, 107B) is vertically extended, and the hollow shafts (107A, 107B) are flexibly connected to each other at their upper ends via an external circulation tube (113) and a bidirectional pump (109), and a drainage tube (115) is flexibly connected to the external circulation tube (113); and the cell compartment (105) is provided with a wall of a support mesh (201) and a flexible filter (203) placed on the wall of the support mesh (201). Claim 2 In claim 1, the bidirectional pump (109) is configured on or in line with the external circulation tube (113), forming a cell maintenance device. Claim 3 A cell maintenance device according to claim 1, wherein the drainage pump (111) is connected to the drainage tube (115). Claim 4 A cell maintenance device according to claim 1, wherein the lower end of each hollow shaft (107A, 107B) having a cell compartment (105) is suspended inside a container (101) filled with a culture medium. Claim 5 In paragraph 4, the cell maintenance device comprises a container (101) having at least one central shaft provided to have at least one impeller (103). Claim 6 In claim 5, the cell maintenance device is provided such that the container (101) has a level sensor for detecting the level of the culture medium within the container (101). Claim 7 In claim 5, each hollow shaft (107A, 107B) having a cell compartment (105) is arranged in a parallel orientation with respect to the central shaft, a cell maintenance device. Claim 8 A cell maintenance device according to claim 1, wherein a sensor (117) is provided along the external circulation tube (113) to detect and measure pressure or vacuum inside the external circulation tube (113) and the cell compartment (105). Claim 9 A cell maintenance device according to claim 1, wherein the flexible filter (203) has a porous structure configured to prevent the infiltration of cells from the culture medium into the cell compartment (105). Claim 10 A cell maintenance device according to claim 1, wherein the size of the pores in the flexible filter (203) is between 0.2 and 10 microns. Claim 11 In paragraph 9, the flexible filter is a cell maintenance device that is expandable and contractible. Claim 12 A cell maintenance device according to claim 9, wherein the thickness of the flexible filter is between 50 and 200 microns. Claim 13 In claim 1, the support mesh (201) is made of a plastic material or a metal material, forming a cell maintenance device. Claim 14 A cell maintenance device according to claim 1, wherein the mesh size of the support mesh (201) is larger than the size of the pores in the flexible filter (203). Claim 15 A cell maintenance device according to claim 1, wherein a plurality of cell compartments (105) are connected in series through hollow shafts (107A, 107B). Claim 16 A cell maintenance device according to claim 1, wherein each cell compartment (105) is rotatably connected to an individual hollow shaft (107A, 107B). Claim 17 In paragraph 2, the bidirectional pump (109) is a cell maintenance device that is a peristaltic pump. Claim 18 In paragraph 2, the bidirectional pump (109) is a magnetic pump, a cell maintenance device. Claim 19 A cell maintenance device according to paragraph 2, wherein a single bidirectional pump (109) is mounted across a plurality of pairs of hollow shafts (107) connected to at least one cell compartment (105). Claim 20 A cell maintenance device according to claim 1, wherein the ultrafilter (119) is arranged in line with the drainage tube (115). Claim 21 A cell maintenance device according to claim 1, wherein the shape of the cell compartment (105) is round, square, rectangular, or any other shape. Claim 22 A method for maintaining cells, comprising: a. suspending at least one pair of hollow shafts (107) having a first hollow shaft (107A) and a second hollow shaft (107B) within a container (101); b. connecting at least one cell compartment (105) to the lower end of each hollow shaft (107A, 107B); c. connecting the first hollow shaft (107A) and the second hollow shaft (107B) at their upper ends via an external circulation tube (113) and a bidirectional pump (109); d. creating a vacuum within the cell compartment (105) connected to the lower end of the first hollow shaft (107A) by operating the bidirectional pump (109) clockwise; e. contracting a flexible filter (203) of the cell compartment (105) connected to the lower end of the first hollow shaft (107A); f. a step of aspirating culture medium from a container (101) through a flexible filter (203) into a cell compartment (105) connected to the lower end of a first hollow shaft (107A) and accumulating cells on the outer surface of the flexible filter (203) of the cell compartment (105) connected to the lower end of the first hollow shaft (107A); g. flowing the aspirated culture medium from the cell compartment (105) connected to the lower end of the first hollow shaft (107A) into the cell compartment (105) connected to the lower end of a second hollow shaft (107B) until a predetermined pressure is created in the subsequent cell compartment (105) or until a predetermined vacuum is created in the preceding cell compartment (105); h. A step of releasing culture medium from a cell compartment (105) connected to the lower end of the second hollow shaft (107B) by passing it through a flexible filter (203); i. a step of creating a vacuum within the cell compartment (105) connected to the lower end of the second hollow shaft (107B) by operating a bidirectional pump (109) in a counter-clockwise direction; j. a step of contracting the flexible filter (203) of the cell compartment (105) connected to the lower end of the second hollow shaft (107B); k.A step of aspirating culture medium from a container (101) through a flexible filter (203) into a cell compartment (105) connected to the lower end of a second hollow shaft (107B) and adhering cells to the outer surface of the flexible filter (203) of the cell compartment (105) connected to the lower end of the second hollow shaft (107B); l. A step of flowing the aspirated culture medium from the cell compartment (105) connected to the lower end of the second hollow shaft (107B) into the cell compartment (105) connected to the lower end of the first hollow shaft (107A), until a predetermined pressure is created in the subsequent cell compartment (105) or until a predetermined vacuum is created in the preceding cell compartment (105); m. A cell maintenance method comprising: a step of releasing a culture medium from a cell compartment (105) connected to the lower end of a first hollow shaft (107A) by passing it through a flexible filter (203); a step of releasing cells from the outer surface of the flexible filter (203) of the cell compartment (105) connected to the lower end of the first hollow shaft (107A); and a step of repeating steps (d) to (n) until an optimal cell density is reached or until the batch is terminated. Claim 23 A cell maintenance method according to claim 22, further comprising the step of withdrawing a used medium not containing cells from a container (101) through at least one hollow shaft (107A, 107B) by means of a drainage pump (111) during or after performing steps (a) to (o). Claim 24 A cell maintenance method according to claim 22, further comprising the steps of: detecting the level of culture medium in a container (101) by means of a level sensor; and adding fresh culture medium from a culture medium supply line by means of a perfusion pump or a medium addition pump until a set level is achieved when a low level is detected. Claim 25 A cell maintenance method according to claim 22, further comprising the step of withdrawing used medium not containing cells from a vessel (101) through at least one hollow shaft (107A, 107B) at a controlled flow rate by a drainage pump (111); and simultaneously adding fresh culture medium from a culture medium supply line at a controlled flow rate by a perfusion pump or a medium addition pump. Claim 26 A cell maintenance method according to claim 22, wherein the predetermined vacuum and pressure of the cell compartments (105) are between 0.1 and 3 bar. Claim 27 A cell maintenance method according to claim 22, further comprising the step of rotating the cell compartment (105) to facilitate the removal of cells from the pores of the flexible filter (203) of the cell compartment (105).

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