JP2019146515A - Continuous culture method and continuous culture apparatus - Google Patents

Abstract

To provide a continuous culture method using a separation membrane in which clog of the separation membrane can reduced.SOLUTION: A continuous culture method includes: a culture process of culturing a microorganism or a cell in culture solution housed in a culture vessel 1; and a membrane separation process of separating a product in culture solution, which is produced by the microorganism or cell by a membrane separation device 3, in which the membrane separation device 3 is installed in the culture vessel 1, and the membrane separation process is a forward osmosis process of bringing culture solution in the culture vessel and draw solution having osmotic pressure higher than the osmotic pressure of the culture solution into contact via a separation membrane, by which the microorganism in the culture solution or the product produced by the cell is allowed to penetrate the separation membrane and move into the draw solution.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method and a continuous culture apparatus for continuously culturing microorganisms or cells.

In recent years, development of biopharmaceuticals that use secretions from cells as drugs, such as antibody drugs and vaccines, has been progressing.
It is known to cultivate microorganisms or cells in a culture solution and to collect the products produced by the microorganisms or cells using a separation membrane.

Patent Document 1 discloses a technique for continuously culturing microorganisms or cells with a continuous culture apparatus using a separation membrane.
The continuous culture apparatus described in Patent Document 1 will be described with reference to FIG.
The continuous culture apparatus includes a culture tank 31 having a separation membrane element 32 therein and a differential pressure control device 33.
The culture medium is supplied continuously or semi-continuously to the culture tank 31 by the culture medium supply pump 37, and if necessary, the fermentation broth in the culture tank 31 is agitated by the agitator 35 in the culture tank 31 and the gas supply device The necessary gas is supplied into the culture tank 31 by 34. If necessary, the pH of the culture medium in the culture tank 31 is adjusted by the pH sensor / control device 39 and the pH adjustment liquid supply pump 38, and the temperature of the fermentation culture liquid in the culture tank 31 is adjusted by the temperature controller 40. Adjust.
From the culture solution, microorganisms and fermentation products are filtered and separated by the separation membrane element 32 installed in the culture tank 31, and the products are taken out from the apparatus system. Filtration / separation by the separation membrane element 32 is performed by using a water head differential pressure with respect to the water surface of the culture tank 31 and can be performed without using any special power. If necessary, a level sensor 36 and a differential pressure control device 33 can be used. The filtration / separation speed of the separation membrane element 32 and the amount of the fermentation broth in the culture tank 31 are appropriately adjusted. Further, the filtration / separation by the separation membrane element can be performed by filtration / separation by suction filtration using a pump or the like, or pressurizing the inside of the apparatus system, if necessary.

Japanese Patent No. 5092487

In a continuous culture apparatus using a conventional membrane separation method, a filtrate containing a product is collected through a separation membrane by pump pressure or suction pressure, and an unfiltered culture solution is refluxed to a culture tank. For this reason, the fouling substance which is the cause of the clogging of the separation membrane is pushed into the separation membrane, and the fouling layer is densified, so that clogging is likely to occur.
For this reason, as a method for preventing clogging, adjustment of the pump operating pressure is performed. Moreover, in order to wash | clean the densified fouling substance, the washing | cleaning using a chemical agent is needed, and it is unpreferable in the culture which dislikes the contamination by a washing | cleaning chemical | drug | medicine.
An object of the present invention is to reduce clogging in a separation membrane in a continuous culture method in which microorganisms or cells are cultured in a culture solution and products produced by the microorganisms or cells are separated and collected by the separation membrane.

(1) It includes a culture step of culturing microorganisms or cells in a culture solution accommodated in a culture tank, and a membrane separation step of separating a product in the culture solution produced by the microorganisms or cells by a membrane separator. A continuous culture method,
The membrane separation device is installed in a culture tank,
In the membrane separation step, the microorganism in the culture solution or the above-mentioned microorganisms in the culture solution is brought into contact with the culture solution in the culture tank and a draw solution having an osmotic pressure higher than that of the culture solution through the separation membrane A continuous culturing method, which is a forward osmosis step in which a product produced by a cell passes through the separation membrane and moves into the draw solution.
(2) The diluted draw solution obtained in the forward osmosis step is supplied to a membrane separator for drawing solution recovery to collect the product contained in the diluted draw solution as a separation membrane permeate and permeate the separation membrane. The continuous culture method according to (1) above, comprising a step of recovering the recovered draw solution that has not been obtained as a draw solution used in the forward osmosis step.
(3) The continuous culture method according to (1) or (2), wherein the pH of the culture solution is adjusted by supplying a pH adjustment solution into the culture tank.
(4) The continuous culture method according to any one of (1) to (3), wherein the osmotic pressure of the culture solution is adjusted by supplying the osmotic pressure adjusting solution into the culture tank.
(5) The continuous culture method according to any one of the above (1) to (4), comprising a washing step of washing the separation membrane.
(6) a culture tank for culturing microorganisms or cells;
A continuous culture apparatus provided with a membrane separation device in which a separation membrane for membrane separation treatment of a culture solution supplied from the culture tank is disposed,
The membrane separation device is installed in a culture tank,
A culture solution and a draw solution having a higher osmotic pressure than the culture solution are brought into contact with each other through a separation membrane, and a product contained in the culture solution produced by the microorganism or the cell is allowed to pass through the separation membrane. A continuous culture apparatus, which is a forward osmosis membrane separation apparatus that is moved into the draw solution to obtain a diluted draw solution containing the product and a concentrated culture solution that has not permeated the separation membrane.
(7) A membrane separator for drawing solution recovery for recovering the draw solution from the diluted draw solution obtained by the forward osmosis membrane separator is provided at the subsequent stage of the forward osmosis membrane separator,
The membrane separation device for collecting the draw solution collects the product contained in the diluted draw solution as a separation membrane permeate and uses the recovered draw solution that has not permeated the separation membrane in the forward osmosis step. The continuous culture apparatus according to the above (6), which is a membrane separation apparatus recovered as a solution.
(8) The continuous culture apparatus according to (6) or (7) above, wherein a pH adjustment liquid supply pipe for supplying a pH adjustment liquid into the culture tank is provided.
(9) The continuous culture apparatus according to any one of (6) to (8), wherein an osmotic pressure adjusting solution supply pipe for supplying the osmotic pressure adjusting solution into the culture tank is provided.
(10) The continuous culture apparatus according to any one of (6) to (9), wherein a cleaning liquid supply pipe for supplying cleaning water to the draw solution side of the separation membrane is provided.

  By using the continuous culture method of the present invention, clogging in the separation membrane can be reduced.

It is a figure which shows an example of embodiment of the continuous culture method of this invention. It is a figure which shows the other example of embodiment of the continuous culture method of this invention. It is a figure explaining the washing | cleaning process in the continuous culture method of this invention. It is a figure explaining a forward osmosis process. It is a figure which shows the change of the relative value of the permeation | transmission speed (Flux) before washing | cleaning in Example 1 and the comparative example 1 after washing | cleaning. It is a figure which shows the conventional membrane separation type continuous culture apparatus.

In the present invention, a forward osmosis method, which is a membrane separation method using an osmotic pressure difference, is used as a means for separating cells and products from a culture solution.
First, the principle of the membrane separation method using the forward osmosis (FO) method will be described with reference to FIG.
As shown in FIG. 4, a culture solution containing water W, cells C, solute S and product P is separated from a draw solution containing water W and draw solute D and having a higher osmotic pressure than the culture solution. Contact through the membrane.
Due to the difference in osmotic pressure between the culture solution and the draw solution, water in the culture solution permeates the separation membrane and moves to the draw solution side using this osmotic pressure difference as a driving force.
At this time, the product P having a size smaller than the pores of the separation membrane permeates the separation membrane and moves to the draw solution side, and the cells C in the culture solution having a size larger than the pores of the separation membrane pass through the separation membrane. Not transparent.
According to the above principle, the product P in the culture solution can be recovered in the draw solution.
Further, the draw solution diluted with the separation membrane permeate is sent to the next product purification step to separate the product.

  In the forward osmosis method, the permeate is extracted from the feed solution to the draw solution due to the osmotic pressure difference. For this reason, since the fouling substance is not pushed into the separation membrane at a high pressure, the fouling layer is not densified, and the separation membrane is not easily clogged. Further, even if fouling occurs, the layer is not densified, so that the membrane performance can be recovered by washing with water. If the cleaning is performed with water, the cleaning can be performed not only when the operation is stopped but also during the operation.

Next, an example of an embodiment of the continuous culture method of the present invention will be described based on FIG.
In the following, the present invention will be described by taking as an example the case of culturing with cells.

[First Embodiment]
(Culture process)
A pump 7 and a medium supply pipe 8 are connected to the culture tank 1, and a medium is continuously or semi-continuously supplied to the culture tank 1 through the medium supply pipe 8 by the pump 7.
The culture tank 1 includes a stirrer 2 so that the culture solution in the culture tank 1 can be stirred by the stirrer 2. Further, the culture tank 1 may be provided with a gas supply pipe 21 to supply a gas necessary for maintaining the atmosphere from the gas supply pipe 21 as necessary.

If necessary, the pH of the culture solution can be adjusted by supplying the pH sensor / control device 11 and the pH adjusting solution to the culture tank 1 by the pump 9.
The culture of microorganisms or cultured cells is usually performed at a pH of 4 to 8 and a temperature of 20 to 40 ° C. The pH of the fermentation broth is usually adjusted to a predetermined value within a pH range of 4 to 8 with an inorganic acid or an organic acid, an alkaline substance, urea, calcium carbonate, ammonia gas, or the like.

  Examples of the microorganisms and cells used in the culture tank 1 include yeast, bacteria, filamentous fungi, radioactive bacteria, animal cells, and insect cells.

  As the components in the culture solution used in the method of the present invention, those usually used as cell culture media can be appropriately used. Specific components include amino acids, vitamins, lipid factors, energy sources, osmotic pressure regulators, iron sources, pH buffering agents, in addition to the above components, for example, trace metal elements, surfactants, Growth cofactors, nucleosides, etc. may be added.

(Forward osmosis process)
In the culture tank 1, a membrane separation module 3 for separating a product produced by a microorganism or a cell is provided. A separation membrane is provided inside the membrane separation module 3, and a culture solution is passed through one chamber through the separation membrane, and a draw solution is fed from the draw solution supply pipe 4 into the other chamber.
In the membrane separation module 3, the product and water in the culture solution are transferred to the draw solution side through the separation membrane by forward osmosis, and the draw solution becomes a diluted draw solution containing the product. This diluted draw solution contains permeate broth and waste products in addition to the draw solution and product.
The diluted draw solution is sent to the next purification step, and is separated into a draw solution containing a permeation culture solution and waste products and a target product by the purification device 30, and the product is recovered.

  The draw solution is not particularly limited as long as the osmotic pressure is higher than that of the culture solution. Examples thereof include a solution containing a molecular polymer. A solution containing ammonia, inorganic salts, oils, or the like may also be used. Moreover, the draw solution may consist of one type or a plurality of draw solutions. The solute of the draw solution may have less diffusion to the feed side when the molecular diameter is larger than the pore diameter of the separation membrane.

  The separation membrane in the membrane separation module 3 is preferably a membrane having separation performance and permeation performance according to the properties and applications of the liquid to be treated, and a forward osmosis membrane, a reverse osmosis membrane, or an inorganic material such as ceramics. Alternatively, a commercially available porous film made of an organic material such as a resin may be used. The material of the separation layer of the separation membrane is not particularly limited, and examples thereof include cellulose acetate, polyamide, polyethyleneimine, polysulfone, and polybenzimidazole materials.

  The separation membrane may be composed of only one type (one layer) of materials used for the separation layer. The separation layer and a support layer that physically supports the separation layer and does not substantially contribute to separation. You may comprise from two or more layers. Examples of the material for the support layer include polysulfone, polyketone, polyethylene, polyethylene terephthalate, and general nonwoven fabrics.

  The separation membrane is preferably a membrane having pores having an average pore diameter of 0.001 μm or more and less than 10 μm in order to effectively separate cells in the culture solution. The average pore diameter is preferably larger than the molecular diameter of the recovery target substance so that the recovery target substance can pass through.

  The form of the separation membrane is not limited, and various types of membranes such as a flat membrane, a tubular membrane, or a hollow fiber membrane can be used. The membrane separation module is, for example, a cylindrical or box-shaped container, and the inside is partitioned into two chambers by the separation membrane by installing the separation membrane inside. Examples of the form of the membrane module include various forms such as a spiral module type, a laminated module type, and a hollow fiber module type. As the membrane separation module, a known separation membrane device can be used, and a commercially available product can also be used.

(Purification process)
The diluted draw solution is extracted from the membrane separation module 3 through the diluted draw solution supply pipe 17 using the pump 18. The extracted diluted draw solution is separated by the purification device 30 into a draw solution containing a permeation culture solution and waste products, and a product as a target substance, and the product is separated and recovered.
The purification method for recovering the product from the diluted draw solution may be any method that can recover the product, and examples thereof include membrane separation, chromatography, and electrophoresis. Since cells and the like are separated in the preceding separation step, membrane filtration using pressure is also possible.

Next, another example of the embodiment of the continuous culture method of the present invention will be described based on FIG.
[Second Embodiment]
In this embodiment, as shown in FIG. 2, a draw solution recovery process is provided between the culture process / forward osmosis process and the purification process.
(Culture process)
In the culture process, the culture medium is continuously or semi-continuously supplied to the culture tank 1 by the pump 7 and the medium supply pipe 8.
Products produced by the cells accumulate in the culture solution.

(Forward osmosis process)
When the concentration of the product in the culture solution becomes stable, the forward osmosis process is started.
The separation membrane provided in the membrane separation module 3 causes the product and water in the culture solution to move to the draw solution side, and the draw solution becomes a diluted draw solution containing the product and is extracted by the pump 18. Then, it is sent to the draw solution recovery step of the next step through the diluted draw solution extraction tube 17. This diluted draw solution contains the permeate broth and waste products in addition to the draw solution and product. The culture solution that has not passed through the separation membrane returns to the culture tank 1 as a concentrated culture solution.

(Draw solution recovery process)
In the draw solution recovery step, the draw solution is recovered by the membrane separation module 25.
A separation membrane is provided inside the membrane separation module 25, and a product in the diluted draw solution is separated as a membrane permeate by a draw solution recovery step in which the diluted draw solution is supplied.
The recovered draw solution that has not permeated the separation membrane is recirculated to the membrane separation module 3 through the reflux pipe 19 as a draw solution by the pump 22 and reused.
In addition, the membrane permeate obtained in the draw solution recovery step is extracted from the membrane separation module 25 by the pump 27 and sent to the next purification step through the membrane permeate discharge pipe 26.

(Purification process)
In the purification step, the membrane permeate that has permeated the separation membrane in the draw solution recovery step is processed in the purification device 30 by membrane separation, chromatography, electrophoresis, or the like, and the product is obtained from the permeation culture solution and the membrane permeate containing waste products. Are separated and recovered.

In the first and second embodiments, when the forward osmosis process is performed, the fouling substance accumulates on the membrane surface. Therefore, a cleaning process for removing the fouling substance is performed as necessary. There is a need to.
Hereinafter, the cleaning process will be described.

(Washing process)
When the forward osmosis process is performed, fouling substances accumulate on the surface of the separation membrane. Therefore, a cleaning process for removing the fouling substances is performed as necessary.
In membrane separation by the forward osmosis method, since the fouling substance is not firmly fixed to the pores of the separation membrane, the performance of the separation membrane can be easily recovered by washing with water without using a cleaning agent.
The cleaning process will be described with reference to FIG.
In the apparatus shown in FIG. 3, a washing water supply pipe 6 is connected to the draw solution supply pipe 4.
Further, the culture tank 1 is provided with a pH sensor / control device 11, a pump 9 for supplying a pH adjusting solution, and a pH adjusting solution supplying pipe 10 as shown in FIG.
Further, the culture tank 1 is provided with an osmotic pressure sensor / control device 13, a pump 12 for supplying an osmotic pressure adjusting liquid, and an osmotic pressure adjusting liquid supply pipe 14.
The washing water can be supplied to the culture solution side of the separation membrane to wash the separation membrane.

When washing is performed during operation, the washing water is supplied from the washing water supply pipe 6 to the draw solution side of the membrane separation module 3 by the pump 16 and the washing water is pushed into the culture solution. Thereby, the fouling substance on the separation membrane surface is removed.
When washing water is supplied to the draw solution supply pipe 4, the pH and osmotic pressure of the culture solution change. For this reason, the pH is measured by the pH sensor / control device 11 and a control signal is sent to the pump 9 to supply the pH adjusting solution to the culture tank 1 to adjust the pH of the culture solution.
Further, the osmotic pressure of the culture solution is measured by the osmotic pressure sensor / control device 13, and the control signal is sent to the pump 12 for supplying the osmotic pressure adjusting solution to supply the osmotic pressure adjusting solution to the culturing tank 1 to infiltrate the medium. Adjust pressure.
The osmotic pressure adjusting solution may be used not only for washing but also for adjusting the osmotic pressure of the culture solution after membrane separation.

  The present invention will be described below in more detail based on examples, but the technical scope of the present invention is not limited to the following examples.

[Example 1]
As the continuous culture apparatus, an apparatus having the configuration shown in FIG. 1 was used.
A hollow fiber filter (modified polyethersulfone: manufactured by Spectrum) is used as a separation membrane of the forward osmosis membrane separator, and polyethylene oxide (manufactured by Wako Pure Chemical Industries, Ltd., molar mass: 300,000 g / mol, concentration: concentration): 30% by mass, osmotic pressure: 384 mOsm / kg) was used. A serum-free medium containing bovine serum albumin was used as the medium.
Using hybridoma TO405 cells producing HBs antibody, normal osmosis membrane separation was started after culturing in a culture tank by a conventional method for 50 hours.
When the concentration of the components in the culture tank was analyzed every 20 hours after the start of membrane separation, the concentration was stable when the culture time was 120 hours.
The main composition of the culture solution after 120 hours of culture was as follows.
・ Total number of cells: 2 × 10 ⁶ cells / mL
・ Glucose: 5 mmol / L
・ Glutamine: 5 mmol / L
・ Ammonia: 0.5 mmol / L
・ Inorganic salt: 10 g / L
・ Bovine serum albumin: 1 g / L
The permeation rate when the culture solution was subjected to membrane separation treatment at a culture time of 120 hours when the concentration became stable was defined as the permeation rate before washing. In addition, after the membrane separation treatment after 120 hours, the supply of the culture solution is temporarily interrupted, and as shown in FIG. 3, the washing solution is supplied from the draw solution supply pipe to wash the separation membrane, and then the culture solution is supplied again. The permeation rate at the time of membrane separation treatment was defined as the permeation rate after washing. The permeation rate represents the permeation flow rate, and is the permeation flow rate divided by the product of the membrane area and time.
FIG. 5 shows a change in the relative value of the permeation rate (Flux) before and after cleaning. "

[Comparative Example 1]
Continuous culture was carried out in the same manner as in Example 1 except that a suction filtration system using a hollow fiber filter (manufactured by Spectrum) was used as a membrane separator.
The suction filtration system was operated at 7 kPa so that the suction pressure was approximately the same as the draw osmotic pressure in the forward osmosis membrane separation apparatus of Example 1.
The permeation rate when the culture solution was subjected to membrane separation when the culture time was 120 hours was defined as the permeation rate before washing. In addition, after the membrane separation treatment after 120 hours, the supply of the culture solution is temporarily interrupted, and as shown in FIG. 3, the washing solution is supplied from the draw solution supply pipe to wash the separation membrane, and then the culture solution is supplied again. The permeation rate at the time of membrane separation treatment was defined as the permeation rate after washing.
FIG. 5 shows a change in the relative value of the permeation rate (Flux) before and after cleaning.

From the results shown in FIG. 5, in Example 1, the permeation rate easily recovered after washing, but in Comparative Example 1, the permeation rate recovery is not good.
This is because the fouling substance does not adhere firmly to the separation membrane in Example 1 using the forward osmosis separation method, but the fouling substance adheres firmly to the separation membrane in Comparative Example 1 using the suction filtration system. Indicates that
Although this example was performed using hybridoma TO405 cells that produce HBs antibodies, it can also be performed in yeast, bacteria, filamentous fungi, radioactive bacteria, insect cells, and other animal cells.

DESCRIPTION OF SYMBOLS 1 Culture tank 2 Stirrer 3 Membrane separation module 4 Draw solution supply pipe 5 Diluted draw solution extraction pipe 6 Washing water supply pipe 7, 9, 12, 16, 18, 22, 27 Pump 8 Medium supply pipe 10 pH adjustment liquid supply pipe 11 pH sensor / control device 13 Osmotic pressure sensor / control device 14 Osmotic pressure adjusting liquid supply pipe 15 Gas supply pipe 17 Diluted draw solution extraction pipe 19 Reflux pipe 20 Culture liquid 25 Membrane separation module 26 Membrane permeate extraction pipe 30 Purification apparatus

Claims (10)

A continuous culture method comprising a culture step of culturing microorganisms or cells in a culture solution housed in a culture tank, and a membrane separation step of separating a product in the culture solution produced by the microorganisms or cells by a membrane separator. Because
The membrane separation device is installed in a culture tank,
In the membrane separation step, the microorganism in the culture solution or the above-mentioned microorganisms in the culture solution is brought into contact with the culture solution in the culture tank and a draw solution having an osmotic pressure higher than that of the culture solution through the separation membrane A continuous culturing method, which is a forward osmosis step in which a product produced by a cell passes through the separation membrane and moves into the draw solution.   The diluted draw solution obtained in the forward osmosis step was supplied to a membrane separator for collecting the draw solution to collect the product contained in the diluted draw solution as a separation membrane permeate and did not permeate the separation membrane. The continuous culture method according to claim 1, further comprising a step of recovering the recovered draw solution as a draw solution used in the forward osmosis step.   The continuous culture method according to claim 1 or 2, wherein the pH of the culture solution is adjusted by supplying a pH adjustment solution into the culture tank.   The continuous culture method according to any one of claims 1 to 3, wherein the osmotic pressure of the culture solution is adjusted by supplying the osmotic pressure adjusting solution into the culture tank.   The continuous culture method according to any one of claims 1 to 4, further comprising a washing step of washing the separation membrane. A culture vessel for culturing microorganisms or cells;
A continuous culture apparatus comprising a membrane separation device in which a separation membrane for membrane separation treatment of a culture solution supplied from the culture tank is disposed,
The membrane separation device is installed in a culture tank,
A culture solution and a draw solution having a higher osmotic pressure than the culture solution are brought into contact with each other through a separation membrane, and a product contained in the culture solution produced by the microorganism or the cell is allowed to pass through the separation membrane. A continuous culture apparatus, which is a forward osmosis membrane separation apparatus that is moved into the draw solution to obtain a diluted draw solution containing the product and a concentrated culture solution that has not permeated the separation membrane. A membrane separation device for collecting a draw solution for collecting a draw solution from a diluted draw solution obtained by the forward osmosis membrane separation device is provided at a subsequent stage of the forward osmosis membrane separation device,
The membrane separation device for collecting the draw solution collects the product contained in the diluted draw solution as a separation membrane permeate and uses the recovered draw solution that has not permeated the separation membrane in the forward osmosis step. The continuous culture apparatus according to claim 6, which is a membrane separation apparatus that recovers as a solution.   The continuous culture apparatus according to claim 6 or 7, further comprising a pH adjustment liquid supply pipe for supplying the pH adjustment liquid into the culture tank.   The continuous culture apparatus according to any one of claims 6 to 8, further comprising an osmotic pressure adjusting liquid supply pipe for supplying the osmotic pressure adjusting liquid into the culture tank.   The continuous culture apparatus according to any one of claims 6 to 9, further comprising a cleaning liquid supply pipe for supplying cleaning water to the draw solution side of the separation membrane.