Bioreactor culture method and sterilizer of bioreactor

Abstract

To solve a problem that the total cost of a conventional single-use bioreactor rises in the case of using a high-cost shaft or the like.SOLUTION: A sterilizer of a bioreactor comprises: cleaning means for cleaning bioreactor auxiliary equipment to be reused after using biochemical reaction, which is installed to a single-use bag of unused biochemical reaction of a bioreactor; and ozone sterilization means for sterilizing the cleaned bioreactor auxiliary equipment in the bag by ozone, which is installed in the bag and to the bag.SELECTED DRAWING: Figure 5

Description

[Technical field]

[0001] The present invention relates to an apparatus and method for sterilizing a bioreactor (biochemical reaction device) that stirs liquids used in the medical field and the like, and that cultivates and stirs parts or individuals of animal and plant cells and tissues, cultures of microorganisms and bacterial cells, etc., a bioreactor culture device having the sterilization apparatus, and a culture method. [Background technology]

[0002] Conventionally, there is a single-use bioreactor in which, for example, a bag (culture vessel) and all bioreactor-related equipment attached (mounted) to the bag, such as an impeller and a shaft, are disposable.

[0003] This single-use bioreactor does not have a stainless steel tank (container), but instead uses a flexible bag, such as a disposable (single-use) bag made of a flexible synthetic resin (plastic such as polyethylene, elastomer), and the bioreactor ancillary equipment such as an impeller and shaft are integrated into the bag. After use in a biochemical reaction such as cultivation, the entire bag is disposable and discarded after one use.

[0004] The bioreactor auxiliary equipment may, for example, be an impeller or a shaft, as well as a sealing means for passing the shaft through the bag in an airtight (liquid-tight) manner so as to be freely rotatable, piping (tubes) for connecting or passing through the bag in an airtight (liquid-tight manner, for supplying or discharging liquid or gas into the bag, or for heating or cooling the contents, or the like, as well as a heating / cooling body, a sparger, a baffle, and the like.

[0005] Furthermore, the single-use bioreactor utilizes bags (containers) whose interiors have been sterilized with gamma sterilization or EG in advance under sealed conditions, so the risk of contamination is extremely low. In addition, there are significant advantages, such as no need for a high-temperature steam sterilization process (SIP) before cultivation, no need for a cleaning in place process (CIP), and the associated validation and utility equipment on the facility side.

[0006] In addition, the piping installed on the bag is also made of flexible tubes, allowing for sterile connection to external pipes, which not only reduces the risk of contamination but also makes operation much easier.

[0007] For example, an example of a single-use bioreactor is disclosed in Patent Document 1. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] JP 2016-212017 A, FIG. 5 Summary of the Invention [Problem to be solved by the invention]

[0009] However, in microbial cultivation, which is often carried out at high aeration rates and high power, and in particular in filamentous fungal cultivation, which requires high aeration and high power mixing with highly viscous fluids, a strong, single-use structure (impeller and shaft) is required to prevent damage due to a weak structure.

[0010] To ensure this, a single-use structure was adopted, but there was an issue that the initial structure was high compared to low-power animal cell culture, etc.

[0011] At the same time, due to cost considerations, there were safety restrictions placed on the culture load, which also posed the risk of problems.

[0012] Therefore, the inventors, through various experiments and investigations, have developed a hybrid bioreactor in which inexpensive bags, tubes, etc. (single-use parts) of a conventional single-use bioreactor are single-use, and costly impellers, shafts, etc. (reusable parts) are reused. As for sterilization of the reused parts, only steam sterilization (SIP), which is the most costly utility, is eliminated, and cleaning processes such as CIP are carried out as usual, while sterilization is carried out using ozone. [Means for solving the problem]

[0013] In order to achieve the above-mentioned object, the bioreactor sterilization device of the present invention is characterized by comprising a cleaning means for cleaning bioreactor auxiliary equipment that is attached to a single-use bag that has not been used in a biochemical reaction of the bioreactor and that is reused after the biochemical reaction, and an ozone sterilization means for sterilizing the inside of the bag and the cleaned bioreactor auxiliary equipment inside the bag with ozone, the ozone sterilization means being attached to the bag.

[0014] The reusable bioreactor auxiliary equipment is characterized in that it includes an impeller and a shaft.

[0015] The bag is also characterized in that it has unused single-use bioreactor ancillary equipment for biochemical reactions, and the sterilization means is a means for sterilizing the unused single-use bioreactor ancillary equipment in the bag and the cleaned bioreactor ancillary equipment attached to the bag with ozone.

[0016] The single-use bioreactor auxiliary equipment is characterized in that it is a tube.

[0017] The bioreactor culture apparatus having the sterilization device of the present invention is characterized by comprising a cleaning means for cleaning bioreactor ancillary equipment that is attached to a single-use bag that has not been used for a biochemical reaction of the bioreactor and that is reused after the biochemical reaction, an ozone sterilization means for sterilizing the inside of the bag and the cleaned bioreactor ancillary equipment attached to the bag with ozone, and a culture means for culturing the contents supplied into the bag.

[0018] The bag is also characterized in that it has a single-use bioreactor ancillary equipment that has not been used for biochemical reactions, and the sterilization means is a means for sterilizing the inside of the bag and the single-use bioreactor ancillary equipment that has not been used for biochemical reactions and the cleaned bioreactor ancillary equipment inside the bag and attached to the bag with ozone.

[0019] The method for sterilizing a bioreactor of the present invention is characterized by comprising a first step of cleaning bioreactor ancillary equipment that is attached to a single-use bag unused for a biochemical reaction of the bioreactor and that is to be reused after use in a biochemical reaction; a second step of attaching the cleaned bioreactor ancillary equipment to the bag; and a third step of sterilizing the inside of the bag and the cleaned bioreactor ancillary equipment attached to the bag with ozone.

[0020] The method for sterilizing a bioreactor of the present invention is characterized by comprising a first step of cleaning the bioreactor ancillary equipment to be reused after a biochemical reaction, which is attached to a single-use bag of a bioreactor having unused single-use biochemical reaction ancillary equipment, a second step of attaching the cleaned bioreactor ancillary equipment to the bag, and a third step of sterilizing the inside of the bag and the single-use bioreactor ancillary equipment not used for a biochemical reaction and the cleaned bioreactor ancillary equipment attached to the bag with ozone.

[0021] The bioreactor culture method of the present invention is characterized by comprising a first step of supplying a culture medium into a sterilized hybrid bioreactor bag comprising a single-use bag unused for a biochemical reaction of a bioreactor and a reusable bioreactor auxiliary equipment unused for a biochemical reaction attached to the bag; a second step of culturing the culture medium; a third step of discharging the cultured culture medium; a fourth step of cleaning the inside of the bag and the reusable bioreactor auxiliary equipment attached to the bag after a biochemical reaction in the bag with a cleaning liquid; a fifth step of discarding the bag and installing the cleaned reusable bioreactor auxiliary equipment in a new single-use bag unused for a biochemical reaction; a sixth step of sterilizing the inside of the bag and the cleaned bioreactor auxiliary equipment attached to the bag with ozone; a seventh step of supplying a culture medium into the bag; and an eighth step of repeating the second to seventh steps as many times as necessary.

[0022] The bioreactor culture method of the present invention includes a first step of supplying a culture solution into a bag of a sterilized hybrid bioreactor, the bag being made of a single-use bag of a bioreactor that has been unused for a biochemical reaction and a reusable bioreactor auxiliary equipment that has been unused for a biochemical reaction and is attached to the bag; a second step of culturing the culture solution; a third step of discharging the cultured culture solution; a fourth step of cleaning the inside of the bag and the reusable bioreactor auxiliary equipment attached to the bag after the biochemical reaction in the bag with a cleaning solution; and a fourth step of cleaning the sintering process of ... a fifth step of discarding the reused bag and bioreactor ancillary equipment, and installing the cleaned reused bioreactor ancillary equipment in a new single-use bag that has not been used for biochemical reactions and that has a new single-use bioreactor ancillary equipment that has not been used for biochemical reactions; a sixth step of sterilizing the single-use bioreactor ancillary equipment and the cleaned bioreactor ancillary equipment in the bag and installed in the bag with ozone; a seventh step of supplying a culture medium into the bag; and an eighth step of repeating the second to seventh steps as many times as necessary.

[0023] The bioreactor culture method of the present invention is characterized by comprising a first step of supplying a culture medium into a sterilized hybrid bioreactor bag comprising a single-use bag unused for a biochemical reaction of a bioreactor and a reusable bioreactor auxiliary equipment unused for a biochemical reaction attached to the bag; a second step of culturing the culture medium; a third step of discharging the cultured culture medium; a fourth step of discarding the bag and cleaning the reusable bioreactor auxiliary equipment after the biochemical reaction with a cleaning liquid; a fifth step of attaching the cleaned reusable bioreactor auxiliary equipment to a new single-use bag unused for a biochemical reaction; a sixth step of sterilizing the inside of the bag and the cleaned bioreactor auxiliary equipment attached to the bag with ozone; a seventh step of supplying a culture medium into the bag; and an eighth step of repeating the second to seventh steps as many times as necessary.

[0024] The bioreactor culture method of the present invention includes a first step of supplying a culture solution into a bag of a sterilized hybrid bioreactor having a single-use bag of a bioreactor unused for a biochemical reaction and a reusable bioreactor auxiliary equipment unused for a biochemical reaction attached to the bag, the bioreactor having a single-use bag of a bioreactor unused for a biochemical reaction, and a reusable bioreactor auxiliary equipment unused for a biochemical reaction attached to the bag, the first step of culturing the culture solution, the third step of discharging the cultured culture solution, and the third step of discarding the single-use bag and the bioreactor auxiliary equipment and washing the reusable bioreactor auxiliary equipment after the biochemical reaction. The method comprises a fourth step of cleaning the bag with a purification solution; a fifth step of attaching the cleaned reused bioreactor ancillary equipment to a new single-use bag having new single-use biochemical reaction unused bioreactor ancillary equipment; a sixth step of sterilizing the single-use bioreactor ancillary equipment in the bag and the cleaned bioreactor ancillary equipment attached to the bag with ozone; a seventh step of supplying a culture medium into the bag; and an eighth step of repeating the second to seventh steps as many times as necessary. Effect of the Invention

[0025] According to the present invention, compared with conventional single-use bioreactors, the introduction cost and initial cost can be significantly reduced, and it is possible to introduce the bioreactor into a culture system that requires strong agitation, such as filamentous fungal culture. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 is an explanatory diagram of single-use components consisting of single-use bags and tubes of the hybrid bioreactor of the present invention. [Diagram 2] FIG. 2 is an explanatory diagram of the single-use tube in FIG. [Diagram 3] FIG. 2 is an explanatory diagram of reused parts of the hybrid bioreactor of the present invention, including an impeller, a shaft, piping, etc. [Figure 4] FIG. 4 is an explanatory diagram of the impeller and shaft in FIG. 3 that are reused. [Diagram 5] FIG. 1 is an explanatory diagram of a bioreactor culture apparatus having a sterilization device of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Examples of modes for carrying out the present invention are given below. EXAMPLES

[0028] A first embodiment of the present invention will be described with reference to FIGS.

[0029] (1. Description of Hybrid Bioreactor)

[0030] In the present invention, a hybrid bioreactor consisting of single-use parts and reused parts is used.

[0031] As described below, the hybrid bioreactor is used for culturing, and the reusable parts of the hybrid bioreactor are sterilized by ozone sterilization, eliminating the need for costly steam sterilization (SIP), thereby achieving overall cost reduction.

[0032] That is, in the present invention, for example, as shown in Figs. 1 and 2, of the conventional single-use bioreactor, a low-cost bag (culture vessel) 1 and a tube 2 that is connected to or penetrates the bag 1 are made single-use, and as shown in Figs. 3 and 4, among the bioreactor auxiliary equipment, an impeller 3 and a shaft 4 that are particularly expensive are made of, for example, stainless steel and reused.

[0033] In addition to the impeller 3 and the shaft 4, the reusable parts may be, for example, a sealing means for the shaft 4 in the bag, a pipe 5 for passing a liquid or gas for heating or cooling the contents, a heating or cooling body (not shown) for heating or cooling the contents, a sparger 6, and a baffle 7, which may be made of stainless steel.

[0034] The single-use parts include, in addition to the bag 1 and the tube 2, a sealing means for the shaft, the piping 5, the heating / cooling body, the sparger 6, the baffle 7, and the like.

[0035] Incidentally, which parts of the bioreactor auxiliary equipment are single-use parts or reusable parts is determined depending on the cost, usage mode, etc. of each part.

[0036] (2. Description of bioreactor culture device with bioreactor sterilization device)

[0037] Next, a bioreactor culture apparatus having a sterilization device for the hybrid bioreactor, which performs culture and sterilization, will be described.

[0038] In the following explanation, the bag and tube are considered as single-use parts, and the impeller, shaft, sparger, pipes, and heating / cooling body are considered as reusable parts, but the same applies to other combinations.

[0039] As shown in FIG. 5, the bioreactor culture apparatus 8 having the sterilization device comprises, for example, a hybrid bioreactor comprising a bag and bioreactor auxiliary equipment, a cylindrical support housing 9 into which the hybrid bioreactor is inserted from above and supported, a culture medium supply and discharge means comprising a culture medium supply tank (not shown) and a culture medium discharge tank (not shown) for supplying and discharging a culture medium and the like comprising a culture medium and cells (bacterial strain) into the bag, each of which is connected to an end of a tube 2 provided in the bag 1 of the hybrid bioreactor, an ozone sterilization means comprising an ozone generator 10 for supplying and discharging ozone into the bag, a cleaning means comprising a cleaning liquid supply device 11 for supplying and discharging a cleaning liquid into the bag and a cleaning agent disposal device 12, a temperature regulator (temperature adjuster) 13 for adjusting the temperature inside the bag, which is connected to an end of the tube 2 or a pipe or the heating and cooling body, a sparger gas supply device (not shown) connected to an end of the sparger, and a control device 14 for controlling each of the above devices.

[0040] The end of each of the tubes has a closed sterile plug that can be connected in a sterile state to an external tube for supplying liquid, gas, etc. into the container, and is also provided with an opening and closing valve.

[0041] Instead of providing an on-off valve on the tube, an on-off valve may be provided on the outer tube side.

[0042] Also, for example, an opening / closing valve may be provided at a desired location in the piping or in an external pipe connected to the piping.

[0043] Furthermore, a rotating means such as a motor is provided on the base of the shaft that protrudes outside the bag.

[0044] Next, a method for using the present invention will be described.

[0045] First, an unused and sterilized hybrid bioreactor is inserted into the support housing 9 from above, fixed therein, and the cover is closed.

[0046] Then, each tube 2 of the hybrid bioreactor is connected to each external pipe such as the culture medium supply tank, the culture medium discharge tank, the ozone generator 10, the detergent supply device 11, the detergent disposal device 12, and the like.

[0047] In addition, the parts of the pipes 5 for flowing a liquid for heating and cooling the contents, the heating and cooling bodies, etc., that protrude outside the bag are connected to the temperature regulator 13, and the part of the sparger that protrudes outside the bag is connected to a sparger gas supply device.

[0048] At this stage, the valves of the tubes, pipes, etc. are closed.

[0049] Then, the on-off valve of the tube connected to the culture medium supply tank is opened, and the culture medium (contents) such as the medium and cells (bacterial strain) is filled into the bag. Then, the on-off valve is closed, and the agitator or the like is operated as necessary to carry out a biochemical reaction such as cultivation.

[0050] After the biochemical reaction such as cultivation is completed, the open / close valve of the tube connected to the cleaning agent disposal device is opened, and the culture medium is extracted into the culture medium discharge tank by suction or the like.

[0051] Then, for example, in CIP cleaning, the on-off valve is closed, and the on-off valves connected to the cleaning agent supply device 11 and the cleaning agent disposal device 12 are opened and closed as necessary to introduce the cleaning liquid from the cleaning liquid introduction tube, clean the inside of the bag and the associated equipment such as the impeller, piping, baffles, etc., and then discharge the cleaning liquid and collect it in the cleaning agent disposal device.

[0052] Next, the hybrid bioreactor is removed from the support housing 9, and single-use parts of the hybrid bioreactor, such as a bag, are removed and discarded. In addition, the used and washed reusable parts, such as the impeller, removed from the hybrid bioreactor are attached to a new unused bag.

[0053] In addition to introducing a cleaning solution into the bag and cleaning the reused parts by CIP, the hybrid bioreactor may be removed from the support housing 9 after a biochemical reaction such as culture, and single-use parts such as the bag of the hybrid bioreactor may be removed and discarded, and the reused parts may be washed separately, and the washed reused parts after the biochemical reaction may be attached to a new, unused bag.

[0054] Then, in the same manner as above, the washed hybrid bioreactor is inserted from above into the support housing 9 and fixed therein, and each of the tubes, piping, etc. is connected to the necessary external pipes, etc.

[0055] Then, the on-off valve of the tube connected to the ozone generator 10 is opened to introduce ozone into the bag, and the inside of the bag and the bioreactor auxiliary equipment in the bag are sterilized with ozone.

[0056] The bioreactor-related equipment in the bag includes the tubes and piping extending outside the bag up to the opening and closing valves.

[0057] The ozone sterilization concentration is then controlled by the controller so that sterilization is carried out, for example, overnight.

[0058] Then, for example, after sterilization on the following day, the on-off valve is closed, and the bag is filled with a culture solution consisting of a medium and cells (bacterial strain), etc., in the same manner as above, and culture is carried out. Then, the culture solution is discharged, washed, and sterilized overnight in the same manner as above.

[0059] Repeat this process.

[0060] According to the present invention, by using a hybrid bioreactor, it is possible to reduce the overall cost by sterilizing the reusable parts by cleaning and ozone sterilization without performing costly steam sterilization (SIP).

[0061] As a result, we have succeeded in significantly reducing the introduction and running costs of PILOT equipment, especially in the hundreds of liter class, as well as the effort required for sterilization validation and the risk of contamination.

[0062] The bioreactor vessel of the present invention is used in the medical product and food industries, etc. [Explanation of symbols]

[0063] 1 bag 2 Tubes 3 Impeller 4 Shaft 5 Piping 6 Sparger 7 Baffle 8. Bioreactor Equipment 9. Housing 10 Ozone Generator 11 Detergent supply device 12 Cleaning agent disposal device 13 Temperature controller 14 Control device

Claims

1. A cleaning means for cleaning bioreactor auxiliary equipment that is to be reused after biochemical reaction, the cleaning means being attached to a single-use bag that has not been used in a biochemical reaction of the bioreactor; A bioreactor sterilization apparatus comprising an ozone sterilization means for sterilizing the inside of the bag and the cleaned bioreactor auxiliary equipment inside the bag with ozone, the ozone sterilization means being attached to the bag.

2. 2. The bioreactor sterilization apparatus according to claim 1, wherein the bioreactor auxiliary equipment that is reused is an impeller and a shaft.

3. The bag has a single-use bioreactor accessory that has not been used for biochemical reactions, The bioreactor sterilization apparatus according to claim 1, characterized in that the sterilization means is a means for sterilizing the interior of the bag and the unused single-use bioreactor auxiliary equipment and the cleaned bioreactor auxiliary equipment attached to the bag by ozone.

4. The bioreactor sterilization apparatus according to claim 3, wherein the single-use bioreactor auxiliary equipment is a tube.

5. A cleaning means for cleaning bioreactor auxiliary equipment that is to be reused after biochemical reaction, the cleaning means being attached to a single-use bag that has not been used in a biochemical reaction of the bioreactor; an ozone sterilization means for sterilizing the inside of the bag and the cleaned bioreactor auxiliary equipment in the bag by ozone, the ozone sterilization means being attached to the bag; A bioreactor culture apparatus having a sterilization device, further comprising a culture means for culturing the contents supplied into the bag.

6. The bag has a single-use bioreactor accessory that has not been used for biochemical reactions, The bioreactor culture apparatus having a sterilization device according to claim 5, characterized in that the sterilization means is a means for sterilizing the inside of the bag and the unused single-use bioreactor auxiliary equipment and the cleaned bioreactor auxiliary equipment in the bag, which are attached to the bag, by ozone.

7. A first step of cleaning bioreactor auxiliary equipment that is attached to a single-use bag that has not been used in a biochemical reaction of the bioreactor and is to be reused after the biochemical reaction; a second step of loading the cleaned bioreactor accessories into the bag; a third step of sterilizing the inside of the bag and the cleaned bioreactor auxiliary equipment attached to the bag with ozone; A method for sterilizing a bioreactor, comprising:

8. A first step of cleaning a bioreactor auxiliary equipment that is to be reused after a biochemical reaction and is attached to a single-use bag that is unused for a biochemical reaction of a bioreactor having unused single-use biochemical reaction auxiliary equipment; a second step of loading the cleaned bioreactor accessories into the bag; a third step of sterilizing the inside of the bag and the unused single-use bioreactor auxiliary equipment and the cleaned bioreactor auxiliary equipment in the bag by ozone; A method for sterilizing a bioreactor, comprising:

9. A sterilized hybrid bioreactor comprising a single-use bag unused for biochemical reactions in a bioreactor and a bioreactor accessory device attached to the bag that is unused for biochemical reactions and is reusable. A first step of supplying a culture medium into the bag; A second step of culturing the culture medium; A third step of discharging the culture medium; a fourth step of cleaning the inside of the bag and the bioreactor auxiliary equipment attached to the bag, which is to be reused after the biochemical reaction in the bag, with a cleaning solution; A fifth step of discarding the bag and installing the cleaned and reused bioreactor auxiliary equipment in a new single-use bag that has not been used in a biochemical reaction; a sixth step of sterilizing the inside of the bag and the cleaned bioreactor auxiliary equipment attached to the bag with ozone; A seventh step of supplying a culture medium into the bag; and an eighth step of repeating the second to seventh steps as many times as necessary.

10. A sterilized hybrid bioreactor having unused single-use bioreactor ancillary equipment for biochemical reactions, the hybrid bioreactor comprising a single-use bag for biochemical reactions unused and a bioreactor ancillary equipment for biochemical reactions unused and reusable attached to the bag. A first step of supplying a culture medium into the bag; A second step of culturing the culture medium; A third step of discharging the culture medium; a fourth step of cleaning the inside of the bag and the bioreactor auxiliary equipment attached to the bag, which is to be reused after the biochemical reaction in the bag, with a cleaning solution; A fifth step of discarding the single-use bag and bioreactor ancillary equipment, and installing the cleaned reused bioreactor ancillary equipment in a new single-use bag unused for biochemical reactions, the new single-use bag having unused biochemical reaction bioreactor ancillary equipment; a sixth step of sterilizing the inside of the bag and the single-use bioreactor auxiliary equipment in the bag and the cleaned bioreactor auxiliary equipment attached to the bag with ozone; A seventh step of supplying a culture medium into the bag; and an eighth step of repeating the second to seventh steps as many times as necessary.

11. A sterilized hybrid bioreactor comprising a single-use bag unused for biochemical reactions in a bioreactor and a bioreactor accessory device attached to the bag that is unused for biochemical reactions and is reusable. A first step of supplying a culture medium into the bag; A second step of culturing the culture medium; A third step of discharging the culture medium; A fourth step of discarding the bag and cleaning the bioreactor auxiliary equipment for reuse after the biochemical reaction with a cleaning solution; A fifth step of installing the cleaned reused bioreactor auxiliary equipment in a new unused single-use bag for biochemical reaction; a sixth step of sterilizing the inside of the bag and the cleaned bioreactor auxiliary equipment attached to the bag with ozone; A seventh step of supplying a culture medium into the bag; and an eighth step of repeating the second to seventh steps as many times as necessary.

12. A sterilized hybrid bioreactor having unused single-use bioreactor ancillary equipment for biochemical reactions, the hybrid bioreactor comprising a single-use bag for biochemical reactions unused and a bioreactor ancillary equipment for biochemical reactions unused and reusable attached to the bag. A first step of supplying a culture medium into the bag; A second step of culturing the culture medium; A third step of discharging the culture medium; A fourth step of discarding the single-use bag and bioreactor auxiliary equipment and cleaning the reused bioreactor auxiliary equipment after the biochemical reaction with a cleaning solution; A fifth step of attaching the cleaned reused bioreactor ancillary equipment to a new unused single-use bag having a new unused single-use bioreactor ancillary equipment; a sixth step of sterilizing the inside of the bag and the single-use bioreactor auxiliary equipment in the bag and the cleaned bioreactor auxiliary equipment attached to the bag with ozone; A seventh step of supplying a culture medium into the bag; and an eighth step of repeating the second to seventh steps as many times as necessary.

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