ATF control device

Abstract

The utility model provides an ATF control device. The ATF control device comprises at least one peristaltic pump, a pipeline used for connecting the peristaltic pump and an ATF column, a pressure sensor arranged on the pipeline, and a control system connected with the peristaltic pump. The ATF control device can simulate the algorithm logic of a commercial ATF controller, provide stable air pressure in real time and complete alternate tangential flow filtration of cell sap, and is used for replacing the commercial ATF controller on the market at present, so that the research and development cost of a perfusion culture process is reduced, and the COG level of the process is further reduced.

Description

Technical Field

[0001] The utility model belongs to the field of cell culture devices and relates to an ATF control device. Background Art

[0002] Among various cell culture models, higher cell yield and quality are the main reasons why the development and optimization of perfusion culture processes have become the focus of current cell process research. To achieve this goal, perfusion of fresh culture medium and cell retention are the core contents of this process. Perfusion of fresh culture medium can generally be achieved by controlling the flow of the pump at the front end of the bioreactor through the Delta V system to achieve medium replacement, while cell retention requires a more complex cell retention device. The importance of cell retention devices lies in their ability to simultaneously harvest the target product during the perfusion process and minimize the loss of suspended cells. In the development and progress of perfusion processes, cell density and protein concentration in the culture process have become increasingly higher, which undoubtedly prompts changes and innovations in cell retention equipment. The most fundamental requirement is to minimize the loss of suspended cells while harvesting as much target product as possible.

[0003] After years of development, cell retention technology can be divided into gravity sedimentation, centrifugal sedimentation, acoustic sedimentation and hollow fiber column filtration according to the working principle. The retention device designed based on the principle of hollow fiber columns has become the most widely used cell retention device in cell perfusion processes due to its high cell retention rate, simple operation and easy scale-up. Among them, according to the filtration method, it can also be divided into tangential flow filtration (TFF) and alternating tangential flow (ATF) mode. Compared with TFF, the presence of the diaphragm in the ATF mode greatly reduces the shear force of liquid flow on cells. At the same time, the alternating reciprocating motion of the culture medium in the hollow fiber column also plays a role in flushing the column to a certain extent, reducing the risk of fiber column clogging, which makes the ATF mode more widely used in perfusion culture.

[0004] In the ATF system, the alternating reciprocating motion of the cell fluid is achieved through the periodic concave and convex motion of the diaphragm pump in the ATF controller. Specifically, the periodic concave and convex motion of the diaphragm pump can be divided into two cycles: the pressure cycle and the exhaust cycle. The diaphragm pump contains a silicone valve that divides the entire diaphragm pump chamber into two areas (the liquid chamber and the air chamber). The liquid chamber is connected to the internal fiber area of ​​the hollow fiber column module and is connected to the culture fluid in the tank; the air chamber is connected to the air column inside the ATF controller. First, the pressure regulating valve inside the ATF controller applies positive compressed air to the air chamber, causing the valve inside the pump to move upward due to the pressure from the compressed air of the ATF controller, injecting the cell fluid in the diaphragm pump into the tank. This period is called the pressure cycle. Similarly, when the pressure sensor senses that the pressure has stabilized, the pressure regulating valve switches to a negative pressure channel. This causes the air in the air chamber of the diaphragm pump to be pumped out, and the valve then moves downward, pumping the cell fluid from the tank toward the hollow fiber column for filtration into the liquid chamber. This period is known as the exhaust cycle. Generally speaking, it primarily involves using compressed air and a vacuum system as driving forces, controlling the alternating flow of air in and out of the bottom of the diaphragm pump through the pressure sensor and pressure regulating valve, thereby achieving alternating tangential flow filtration of the culture fluid through the hollow fiber column.

[0005] Currently, mainstream commercial ATF controllers are primarily closed-loop instruments consisting of a pressure sensor, a proportional pressure regulator, and a central control system. These devices require external compressed air and vacuum systems for operation. While these controllers provide stable air pressure and utilize precise algorithms for real-time pressure control, their complex internal structure and control system maintenance lead to high procurement and maintenance costs. The licensing fees for the ATF control system software are also significant. As perfusion processes scale up, the ATF controllers used must be replaced as culture volumes and gas throughput increase. Generally, small-scale cultures in laboratories typically range from 3L to 7L, and 15L, requiring ATF controllers of corresponding sizes. However, due to the low frequency of use of 15L reactors, ATF controllers often sit unused for extended periods, resulting in a significant waste of resources. Furthermore, the ATF requires compressed air and vacuum systems, which impose unnecessary costs on perfusion processes and limit the convenience and usability of ATF controllers. As perfusion processes mature and cell lines are developed, cell growth becomes more rapid, leading to ever-increasing cell culture densities. The emergence of the new ultra-intensified intermittent perfusion fed-batch process has further raised the upper limit of cell culture density. Consequently, with the increasing demand for perfusion processes and increasing culture densities, the disadvantages of ATF controllers have gradually widened, leading to a growing demand for cost-effective, simple alternatives to commercial ATF controllers. Utility Model Content

[0006] In order to solve the technical problems existing in the prior art, the utility model provides an ATF control device, which can simulate the algorithm logic of a commercial ATF controller, provide stable air pressure in real time, and complete alternating tangential flow filtration of cell fluid. It is used to replace the commercial ATF controllers currently on the market, so as to reduce the R&D cost of perfusion culture process and further reduce the process COG level.

[0007] In order to achieve the above technical effects, the present invention adopts the following technical solutions:

[0008] The utility model provides an ATF control device, which comprises at least one peristaltic pump, a pipeline for connecting the peristaltic pump and an ATF column, a pressure sensor arranged on the pipeline, and a control system connected with the peristaltic pump.

[0009] As a preferred technical solution of the present invention, the ATF control device includes one or two peristaltic pumps, and the peristaltic pumps are bidirectional peristaltic pumps.

[0010] As a preferred technical solution of the present invention, the ATF control device is used to directly adjust the rotation direction and speed of the peristaltic pump.

[0011] As a preferred technical solution of the present invention, the pipeline includes a hose and a wear-resistant pipe that are connected to each other.

[0012] As a preferred technical solution of the present invention, one end of the hose of the pipeline is connected to the ATF column, and one end of the wear-resistant tube of the pipeline is connected to the peristaltic pump.

[0013] As a preferred technical solution of the present invention, a pressure sensor is provided at the connection between the hose and the ATF column.

[0014] As a preferred technical solution of the present invention, the pressure sensor and the control system are connected via a wired network or a wireless network.

[0015] As a preferred technical solution of the present invention, when the ATF control device includes a peristaltic pump, the control system is used to control the conversion, speed and rotation time of the bidirectional alternating rotation of the peristaltic pump.

[0016] As a preferred technical solution of the present invention, when the ATF control device includes two peristaltic pumps, the control system is used to independently control the start and stop sequence, rotation speed and rotation time of the peristaltic pumps.

[0017] As a preferred technical solution of the present invention, the control system includes an alarm component.

[0018] Compared with the prior art, the present invention has at least the following beneficial effects:

[0019] (1) The utility model provides an ATF control device, which simplifies the ATF control system and improves the flexibility and control space of the ATF controller;

[0020] (2) The present invention provides an ATF control device, which can reduce the operating cost of the ATF controller and reduce the COG of the perfusion process;

[0021] (3) The present invention provides an ATF control device, which can increase the upper limit of cell culture density and provide more possibilities for cell culture;

[0022] (4) The present invention provides an ATF control device that can intelligently regulate ventilation rate and time, thereby reducing the risk of ATF blockage;

[0023] (5) The utility model provides an ATF control device, which is easy to scale up and can be scaled up proportionally according to the culture volume and ventilation volume. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1a A schematic structural diagram of the ATF control device provided in Example 1 of the present utility model;

[0025] Figure 1b This is a schematic diagram of the interface of the control system in the ATF control device provided in Example 1 of the present utility model;

[0026] Figure 2a A schematic structural diagram of the ATF control device provided in Example 2 of the present utility model;

[0027] Figure 2b This is a schematic diagram of the interface of the control system in the ATF control device provided in Example 2 of the present utility model;

[0028] Figure 3 A schematic structural diagram of a cell culture device provided in Application Example 1 of the present utility model;

[0029] Figure 4 This is a comparison chart of the live cell density in culture at the N-1 stage between a commercial ATF controller and a self-developed ATF controller;

[0030] Figure 5 This is a comparison chart of the culture viability at the N-1 stage between commercial ATF controllers and self-developed ATF controllers;

[0031] Figure 6 This is a comparison chart of the total viable cell density in the N-1 stage of culture between a commercial ATF controller and a self-developed ATF controller;

[0032] Figure 7 This is a comparison chart of the N-1 stage culture growth rate between the commercial ATF controller and the self-developed ATF controller;

[0033] Figure 8 This is a comparison chart of the live cell density in N-stage culture between a commercial ATF controller and a self-developed ATF controller;

[0034] Figure 9 This is a comparison chart of the N-stage culture viability between commercial ATF controllers and self-developed ATF controllers;

[0035] Figure 10 This is a comparison chart of the total viable cell density in N-stage culture between a commercial ATF controller and a self-developed ATF controller;

[0036] Figure 11 This is a comparison chart of glucose concentration in N-stage culture between a commercial ATF controller and a self-developed ATF controller;

[0037] Figure 12 This is a comparison chart of lactate concentration in N-stage culture between a commercial ATF controller and a self-developed ATF controller;

[0038] Figure 13 This is a comparison chart of sodium ion concentration in N-stage culture between commercial ATF controller and self-developed ATF controller;

[0039] Figure 14 This is a comparison chart of the osmotic pressure concentration in N-stage culture between the commercial ATF controller and the self-developed ATF controller;

[0040] Figure 15 This is a comparison chart of target protein concentration in N-stage culture between commercial ATF controller and self-developed ATF controller;

[0041] In the figure: 1-fresh perfusion medium, 2-bioreactor, 3-ATF column, 4-ATF controller, 41-first peristaltic pump, 42-second peristaltic pump, 43-pipeline, 44-pressure sensor, 45-control system, 411-bidirectional peristaltic pump, 5-harvest medium.

[0042] The following is a further detailed description of the present invention. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims. DETAILED DESCRIPTION

[0043] The technical solution of this application is further explained below through specific implementation methods.

[0044] The specific embodiment of the present utility model provides an ATF control device, which includes at least one peristaltic pump, a pipeline for connecting the peristaltic pump and the ATF column, a pressure sensor arranged in the pipeline, and a control system connected to the peristaltic pump.

[0045] In the present invention, power is provided by absorbing or exhausting air through the rotation of a peristaltic pump, replacing the use of compressed air, simplifying the structure of the ATF control device and reducing the procurement cost of the ATF control device. In addition, the pressure provided by the peristaltic pump is much greater than the pressure provided by traditional commercial ATF controllers, thereby being able to adapt to the needs of cell culture with high cell density. The pressure sensor and the peristaltic pump are monitored by the control system, and the speed is adjusted in real time to ensure that the diaphragm pump can perform complete concave and convex motion.

[0046] In a specific embodiment of the present invention, the peristaltic pump and the control system can be connected via a wired network or a wireless network. The control system can be set separately from the peristaltic pump, or the control system can be integrated into the peristaltic pump, that is, the control system is an integral part of the peristaltic pump.

[0047] In a specific embodiment of the present invention, when the ATF control device includes a peristaltic pump, the peristaltic pump is a bidirectional peristaltic pump, and the purpose of controlling air absorption and discharge is achieved by controlling the rotation direction of the peristaltic pump.

[0048] In a specific embodiment of the present invention, when the ATF control device includes two peristaltic pumps, one peristaltic pump is used to control air absorption, and the other peristaltic pump is used to control air discharge.

[0049] In a specific embodiment of the present invention, the model of the peristaltic pump can be selected according to the culture scale of the cells, and is not specifically limited here.

[0050] In a specific embodiment of the present invention, the hose is preferably a silicone tube.

[0051] In a specific embodiment of the present invention, the type of pipeline can be selected according to the culture scale of the cells and the model of the peristaltic pump, so no specific limitation is made here. Specifically, the inner diameter of the pipeline will affect the amount of air passing through per unit time, that is, the flow rate of the air, thereby affecting the pressure provided by the peristaltic pump. If the inner diameter of the pipeline is too small, the pump will not be able to completely fix the pipeline and complete the full flow of air, nor will it be able to provide enough pressure to allow the diaphragm pump to complete the up and down movement; similarly, if the inner diameter of the pipeline is too large, the pump will be subjected to excessive extrusion and deformation, and excessive pressure will also challenge the bearing capacity of the diaphragm pump and increase the risk of rupture. Therefore, the size of the pipeline needs to be selected according to the culture scale of the cells and the applicability of the peristaltic pump model.

[0052] In a specific embodiment of the present invention, the pressure sensor is arranged at the connection between the silicone tube and the ATF column, which can be considered to be equivalent to the pressure exerted on the diaphragm pump.

[0053] In a specific embodiment of the present invention, the cross-section of the control system includes the start button of the ATF controller (ATF START), the alarm component (ALARM), the pump speed setting (Pump 1speed & Pump 2speed), the rotation time setting (CWTIME & CCW TIME) and the real-time operation status display of the peristaltic pump.

[0054] In one embodiment of the present invention, when the ATF control device includes a peristaltic pump, the control system is used to control the rotation direction, speed, and rotation time of the peristaltic pump. Specifically, the rotation time and speed are set in the control system according to the model of the ATF column. The peristaltic pump rotates clockwise according to the set rotation time and speed. After the clockwise rotation ends, the peristaltic pump immediately rotates counterclockwise according to the set rotation time and speed, and the peristaltic pump performs a reciprocating motion in this manner. The control system adjusts the rotation speed of the peristaltic pump based on the detection results of the pressure sensor to ensure appropriate pressure supply.

[0055] In one embodiment of the present invention, when the ATF control device includes two peristaltic pumps, the control system is used to independently control the start and stop sequence, rotation speed, and rotation time of the peristaltic pumps, while also being able to regulate the rotation direction and speed in real time through the peristaltic pumps. Specifically, the control system sets the rotation time and rotation speed according to the model of the ATF column. The first peristaltic pump rotates clockwise according to the set rotation time and rotation speed. After the first peristaltic pump completes rotation, the second peristaltic pump immediately rotates counterclockwise according to the set rotation time and rotation speed, and the two pumps perform reciprocating motion in this manner. The control system adjusts the rotation speeds of the first and second peristaltic pumps based on the pressure sensor detection results to ensure appropriate pressure supply.

[0056] In a specific embodiment of the present invention, the rotation time can be set according to the model of the ATF hollow fiber column, which is usually related to the volume of the diaphragm pump chamber, that is, the displacement volume of the diaphragm pump.

[0057] In a specific embodiment of the present invention, the alarm component in the control system monitors the data of the rotation speed and pressure sensor, thereby greatly reducing the experimental abnormalities caused by a series of equipment factors such as loose pump head and pipeline rupture of the peristaltic pump.

[0058] In order to better illustrate the present invention and facilitate understanding of the technical solution of the present invention, typical but non-limiting embodiments of the present invention are as follows:

[0059] Example 1

[0060] This embodiment provides an ATF control device, the structure of which is as follows: Figure 1a and 1bAs shown, the ATF control device includes two peristaltic pumps, namely a first peristaltic pump 41 and a second peristaltic pump 42, both of which are unidirectional peristaltic pumps; a pipeline 43 for connecting the first peristaltic pump 41, the second peristaltic pump 42 and the ATF column 3, the pipeline 43 including a silicone tube and a wear-resistant tube connected to each other, one end of the silicone tube of the pipeline 43 is connected to the ATF column 3, and one end of the wear-resistant tube of the pipeline 3 is connected to the first peristaltic pump 41 and the second peristaltic pump 42 respectively; a pressure sensor 44 is arranged in the pipeline 43, specifically, the pressure sensor 44 is arranged at the connection between the silicone tube and the ATF column 3; and a control system 45 electrically connected to the first peristaltic pump 41, the second peristaltic pump 42 and the pressure sensor 44, respectively, the control system 45 is used to independently control the start and stop sequence, speed and rotation time of the first peristaltic pump 41 and the second peristaltic pump 42, and adjust the speed of the first peristaltic pump 41 and the second peristaltic pump 42 according to the pressure monitoring result of the pressure sensor 44.

[0061] Example 2

[0062] This embodiment provides an ATF control device, the structure of which is as follows: Figure 2a and 2b As shown, the ATF control device includes a bidirectional peristaltic pump 411; a pipeline 43 for connecting the bidirectional peristaltic pump 411 and the ATF column 3, the pipeline 43 includes a silicone tube and a wear-resistant tube connected to each other, one end of the silicone tube of the pipeline 43 is connected to the ATF column 3, and one end of the wear-resistant tube of the pipeline 3 is connected to the bidirectional peristaltic pump 411; a pressure sensor 44 is arranged in the pipeline 43, specifically, the pressure sensor 44 is arranged at the connection between the silicone tube and the ATF column 3; and a control system 45 electrically connected to the bidirectional peristaltic pump 411 and the pressure sensor 44, the control system 45 is used to control the rotation direction, speed and rotation time of the bidirectional peristaltic pump 411, and adjust the speed of the bidirectional peristaltic pump 411 according to the pressure monitoring result of the pressure sensor 44.

[0063] Application Example 1

[0064] This application example provides a cell culture device, the structure of which is as follows: Figure 3 As shown, the cell culture apparatus includes a fresh perfusion medium 1, a bioreactor 2, an ATF column 3, an ATF controller 4, and a harvested medium 5. The ATF controller 4 is provided in Example 1. The first peristaltic pump 41 and the second peristaltic pump 42 are both Waston Marlow 232S models. The first peristaltic pump 41 is responsible for rotating clockwise to exhaust air, while the second peristaltic pump 42 is responsible for rotating counterclockwise to inhale air. The pipe 43 is model 17# with an inner diameter of 6.4 mm, suitable for a maximum flow rate of 1400 ml / min provided by Waston Marlow. The ATF system can provide a pressure of approximately 0.8 bar.

[0065] Before the experiment began, the rotation time of the first peristaltic pump 41 and the second peristaltic pump 42 was set in the control system 45. The volume of the bioreactor 2 was 3 L, the model of the ATF column 3 was ATF 2, the volume contained in the diaphragm pump chamber was approximately 100 mL, and the base flow rate was 0.5 L / min. The rotation time of the first peristaltic pump 41 and the second peristaltic pump 42 was set to 0.2 min (12 s). The control system 45 adjusted the rotation speed of the first peristaltic pump 41 and the second peristaltic pump 42 based on the monitoring results of the pressure sensor 44.

[0066] The cell culture device provided in the application example was used for cell culture. A cell line developed by the applicant was used, and the culture process was the UI-IPFB (Ultra-intensified Intermittent Perfusion Fed-Batch) process developed by the applicant. This process is based on IFB and improves the N-1 inoculation to N stage and the N stage culture mode, so that a higher inoculation density can be obtained in the N stage and the cells can be cooled down to enter the plateau phase in advance, further increasing the cell protein production time, thereby obtaining a higher protein yield. The characteristic of this process is that a concentration operation will be performed in the N-1 stage, at which time the cell density will rise to nearly 2.0×108 cells / mL, and the cell density will be maintained at 4.0×108 cells / mL for a long time in the N production culture stage. 7 cells / mL or more greatly increases the pressure of the ATF controller, thereby increasing the risk of ATF column clogging. Therefore, the ATF controller needs to provide a higher pressure to drive the concave and convex movement of the diaphragm pump. The ATF system provided in Example 1 of the present application can provide a pressure of approximately 0.8 bar, which is much greater than the pressure that a traditional commercial ATF controller can provide and is more suitable for this process.

[0067] During the seed chain stage, this cell line was cultured in a suitable flat-bottom shake flask (Nest) during the passage and expansion process, and the culture conditions in the shaker (Kuhner) were 36.5°C, 110 rpm, and 6% CO2 concentration. The culture medium used for passage was the commercial medium CD CHO (Cytiva), and the N-2 stage was adapted in Actipro (Cytiva) for inoculation to N-1. A 3L reactor was used in the N-1 and production culture stages, with an initial culture volume of 1.5L, a pH setting of 6.90±0.25, a dissolved oxygen saturation of 40%, and the N-1 culture temperature was maintained at 36.5°C. When inoculated into the N production culture stage, the temperature was reduced to 33°C on D1. The initial inoculation density of N-1 was 2.0×10 6cells / mL. When the cell culture density reached the expected level on D5 or D6, a 0.2 μm ATF hollow fiber column (RepliGen) was used for concentration, which increased the cell culture density to 1.0-2.0 × 10 cells / mL in a short period of time. 8 cells / mL, and then inoculated into the N production culture stage at a 1:5 dilution ratio of seed solution to culture medium. The ATF hollow fiber column used in the N stage was a 50 kD column from Repligen. The basal medium used in the N-1 and N culture stages was Actipro (Cytiva), and the feed medium was also Cytiva's Cell Boost 7a / b.

[0068] Comparative Application Example 1

[0069] In this application example, the conditions are the same as those in application example 1, except that the ATF controller is a traditional commercial ATF controller. The N-1 stage of the commercial ATF controller is 1.0×10 6 cells / mL inoculation density.

[0070] Figure 4-7 The cell performance at the N-1 production stage is shown. Figure 4 It can be seen that the increase in initial seeding density significantly accelerated the increase in cell culture density. Figure 5 and Figure 7 It also showed that the cell viability and specific growth rate were not affected by high density, which provided more room for increasing the initial inoculation density in the N production stage.

[0071] Figure 8-15 Shown are the cell performances during the N production phase, where Figure 8-10 The cell growth status is described. It can be seen that compared with the historical N-stage cell culture using a commercial ATF controller, the use of the self-developed new ATF controller can adapt to higher inoculation densities while maintaining better cell viability and longer culture time, thereby obtaining a higher total live cell density.

[0072] Figure 11-15 The cell metabolism and protein concentration are described. It can be seen that despite the differences in cell density, the metabolism of glucose and lactate maintains similar trends, while sodium ion and osmotic pressure also show comparable trends. Due to the relatively high total viable cell density, the target protein using the self-developed new ATF controller is also relatively higher.

[0073] The applicant declares that while the above-described embodiments illustrate the detailed structural features of the present invention, the present invention is not limited to these detailed structural features, nor does it imply that the present invention must rely on these detailed structural features in order to be implemented. Persons skilled in the art should understand that any improvements to the present invention, equivalent replacements for selected components, additions of auxiliary components, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0074] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0075] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0076] In addition, the various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. An ATF control device, characterized in that: The ATF control device includes at least one peristaltic pump, a pipeline for connecting the peristaltic pump and the ATF column, a pressure sensor arranged on the pipeline, and a control system connected to the peristaltic pump.

2. The ATF control device according to claim 1, characterized in that: The ATF control device includes one or two peristaltic pumps, and the peristaltic pumps are bidirectional peristaltic pumps.

3. The ATF control device according to claim 1, characterized in that: The ATF control device is used to directly adjust the rotation direction and rotation speed of the peristaltic pump.

4. The ATF control device according to claim 1, characterized in that: The pipeline includes a hose and a wear-resistant pipe connected to each other.

5. The ATF control device according to claim 4, characterized in that: One end of the hose of the pipeline is connected to the ATF column, and one end of the wear-resistant tube of the pipeline is connected to the peristaltic pump.

6. The ATF control device according to claim 4, characterized in that: The pressure sensor is arranged at the connection between the hose and the ATF column.

7. The ATF control device according to claim 1, characterized in that: The pressure sensor is connected to the control system via a wired or wireless network.

8. The ATF control device according to claim 1, characterized in that: When the ATF control device includes a peristaltic pump, the control system is used to control the conversion, rotation speed and rotation time of the bidirectional alternating rotation of the peristaltic pump.

9. The ATF control device according to claim 1, characterized in that: When the ATF control device includes two peristaltic pumps, the control system is used to independently control the start and stop sequence, rotation speed and rotation time of the peristaltic pumps.

10. The ATF control device according to claim 1, characterized in that: The control system includes an alarm component.

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