JP7269839B2 - Incubation device - Google Patents

Description

The present invention relates to a culture device.

In recent years, the distribution of biopharmaceuticals containing biological substances as active ingredients is expanding. Biopharmaceuticals such as antibody drugs are generally produced by culturing floating cells in a culture medium to produce target substances in the culture medium. In various industrial fields, various useful substances are produced by culturing animal cells, plant cells, microbial cells, and the like.

Culture methods include batch culture, fed-batch culture, and perfusion culture. Batch culture is a culture method in which a medium is prepared for each culture and no medium is supplied during the culture. Fed-batch culture is a culture method in which a culture medium is supplied from outside the system during culture, but the culture solution is not discharged until the culture is completed. Perfusion culture is a culture method in which medium is continuously supplied during culture and the same amount of culture solution is continuously discharged.

Batch culture is a method in which seeded cells are cultured until nutrients are depleted, and thus has the advantage of simple operation. Fed-batch culture is a system in which nutrients are supplied to the cells being cultured, so that culture can be carried out for a longer period of time than batch culture, and the amount of substance produced can be increased relatively easily. Perfusion culture can reduce the concentration of waste products while maintaining a high concentration of nutrients, so that the culture conditions can be kept constant and substances can be produced stably.

As a method of operating perfusion culture, it is common to continuously discharge the culture medium in the culture tank together with the cells, send the culture medium containing cells to the cell separator, and return the separated and concentrated cells to the culture tank. is. Cell separators are known that utilize various principles such as centrifugation, gravity separation, and membrane separation to separate cultured cells from substances and waste products produced by the cells.

Patent Document 1 describes a culture technique that employs a cross-flow (or Tangential Flow Filtration, TFF) filtration device as a cell separation device in perfusion culture. In this device, the culture solution is drawn out from the culture tank by the liquid feed pump (cell suspension), and after part of the drawn out cell suspension is filtered by the TFF filtration device, the cells are concentrated by discharging the filtrate. The cell suspension (concentrated culture medium) is returned to the culture medium.

JP 2015-217395 A

In the perfusion culture configured as in Patent Document 1, the medium is continuously supplied during the culture, and the same amount of the culture solution is continuously discharged, so that the concentration can be kept constant, and the cell density can be kept constant. While it can be kept at 200°C, increasing the consumption rate of the medium is a challenge. Furthermore, since it requires large-scale equipment such as a cell separator, it is not suitable for perfusion culture because the cost of equipment increases for small-scale experiments.

Batch culture and fed-batch culture are performed as methods that allow easier culture than perfusion culture and do not require large-scale equipment. Batch culture and fed-batch culture have a problem of lower productivity than perfusion culture.

Therefore, an object of the present invention is to perform culture with high productivity in batch culture or repeat batch culture in which fed-batch culture is repeated.

A culture apparatus according to the present invention for solving the above-mentioned problems is a cell culture apparatus for repeat batch culture in which cells are repeatedly cultured by batch culture or fed-batch culture, comprising: a culture tank for culturing cells; A medium component concentration meter that is connected to a culture tank and measures the concentration of medium components in the culture tank, a cell density meter that is connected to the culture tank and measures the cell density in the culture tank, and , a control device that calculates a medium loss term from the measurement results of the medium component concentration meter and the cell density meter, and controls culture based on the medium loss term, wherein the medium in the repeat batch culture The loss term is the value obtained by dividing the amount of one or more components of the medium discharged to the outside of the culture tank at each culture medium exchange by the average cell density during each operation, and the same cells were cultured by perfusion culture. When the medium loss term in the above is a value obtained by dividing the discharge rate of one or more medium components to the outside of the culture tank by the cell density, the control device determines that the medium loss term in the repeat batch culture is the above It is characterized in that it is operated so as to be smaller than the medium loss term when cultured by perfusion culture .

According to the present invention, culture with high productivity can be performed in batch culture or repeat batch culture in which fed-batch culture is repeated.

1 is a schematic diagram showing a schematic configuration of a culture apparatus according to this embodiment; FIG. FIG. 4 is a diagram showing changes in culture volume and cell density over time. FIG. 10 shows the relationship between viable cell density and medium loss. FIG. 10 shows the relationship between viable cell density and medium loss when using CHO cells. FIG. 4 is a graph showing the relationship between viable cell density and medium loss when using yeast.

EMBODIMENT OF THE INVENTION Hereafter, the form for implementing this invention is demonstrated, referring drawings. In addition, the same code|symbol is attached|subjected about the structure which is common in each following figure, and the overlapping description is abbreviate|omitted.

FIG. 1 is a schematic diagram showing a culture apparatus according to an embodiment of the present invention.
As shown in FIG. 1, the culture apparatus 100 according to the present embodiment includes a culture tank 10, a supplemented medium container 20, a transfer pipe 21, a transfer pump P1, a valve V1, an extraction pipe 22, an extraction It includes a pump P2, a filter F1, and a control device 23. FIG.

The culture apparatus 100 can be applied to batch culture or fed-batch culture by using the culture tank 10 as a culture tank for culturing cells.

Cells to be cultured are derived from animals such as mammals and birds, but are not particularly limited. Specific examples of mammals include primates such as humans, monkeys, chimpanzees, rhesus monkeys, cynomolgus monkeys, baboons, marmosets and squirrel monkeys; ungulates such as cows, pigs, horses, sheep and goats; mice, rats, guinea pigs; Examples include rodents such as hamsters, rabbits, dogs, and cats.

Specific examples of cells to be cultured include iPS cells, ES cells (embryonic stem cells), adult stem cells, bone marrow-derived pluripotent stem cells, germ stem cells, mesenchymal stem cells, hepatic stem cells, cardiac stem cells, and pancreatic stem cells. , kidney stem cells, neural stem cells, muscle stem cells, epidermal stem cells, hair follicle stem cells, hematopoietic stem cells, hepatocytes, cardiomyocytes, pancreatic cells, kidney cells, fibroblasts, epithelial cells, endothelial cells, epidermal cells, cartilage cells, osteoblasts, somatic cells such as synovial cells, and the like.

The culture tank 10 is a tank for growing or maintaining cells, and can be made of appropriate materials such as stainless steel, aluminum alloy, plastic (resin), and glass. Tanks and containers made of stainless steel are excellent in washability and sterilization, and can be used repeatedly. Resin-made tanks and containers can be used as disposable items, and the installation of cleaning equipment, sterilization equipment, etc. can be omitted.

As shown in FIG. 1 , a supplemented medium container 20 is connected to the culture tank 10 . The culture tank 10 and the inlet of the supplemented medium container 20 are connected by a transfer pipe 21 . By driving the transfer pump P<b>1 , the medium is drawn out from the supplemented medium container 20 and supplied to the culture tank 10 through the transfer pipe 21 .

The tank interior of the culture tank 10 of the culture apparatus 100 is connected to the inlet on the primary side of the filter F1 via an extraction pipe 22 . The withdrawal pipe 22 is provided with a withdrawal pump P2 for withdrawing the liquid from the culture tank 10 side and sending it to the filter F1. The withdrawal pipe 22 is a pipe for collecting the target substance after culture, and withdraws the liquid in the culture tank 10 and sends it to the primary side of the filter F1.

A filter having a predetermined pore size is used for the filter F1 according to the size of the target substance to be separated and purified.

The culture tank 10 includes a stirring device 11 for stirring the culture solution, an aeration device 12 for aerating the culture solution with air, oxygen, nitrogen, carbon dioxide, etc., a temperature control device 13 for adjusting the temperature of the culture solution, and medium components. It comprises a densitometer 14 , a cell density meter 15 and a medium loss indicator 16 . A control device 23 that controls the stirring device 11, the aeration device 12, the temperature adjustment device 13, the medium component concentration meter 14, and the cell density meter 15 to adjust the culture environment can be provided.

As the stirring device 11, a shaking stirring device, an air flow stirring device, or the like can be used instead of a mechanical stirring device equipped with stirring blades. As the aeration device 12, in addition to a diffuser pipe and a sparger for aeration in the liquid, a ventilation pipe for aeration in the air can be provided. As the temperature control device 13, for example, a water jacket, an electric heater, or the like can be provided around the tank.

The medium component concentration meter 14 is a device capable of measuring substrate components contained in the medium. Furthermore, the cell density meter 15 can measure the cell density within the culture tank 10 . The values obtained by medium component concentration meter 14 and cell density meter 15 are sent to controller 23 to calculate the value of the medium spoilage term, which can be output to medium loss indicator 16 . This allows calculation of the media loss term and facilitates control of productivity.

The culture apparatus 100 can also include a control device 23 that controls the culture environment of the culture tank 10 and the perfusion culture operation. The control device 23 controls the operation/stop and temperature output of the temperature adjustment device 13, the operation/stop and ventilation amount of the ventilation device 12, the opening/closing of the valve V1, the operation/stop of the transfer pump P1 and the discharge pump P2, the discharge amount, and the like. to control.

A culture method using the culture apparatus 100 according to the present embodiment will be described.
As described above, in the culture apparatus 100, culture is performed by batch culture or fed-batch culture without withdrawing the culture medium until the end of the culture. After the culture is completed, part of the culture medium is removed from the culture tank 10, and new culture medium is supplied. By repeating this operation, cells are cultured (hereinafter referred to as repeat batch method).

(Culture method)
Next, the method and principle by which the culture apparatus 100 of the present embodiment achieves higher productivity than perfusion culture will be described.
As shown in the change in liquid volume over time in Figure 2, in the perfusion culture, the medium is supplied and withdrawn at a constant pace, so compared to the repeat batch method, more medium components are lost over time. there is
In addition, as shown in the change in cell number density over time in FIG. 2, the perfusion culture can keep the cell density at a high level. However, in the repeat batch method, the medium is extracted from the culture vessel and fresh medium is added after each culture is completed (every batch). Growth phase) is withdrawn after completion of culture, resulting in a low cell density.

Therefore, the present invention focuses on the fact that the consumption of the medium can be reduced and the cost can be reduced by increasing the amount of increase in cell density and the amount of purified target product per volume of input medium. In other words, when culturing cells by repeat batch culture, it is possible to operate the culture apparatus in a state where more of the target product can be purified with respect to the input medium than when culturing cells by perfusion culture. Productivity can be improved.

Next, the principle of increasing productivity will be described. The productivity here is the amount of protein produced by cells per input medium. Generally, this production amount can be obtained from the relationship shown in the formula (1).

For repeated culture (repeat-batch culture) in which a part of the cells and the produced protein are periodically collected without using a special continuous cell separation device and supplemented with fresh medium each time, a part of the culture medium is removed from the culture tank. Only Bc is withdrawn and fresh medium is supplied accordingly. At that time, the number of cells is reduced by Bc·Xe due to part of the culture medium being withdrawn. Then, the viable cell density after supplying the fresh medium returns to the value X ₀ at the start of culture. Therefore, (1−Bc)·Xe=X ₀ . Therefore, Equation 1 can be expressed as Equation 2.

In Equation 2, when cells are substantially logarithmically growing (X(t)=X ₀ ·e ^μt , viable cell density grows), it can be expressed as follows.

If we take the limit of the repetition period tc→0,

This is the operation of a chemostat that continuously discharges the culture medium as it is and simultaneously supplies fresh medium at the same flow rate. At this time, the concentration of medium components and the viable cell density are the same at the start and end of each culture.

ここで、
D ：灌流率（Bleeding含む）[d^-1]
Sp ：培養槽内培地成分濃度 [mmol/L]
Xp ：灌流培養における生細胞密度 [cells/L]
：灌流培養における“培地損失項” [mmol/cells/h]

In the case of perfusion culture, the viable cell density is also a constant value Xp according to formula (1), so the protein productivity from the input medium is expressed as follows.

here,
D : Perfusion rate (including bleeding) [d ^-1 ]
Sp : Concentration of medium components in culture tank [mmol/L]
Xp: Viable cell density in perfusion culture [cells/L]
: “Medium loss term” in perfusion culture [mmol/cells/h]

Equations 2 and 5 show that the protein productivity of cells in perfusion and repeat-batch cultures differs with the medium loss term. Therefore, in order to perform culture with higher productivity by repeat batch culture than perfusion culture, it is necessary to pay attention to the medium loss term. In other words, the value of the medium loss term due to perfusion culture may be used as a reference value, and the operation may be performed so that the value of the medium loss term due to repeat batch culture is lower than the reference value.

The medium loss term is preferably controlled by appropriately measuring the medium component concentration and cell density so that the value becomes smaller than the reference value. If the value of the medium loss term in repeat batch culture is higher than the reference value, the value of the medium loss term may be lowered below the reference value by adjusting the medium concentration or increasing the number of cultures. The medium concentration can be adjusted by increasing or decreasing the amount of added medium (adjusting the cell dilution ratio) or extending the culture period.

Comparing Eq. call) only. Therefore, from Equations 2 and 5, the medium loss term in repeat batch culture, that is, the value obtained by dividing the amount of medium components discharged at each culture solution exchange by the average cell density during each operation, is the same cell density. Repeat batch culture with higher productivity than perfusion culture can be performed by operating so that the medium loss term in the conventional perfusion culture, that is, the value obtained by dividing the product of the reflux rate and the medium component concentration by the cell density, is smaller. can.

More specifically, it is preferred to compare medium loss terms during logarithmic growth phase. Comparing Equation 3 and Equation 5, which represent the productivity of the logarithmic growth phase by the repeat batch method,
In the case of the logarithmic growth phase, from equations 3 and 5, the medium loss term in repeat batch culture, that is, the product of the specific growth rate and the medium component concentration at the end of each culture divided by the cell density at the end of each culture , by operating the medium loss term in perfusion culture using the same cells, i.e., the product of the perfusion rate and the medium component concentration divided by the cell density, the repeat batch is more productive than the perfusion culture. Cultivation can be performed.

As described above, since the protein productivity of cells depends on the medium loss term, a scaled-down experiment can be performed by setting the medium loss term due to repeat batch culture to a value equivalent to the reference value. That is, by setting conditions equivalent to perfusion culture in repeat batch culture, it is possible to perform simpler experiments with reduced facility costs.

Comparing Eq. call) only. Therefore, from Equations 2 and 5, the medium loss term in repeat batch culture, that is, the value obtained by dividing the amount of medium components discharged at each culture solution exchange by the average cell density during each operation, is the same cell density. Repeat batch culture with the same productivity as perfusion culture can be performed by operating so that the medium loss term in perfusion culture, that is, the value obtained by dividing the product of the reflux rate and the concentration of medium components by the cell density, is the same. . By doing so, unlike perfusion culture, which requires large-scale equipment, culture can be performed with simple equipment, so that the cost of equipment and culture medium can be reduced.

More specifically, it is preferred to compare medium loss terms during logarithmic growth phase. Comparing Equation 3 and Equation 5, which represent the productivity of the logarithmic growth phase by the repeat batch method,
In the case of the logarithmic growth phase, from equations 3 and 5, the medium loss term in repeat batch culture, that is, the product of the specific growth rate and the medium component concentration at the end of each culture divided by the cell density at the end of each culture , the repeat batch culture with the same productivity as the perfusion culture by operating to be the same as the medium loss term in the perfusion culture using the same cells, that is, the product of the reflux rate and the medium component concentration divided by the cell density. It can be performed. By doing so, unlike perfusion culture, which requires large-scale equipment, culture can be performed with simple equipment, so that the cost of equipment and culture medium can be reduced.

As described above, according to the culture method using the culture apparatus 100 according to the present embodiment, it is possible to control the culture so as to increase productivity by calculating the medium loss, and the perfusion culture in the repeat batch method. Cultivation can be performed with the above productivity.

(Example)
EXAMPLES The present invention will be specifically described below by showing examples, but the technical scope of the present invention is not limited to these.

Using the culture apparatus 100 shown in FIG. 1 as the culture apparatus, culture was performed by the repeat batch method, and a productivity comparison test with perfusion culture was performed. Equipment for perfusion culture includes a commonly used cell separation device.
<Glucose>

First, a liquid medium containing glucose was prepared. Cells were seeded in this liquid medium and subjected to stirring culture in a batch culture method in a culture tank.

Next, similar cells were used for comparison and cultured under the following parameters in a conventional perfusion culture. B: Bleeding rate 0.4 [d-1], CS/X: Medium specific consumption rate 1.25×10 ^-9 [mmol/cell/d], D: Perfusion rate 1 [d-1], Si: Glucose concentration in fresh medium 50 [mmol/L], Sp: medium components in the culture medium (glucose concentration as a representative value) 25 [mmol/L], X: cell density 2×10 ¹⁰ [cells/L]
μ: Specific growth rate 0.4 [d-1]

In the steady-state operation of the chemostat, the medium exchange and cell dilution are the same, so the perfusion rate D is the same as the cell specific growth rate μ, and the relationship Xp=μ(Si-Sp)/Cs/x is established. In the perfusion culture, the discharged culture medium and cells are separated, and the cells are left in the culture tank, so the culture medium can be exchanged at a speed higher than the specific growth rate of the cells. As shown by the thick line in Fig. 3, the cell density can be increased from the chemostat by exchanging the culture medium at a speed higher than the specific growth rate. ) is required and the value of the medium loss term is the same as in the chemostat.

In the setting example of this embodiment, in which the concentration of the medium components in the culture solution is half that in the fresh medium, the medium loss term in perfusion culture is equal to the medium specific consumption rate of the cells, and is not negligible. In addition, an increase in cell density requires not only an increase in medium exchange rate (perfusion rate), but also oxygen supply, carbon dioxide removal, mixing, and enhancement of cell separation ability, so there is a certain upper limit.

On the other hand, in repeat-batch culture, as shown in FIG. 3, when the average medium component concentration is 25 mmol/L, which is the same as perfusion culture, culture with less medium loss than perfusion culture is possible. The medium loss term can be further reduced by lengthening the repetition period tc within a range that does not interfere with repeated culture.

In this example, as shown in FIG. 3, the medium loss term can be reduced to about half that of perfusion culture. Therefore, economical operation with reduced costs is possible.

In perfusion culture, the medium components in the extracted culture solution are always constant. This indicates that it is not possible to drive to reduce.

However, as shown in Figure 3, when the average medium component concentration is higher (28 mmol/L) than in the perfusion culture (25 mmol/L), the medium loss term is larger than in the perfusion culture in the early stages of operation. becomes. Therefore, in order to operate the medium loss term at a lower value than perfusion culture, the glucose concentration should be 4.4 mmol / L (lower limit to maintain productivity, Biotechnol Prog. 2017 May;33(3):771-785 ) to 25 mmol/L.

<CHO cells>
FIG. 4 shows the results of a comparative test using CHO cells as the cells and glutamine as the medium.
If the glutamine concentration exceeds 0.4 mM, the production rate of by-products including lactic acid increases, affecting protein production and quality. Therefore, culture was performed at a glutamine concentration of 0.4 mM or less. In this case, in repeat batch culture, the initial glutamine concentration is the maximum of 0.4 mM (Smax), and the glutamine concentration after growth is about 0 mM. Therefore, the cultivable range in the repeat batch is the area shown in the figure. Production by perfusion culture often continues for 30 days or longer. In repeat batches of animal cells, one cycle is about one day, so 30 or more cultures are performed. If necessary, the amount of added medium can be adjusted or the repeat batch cycle can be changed.

<Yeast>
FIG. 5 shows the results of a comparative test in which the alcohol concentration, which is a metabolite, affects the cell viability in yeast. Above 16% alcohol concentration the yeast begins to die and at 20% it is completely dead. Therefore, it is necessary to culture so that the alcohol concentration is 16% or less. Therefore, in the repeat batch culture, the alcohol concentration after growth was controlled to 16 or less. Therefore, the cultivable range in the repeat batch is the area shown in the figure.

Although the present invention has been described above, the present invention is not limited to the above-described embodiments and modifications, and various changes are possible without departing from the scope of the present invention.

Although the economic comparison focused on the consumption of one nutrient component in the medium was performed, for example, a complex and exhaustive comparison focusing on multiple nutrient components is also possible. It is also possible to make a comparison by replacing the medium loss term with the waste product discharge term by focusing on the toxicity that has the opposite effect to the nutritional component, the cell metabolism component that has a suppressive effect, and the accumulation and discharge of the product.

REFERENCE SIGNS LIST 100 culture device 10 culture tank 11 agitator 12 aeration device 13 temperature control device 14 medium component concentration meter 15 cell density meter 16 medium loss indicator 23 control device

Claims (4)

An apparatus for culturing cells by repeat-batch culture in which cells are repeatedly cultured by batch culture or fed-batch culture,
a culture tank for culturing cells;
a medium component concentration meter that is connected to the culture tank and measures the concentration of medium components in the culture tank;
A cell density meter connected to the culture tank and measuring the cell density in the culture tank;
a control device that calculates a medium loss term from the measurement results of the medium component concentration meter and the cell density meter in each repetition, and controls the culture based on the medium loss term ;
The medium loss term in the repeat batch culture is a value obtained by dividing the amount of one or more components of the medium discharged to the outside of the culture tank at each time of culture solution exchange by the average cell density during each time of operation,
When the medium loss term when the same cells are cultured by perfusion culture is a value obtained by dividing the discharge rate of one or more medium components out of the culture tank by the cell density,
The control device is
Operate so that the medium loss term in the repeat batch culture is smaller than the medium loss term in the perfusion culture.
A culture apparatus characterized by: An apparatus for culturing cells by repeat-batch culture in which cells are repeatedly cultured by batch culture or fed-batch culture,
a culture tank for culturing cells;
A medium component concentration meter that is connected to the culture tank and measures the concentration of medium components in the culture tank;
A cell density meter connected to the culture tank and measuring the cell density in the culture tank;
a control device that calculates a medium loss term from the measurement results of the medium component concentration meter and the cell density meter in each repetition, and controls the culture based on the medium loss term ;
The medium loss term in the repeat batch culture is the product of the specific growth rate when the cultured cells are in the logarithmic growth phase and the output amount at the end of each culture divided by the cell density at the end of each culture,
When the medium loss term when the same cells are cultured by perfusion culture is a value obtained by dividing the discharge rate of one or more medium components out of the culture tank by the cell density,
The control device is
Operate so that the medium loss term in the repeat batch culture is smaller than the medium loss term in the perfusion culture.
A culture apparatus characterized by: An apparatus for culturing cells by repeat-batch culture in which cells are repeatedly cultured by batch culture or fed-batch culture,
a culture tank for culturing cells;
a medium component concentration meter that is connected to the culture tank and measures the concentration of medium components in the culture tank;
A cell density meter connected to the culture tank and measuring the cell density in the culture tank;
a control device that calculates a medium loss term from the measurement results of the medium component concentration meter and the cell density meter in each repetition, and controls the culture based on the medium loss term ;
The medium loss term in the repeat batch culture is a value obtained by dividing the amount of one or more components of the medium discharged to the outside of the culture tank at each time of culture solution exchange by the average cell density during each time of operation,
When the medium loss term when the same cells are cultured by perfusion culture is a value obtained by dividing the discharge rate of one or more medium components out of the culture tank by the cell density,
The control device is
The medium loss term in the repeat batch culture is controlled to be the same as the medium loss term in the perfusion culture.
A culture apparatus characterized by: An apparatus for culturing cells by repeat-batch culture in which cells are repeatedly cultured by batch culture or fed-batch culture,
a culture tank for culturing cells;
a medium component concentration meter that is connected to the culture tank and measures the concentration of medium components in the culture tank;
A cell density meter connected to the culture tank and measuring the cell density in the culture tank;
a control device that calculates a medium loss term from the measurement results of the medium component concentration meter and the cell density meter in each repetition, and controls the culture based on the medium loss term ;
The medium loss term in the repeat batch culture is the product of the specific growth rate when the cultured cells are in the logarithmic growth phase and the output amount at the end of each culture divided by the cell density at the end of each culture,
When the medium loss term when the same cells are cultured by perfusion culture is a value obtained by dividing the discharge rate of one or more medium components out of the culture tank by the cell density,
The control device is
Operate so that the medium loss term in the repeat batch culture is the same as the medium loss term in the perfusion culture.
A culture apparatus characterized by:

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