KR101367870B1 - Cell culture device of perfusion type - Google Patents

Abstract

Provided is a perfusion cell culture device using an alternating tangential flow (ATF) system. The perfusion cell culture device includes an incubator for storing cell culture solution; A filter module including a housing having a first port, a second port, and an outlet port, and a plurality of hollow fibers installed inside the housing; A connecting tube installed between the incubator and the first port; A diaphragm pump connected to the second port and providing a pumping pressure into the hollow fiber; And a recovery device having a non-return valve connected to the outlet port and a pinch valve provided at the front or rear end of the non-return valve.

Description

Perfusion cell culture device {CELL CULTURE DEVICE OF PERFUSION TYPE}

The present invention relates to a perfusion cell culture device, and more particularly, to a perfusion cell culture device using an alternating tangential flow (ATF) system.

In the production of protein therapeutics using animal cells, perfusion culture methods are widely used when high concentrations of cells are required or target proteins are unstable (Adv Biochem Engin. 2002 (74), 129-169).

In the perfusion culture method, an alternating tangential flow (ATF) system using a filter module made of hollow fiber for circulating cell culture and separating cells and cell cultures has been developed and used (US Pat. No. 6,544,424). In the perfusion culture method using the ATF system, the cell culture fluid flows out of the hollow fiber of the filter module by the alternating tangential flow induced by the diaphragm pump, and the discharged cell culture fluid is recovered by using a pump connected to the filtration valve. to be.

The perfusion culture method using the ATF system is known to be able to maintain a very high cell concentration and to obtain a high concentration of biological material compared to other perfusion culture methods (BIOPHARM INT, 2011 (24), 54-60). , GEN, 2010 (30), 7, Apr. 1). In addition, the perfusion culture method using the ATF system is known to reduce the aggregation of cells during the culturing process, in particular the aggregation of cells having an inherent tendency to form aggregates in cell culture (International Publication No. WO05 / 095578).

In the perfusion culture apparatus using the ATF system, a cell culture liquid is recovered by using a pump. However, in the apparatus with a pump, the cell culture liquid forced out of the hollow yarn is forcibly recovered only during the operation of the pump regardless of the period in which positive transmembrane pressure is formed by the diaphragm pump in the hollow fiber. In this recovery structure, the pressure formed in the hollow fiber is not effectively used as the intermembrane pressure. Therefore, in the case of a biological material having a large molecular weight and a hydrodynamic radius, a recovery rate is sharply lowered.

The present invention is to provide a perfusion cell culture apparatus that can obtain a biological material with a high recovery rate regardless of molecular weight by improving the recovery structure of the cell culture.

Perfusion cell culture apparatus according to an embodiment of the present invention comprises a culture medium for storing the cell culture; A filter module including a housing having a first port, a second port, and an outlet port, and a plurality of hollow fibers installed inside the housing; A connecting tube installed between the incubator and the first port; A diaphragm pump connected to the second port and providing a pumping pressure into the hollow fiber; And a recovery device provided with a non-return valve connected to the outlet port and a pinch valve provided at the front end or the rear end of the non-return valve so as to coincide with the period in which the interlurgical pressure is formed in the hollow fiber by the diaphragm pump and the recovery period.

The first port and the second port face each other along one direction, and the outlet port may be formed along a direction crossing one direction.

Hollow yarns are installed side by side in one direction and may be divided into a lumen side corresponding to the hollow and a shell side outside the hollow fiber wall. The first port and the second port may be connected to the inside of the hollow yarns, and the outlet port may be connected to the outside of the hollow yarns.

A recovery pipe may be mounted at the outlet port, a backflow prevention valve and a pinch valve may be installed at the recovery port, and a filtration valve may be positioned in front of the nonreturn valve.

The pinch valve is connected to the timer and the control unit, and may operate only for a time set in the timer to open the return pipe. The incubator is equipped with a load cell for detecting a change in the weight of the cell culture medium, the control unit may be electrically connected to the load cell to control the operation of the pinch valve according to the weight of the cell culture medium.

In another aspect, there is provided a cell culture method comprising inoculating cells into the perfusion cell culture device to perform perfusion culture.

In the perfusion cell culture apparatus of this embodiment, the recovery device coincides with the period during which the positive intermembrane pressure is formed in the hollow fiber and the period during which the target material is recovered through the pores of the hollow fiber, and as a result, the molecular weight and the hydrodynamic radius are large. The target substance can be easily recovered.

1 and 2 is a block diagram showing a perfusion cell culture apparatus according to an embodiment of the present invention.
3 is a CHO (DG44) cell in which the coagulation factor 8 gene (B-domain deleted clotting factor VIII) and the von Willebrand factor gene are simultaneously transformed using a conventional perfusion cell culture apparatus ( It is a graph showing the concentration of viable cells when cultured KCLRF-BP-172) and the amount of recombinant blood coagulation factor 8 in the cell culture medium of the incubator.
Figure 4 is a culture of CHO (DG44) cells (KCLRF-BP-172) transformed simultaneously with the coagulation factor 8 gene and von Willibrand factor gene using a perfusion cell culture apparatus according to an embodiment of the present invention It is a graph showing the concentration of surviving cells in one case and the amount of recombinant coagulation factor 8 in the cell culture medium of the incubator.
5 is a CHO (DG44) cell in which the coagulation factor 8 gene and the von Willebrand factor gene are simultaneously transformed using a conventional perfusion cell culture device and a perfusion cell culture device according to an embodiment of the present invention. It is a graph which shows the quantity (recovery rate) of the 8th factor of recombinant blood coagulation in collect | recovered recovered cell culture liquid in case of culturing KCLRF-BP-172).
FIG. 6 shows that the coagulation factor 8 present in the cell culture medium is present in the form of binding to von Willebrand factor as well as the form not bound to von Willibrand factor, and A represents an anti-von Willibrand antibody. Western blot was used, and B is the result of Western blot using the anticoagulation factor 8 antibody.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, which will be readily apparent to those skilled in the art to which the present invention pertains. The present invention may be embodied in many different forms and is not limited to the embodiments described herein.

1 and 2 is a block diagram showing a perfusion cell culture apparatus according to an embodiment of the present invention.

1 and 2, the perfusion cell culture apparatus 100 of the present embodiment includes a cultivator 10 for storing a cell culture solution, a filter module 20 composed of a housing 21 and a plurality of hollow fibers 22. And a diaphragm pump 30 connected to the filter module 20 to provide a pumping pressure, and a recovery device 40 for recovering the cell culture liquid flowing out of the filter module 20.

The incubator 10 receives and stores the culture solution by the culture solution supply pump 11, and cultures the biological material in the stored culture solution. The incubator 10 is covered with a cover plate 12, and a stirrer 13 for stirring the cell culture solution may be installed on the cover plate 12. In addition, a load cell 14 for detecting a change in weight of the cell culture solution may be installed at the bottom of the incubator 10.

In the perfusion cell culture apparatus 100 of the present embodiment, the culture material (target material) may be a biological material having a molecular weight of 100 kDa or more, such as 100 to 100,000 kDa. In particular, the molecular weight of the single molecule, such as the von Willebrand factor (von Willebrand factor) may form a polymer having a molecular weight of 260kDa or about 20,000kDa, so that the hydrodynamic radius may be increased to 0.4㎛ or more.

The housing 21 of the filter module 20 forms a first port 23 and a second port 24 facing each other along one direction (vertical direction with reference to the drawings). The first port 23 is provided with a connection tube 51 connected to the incubator 10 to allow movement of the cell culture solution between the incubator 10 and the housing 21. The diaphragm pump 30 is connected to and installed in the second port 24 to provide pumping pressure into the housing 21. In addition, the outlet port 25 is formed in the housing 21 along a direction (horizontal direction based on the drawing) that intersects the above-described one direction.

1 and 2, the first port 23 and the connection pipe 51 are positioned above the housing 21, and the second port 24 and the diaphragm pump 30 are positioned below the housing 21. Although illustrated as an example, the reverse is also possible. That is, the diaphragm pump 30 may provide a pumping pressure at the upper side of the housing 21, and the connector 51 may be connected to the lower side of the housing 21.

The plurality of hollow fibers 22 are installed side by side in the form of a bundle in parallel with the above-described one direction (vertical direction with reference to the drawings) inside the housing. In this case, the plurality of hollow fibers 22 are positioned at a predetermined distance from the inner wall of the housing 21.

The hollow fiber 22 forms a hollow penetrating the inside along the longitudinal direction, and numerous voids are formed in the wall of the hollow yarn. The pores of the hollow fiber 22 may have a size in the range of 0.2 μm to 0.5 μm. The hollow fiber 22 is divided into a lumen side corresponding to the hollow and a shell side outside the wall of the hollow fiber 22. The first port 23 and the second port 24 are connected to the inside of the hollow fiber 22, the outlet port 25 is connected to the outside of the hollow fiber 22.

The diaphragm pump 30 includes a first chamber 31 and a second chamber 32 separated by the diaphragm 33. The first chamber 31 is connected to the alternate tangential flow control unit 35 (hereinafter referred to as an 'ATF control unit') to receive compressed air periodically therefrom. The second chamber 32 is connected to the inside of the hollow fiber 22 by the second port 24.

When compressed air is provided to the first chamber 31 and the first chamber 31 expands due to the deformation of the diaphragm 33 (see FIG. 1), the cell culture medium filling the second chamber 32 is filtered. It is pushed in the direction of) and moves to the incubator 10 through the hollow fiber 22 inside. On the contrary, when the compressed air of the first chamber 31 is recovered, the second chamber 32 is expanded due to the deformation of the diaphragm 33 (see FIG. 2), and the cell culture solution stored in the incubator 10 moves inside the hollow fiber 22. It moves to the 2nd chamber 32 via.

As the injection and recovery of the compressed air to the first chamber 31 is repeated, the cell culture medium continuously reciprocates inside the hollow fiber 22. At the same time, the cell culture solution containing the target substance flows out through the pores of the hollow fiber 22 by the transmembrane pressure applied to the hollow fiber 22. This flow of cell culture is called an alternating tangential flow (ATF). The cell culture solution containing the target substance is supplied to the recovery device 40 via the outlet port 25.

The outlet port 25 is equipped with a recovery pipe 43 connected to a recovery tank (not shown), and the recovery device 40 including a non-return valve 41 and a pinch valve 42 in the recovery pipe 43. Is installed. The filtration valve 44 may be located in front of the non-return valve 41. The pinch valve 42 may be located in front of or behind the non-return valve 41. 1 and 2 illustrate the case where the pinch valve 42 is located behind the non-return valve 41 as an example.

The non-return valve 41 is a device that allows the cell culture fluid flowing out of the hollow fiber 22 to flow in only one direction (recovery direction) by the alternating tangential flow. In the case of FIG. 1, the diaphragm pump 30 is always operated. It is open and works to close in the case of FIG. The pinch valve 42 is a device for opening and closing the recovery pipe 43 to control the recovery time of the spilled cell culture fluid to the recovery tank to adjust the recovery amount of the cell culture fluid, that is, the perfusion rate.

In the perfusion cell culture apparatus 100 of the present embodiment, the target substance may be a substance in which a molecular polymer (vWF / FVIII complex) of the recombinant coagulation factor 8 is bound to the von Willebrand factor multimer. It is reported that von Willibrand factor multimers can increase molecular weight up to 20,000 kDa and hydrodynamic radius can increase to 0.45 μm or more.

This target material may or may not be able to pass through the pores (0.2 μm to 0.5 μm) of the hollow fiber 22 because of its large hydrodynamic radius. In addition, not only living cells but also cell disruption materials are present in the cell culture, and these materials become partial clogging, which makes the target material more difficult to pass through the pores.

As such, the amount of the target substance that passes through the pores of the hollow fiber 22 may vary according to the molecular weight or distribution of the hydrodynamic radius of the target substance and the degree of clogging of the pores. However, if the hydrodynamic radius of the target material is similar to or slightly smaller than the size of the pore and thus cannot pass through the pore, a transmembrane pressure may be applied to the target material that could not pass through the pore.

The von Willibrand polymer has a property that changes its shape depending on shear stress such as pressure. In other words, it exists in a globular form and changes its shape into a linear form when subjected to shear stress. As a result, the circular shape may not pass through the voids, but may change into a linear shape and pass through the hollow fiber 22 pores having the same size.

The non-return valve 41 is temporarily open and is always open in the case of FIG. 1 when the diaphragm pump 30 is operated, unlike a conventional pump (for convenience, referred to as a "recovery pump") for forcibly recovering the spilled cell culture solution. In the case of operating to close because the diaphragm pump 30 minimizes the loss of filtration pressure.

In the conventional recovery pump application, the intermembrane pressure works efficiently only when the recovery pump is operated irrespective of the period during which the intermembrane pressure by the diaphragm pump 30 is formed. That is, since the recovery pipe 43 is closed when the recovery pump is not operated, it is difficult for the target substance to pass through the hollow fiber 22 void even if the diaphragm pump 30 works. Therefore, the recovery rate of the target substance is lowered.

However, since the non-return valve 41 of the embodiment is always open in FIG. 1 and remains closed in FIG. 2 when the diaphragm pump 30 is operated, the intermembrane pressure generated by the diaphragm pump 30 may be a target material. 100% is used to pass through the yarn (22) voids. Therefore, the non-return valve 41 can maximize the intermembrane pressure by minimizing the loss of the filtration pressure, and as a result, it is possible to increase the recovery of the target material having a large molecular weight and a hydrodynamic radius.

In addition, the non-return valve 41 prevents reverse flow when the cell culture fluid flows back into the hollow fiber 22 by the alternating tangential flow, thereby minimizing the amount of recovered cell culture fluid to be re-introduced into the filter module 20. In addition, the cell culture fluid in the incubator 10 is introduced into the filter module 20 to enable perfusion culture.

The pinch valve 42 is variously configured in a pneumatic or other manner, and consists of a two-way pinch valve. The pinch valve 42 operates to close and recover the recovery pipe 43 for a predetermined time so that the cell culture fluid can flow out into the recovery tank, thereby functioning to regularly recover a certain amount of cell culture fluid every day. Therefore, the pinch valve 42 is connected to the timer 45 and the controller 46, and may operate only for a time set in the timer 45 to open the recovery pipe 43.

If it is assumed that the non-return valve 41 is installed in the recovery pipe 43 without the pinch valve 42, the cell culture fluid passing through the hollow fiber 22 pores is continuously discharged to the recovery tank so that the incubator 10 may be installed. All of the cell cultures in the cell will be drained. Therefore, by providing the pinch valve 42 in the recovery pipe 43, it can prevent that all the cell culture liquid in the incubator 10 escapes.

In addition, the controller 46 may be electrically connected to the load cell 14 to control the operation of the pinch valve 42 in accordance with the weight of the cell culture solution introduced into the incubator 10. In this case, the recovery amount of the cell culture can be easily adjusted.

As described above, in the present embodiment, the recovery device 40 includes a time point (period) at which positive intermembrane pressure is formed in the hollow fiber 22 and a time point (period) at which the target material is recovered through the voids of the hollow fiber 22. As a result, it is possible to easily recover a target substance having a large molecular weight and a hydrodynamic radius.

In another aspect, there is provided a cell culture method comprising inoculating cells into the perfusion cell culture device to perform perfusion culture. The cell culture method may be followed by other conventional culture steps and conditions for the cells to be cultured, except for using the perfusion cell culture device according to the present invention as a culture device.

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, Of course.

Example

< Example  1> Check the non-return valve and the pinch valve The number of times to include  Fabrication of the device

Conventionally, perfusion culture was performed by connecting an ATF 4 system (Refine Technology, USA) to a BioFlo 310 (New Brunswick Scientific, USA) bioreactor. In this perfusion culture, the cell culture fluid was recovered using a pump mounted on the BioFlo 310 bioreactor, and the medium was supplied into the cell culture fluid using a medium supply pump. The recovery device including the non-return valve and the pinch valve according to the present invention replaces the pump mounted on the BioFlo 310 and performs two functions of recovery of cell culture and control of perfusion rate. The non-return valve (code 418, Kartell spa, Italy) is mounted on a 3/16 inch Sani-Tech silicone tube (Saint-Gobain performance plastics, USA) connected to the filtration valve of the ATF 4 system. A 3/16 inch Sani-Tech silicone tube connected to the non-return valve was mounted in the pinch valve to open and close according to the operation of the pinch valve. The non-return valve minimizes the amount of the cell culture solution in the incubator when the cell culture fluid leaked out of the hollow fiber membrane (Refine Technology, USA) by the alternating tangential flow flows back into the hollow fiber membrane and closes the valve. By allowing the filter module to be introduced, it functions to enable perfusion culture. Fresh medium was fed into the cell culture at a constant rate using a medium feed pump mounted on BioFlo 310. In addition, the load cell installed under the incubator of the BioFlo 310 sends a signal to the control unit connected to the pinch valve when the weight of the incubator exceeds a certain level according to the medium supply. The control unit sends a signal received from the load cell to the timer to open the pinch valve for a predetermined time to adjust the cell culture recovery amount, that is, perfusion rate (see FIGS. 1 and 2).

< Example  2> Recombinant coagulation factor 8 ( Factor VIII A) producing cells and Stem Cell Culture

In order to confirm the recovery rate of the biological material having a large molecular weight, seed cell culture of production cells expressing recombinant coagulation factor 8 and von Willibrand factor at the same time was performed. As production cells, B-domain deleted clotting factor VIII and von Willebrand factor genes were simultaneously transformed with CHO (DG44) cells (disclosed). Strain, Accession No. KCLRF-BP-172, Patent Publication No. 10-2010-0062340). Recombinant coagulation factor 8 (remove 765th to 1658th amino acid sequence of GenBank accession no. NP-000123 protein) consists of a heavy chain having a molecular weight of 90 kDa and a light chain having a molecular weight of 80 kDa. The Brand Factor (GenBank accession no. P04275) can form a polymer having a single molecule of 260 kDa and a molecular weight of up to about 20,000 kDa.

In a disposable Erlenmeyer flask containing 500 mL of medium (suspension cell culture medium; EX-CELL CHO DHFR- medium; SAFC biosciences), the CHO (DG44) cells (Accession No. KCLRF-BP-172) were placed at 5x10 ⁵ cells / mL. Inoculated in amounts and incubated for 4 days with stirring on a stirrer in a CO ₂ incubator maintained at 34 ° C. and 5% concentration of carbon dioxide.

Recombinant hemagglutinin factor 8 and von Willibrand factor expressed in the CHO cells are secreted into the cell culture and present in a form in which the recombinant hemagglutinin factor 8 is bound to the von Willibrand polymer.

6 is a diagram showing that the coagulation factor 8 present in the cell culture fluid is present in the form of binding to von Willebrand factor as well as the form not bound to von Willebrand factor. A is a Western blot using an anti-von Willibrand antibody, B is a Western blot using an anticoagulant factor 8 antibody. Coagulation factor 8 of the single-chain form has a molecular weight of 170 kDa, but it can be seen that the coagulation factor 8 having a molecular weight higher than that of von Willibrand is detected.

< Example  3> With conventional pump ATF system Using Perfusion  culture

The cell culture solution of the seed cell culture obtained in Example 2 was inoculated into a BioFlo 310 bioreactor equipped with a pump connected to the ATF system and equipped with a pump for recovery of the leaked cell culture, and then perfusion culture was performed. At this time, the cell inoculation concentration was 5x10 ⁵ cells / mL, the culture temperature was 34 ^o C, the medium was used EX-CELL CHO DHFR- medium (SAFC biosciences). Perfusion rate was carried out perfusion culture while maintaining the ratio of the volume of cell culture recovered every day divided by the working volume of the bioreactor to 0.5 to 4. Cell concentration maintenance was performed using a method of bleeding a certain amount of the cell culture of the bioreactor. Cell concentration in cell culture was measured using Cedex XS (Roche diagnostics Ltd., USA), an automatic cell counting instrument. In addition, the amount of recombinant coagulation factor 8 in the recovered cell culture was measured using a chromogenic assay using ACL TOP 500 (Instrumentation Laboratory, USA).

The viable cell concentration obtained in the above experiment and the amount of the recombinant coagulation factor 8 in the cell culture medium of the incubator are shown in FIG. 3, and the amount (recovery rate) of the recombinant coagulation factor 8 in the recovered cell culture medium is shown in FIG. 5. As can be seen in Figures 3 and 5, since the recombinant coagulation factor 8 produced in the cell culture medium does not pass through the hollow fiber membrane, the amount of recombinant coagulation factor 8 accumulated in the cell culture medium of the incubator is continuously maintained. Increasingly, the amount of recombinant coagulation factor 8 in the recovered cell culture was continuously decreasing. As shown in FIG. 3, the recovery rate of the recombinant coagulation factor 8 in the perfusion culture method using the conventional peristaltic pump showed a tendency to decrease rapidly as the culture continued, and the average recovery rate was 11%. Giving.

< Example  4> Equipped with non-return valve and pinch valve ATF system Using Perfusion  culture

The cell culture medium in which the seed cell culture was completed was inoculated into a BioFlo 310 bioreactor (see FIGS. 1 and 2) connected to an ATF system equipped with a non-return valve and a pinch valve prepared in Example 1, and the same as Example 3 above. Perfusion culture was carried out under the conditions (cell inoculation concentration 5x10 ⁵ cells / mL, culture temperature 34 ^° C, medium EX-CELL CHO DHFR- medium (SAFC biosciences)). Perfusion rate was carried out perfusion culture while maintaining the ratio of the volume of cell culture recovered every day divided by the working volume of the bioreactor to 0.5 to 4. Cell concentration retention, cell concentration measurement, and measurement of the amount of recombinant coagulation factor 8 in the recovered cell culture were carried out in the same manner as in Example 3.

The viable cell concentration obtained in the experiment and the amount of the recombinant coagulation factor 8 in the cell culture medium of the incubator are shown in FIG. 4, and the amount (recovery rate) of the recombinant coagulation factor 8 in the recovered cell culture medium is shown in FIG. 5. As can be seen in Figures 4 and 5, the cell concentration was similar to the method using a conventional pump according to Example 2, but in the perfusion culture method using a cell culture liquid recovery apparatus according to the present invention The amount of accumulation of the recombinant coagulation factor 8 in the incubator is greatly reduced compared to the conventional method, and shows that most of the produced recombinant coagulation factor 8 is recovered. As shown in Figures 4 and 5, the average recovery rate of the recombinant coagulation factor 8 in the perfusion culture method using the recovery device according to the present invention is 67%, which is 6 times better than the perfusion culture method using a conventional pump. Recovery rate was obtained.

Claims (7)

An incubator for storing cell culture;
A filter module including a housing having a first port, a second port, and an outlet port, and a plurality of hollow fibers installed in the housing;
A connecting tube installed between the incubator and the first port;
A diaphragm pump connected to the second port and providing a pumping pressure into the hollow fiber; And
A recovery device provided with a non-return valve connected to the outlet port and a pinch valve provided at a front end or a rear end of the non-return valve to match a period during which the inter-fiber pressure is formed in the hollow fiber by the diaphragm pump and a recovery period
Perfusion cell culture apparatus comprising a. The method of claim 1,
The first port and the second port face each other in one direction,
The outflow port is formed in a perfusion cell culture device along a direction crossing the one direction. 3. The method of claim 2,
The hollow yarn is installed in parallel with the one direction, and divided into a lumen side corresponding to the hollow and a shell side outside the hollow fiber wall.
The first port and the second port is connected to the inside of the hollow yarns,
The outflow port is connected to the outside of the hollow fiber perfusion cell culture apparatus. The method of claim 1,
A recovery pipe is mounted to the outlet port,
The non-return valve and the pinch valve are installed in the recovery pipe,
A perfusion cell culture device, wherein the filtration valve is located in front of the non-return valve. 5. The method of claim 4,
The pinch valve is connected to the timer and the control unit, the perfusion cell culture apparatus to open the recovery tube by operating only for the time set in the timer. The method of claim 5,
The incubator is equipped with a load cell for detecting a change in the weight of the cell culture,
The control unit is electrically connected to the load cell and the perfusion cell culture apparatus for controlling the operation of the pinch valve in accordance with the weight of the cell culture. Inoculating the perfusion cell culture device of any one of claims 1 to 6 comprising the step of performing perfusion culture,
Cell culture method.

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