Patent Application
20230024632
The present invention relates to a synthesis device and a synthetic method for chemically synthesizing proteins, peptides, nucleic acids, and the like.
One method for chemically synthesizing proteins, peptides, nucleic acids, and the like is to sequentially supply a plurality of types of solution (reagent) to a reaction vessel, and allow the reaction to proceed in the reaction vessel. For example, in the case of synthesizing a nucleic acid, a large number of granular carriers (beads) are provided in a reaction vessel, detritylation, coupling, oxidation, and capping are repeatedly performed while sequentially supplying solutions to the reaction vessel, and bases are bonded one after another from the carriers.
Dozens of kinds of solution may be used here, and these solutions are selectively sent to the reaction vessel, and the target substance (nucleic acid) is produced by the molecular material contained in the solutions. One known device for performing such chemical synthesis is, for example, the synthesis device described in Japanese Laid-Open Patent Application Publication No. 2019-170187 (Patent Literature 1).
A large number of carriers are contained in the reaction vessel provided to the synthesis device. A solution is supplied into the reaction vessel through a port provided at either the upper and lower part of the reaction vessel, and the solution that has passed through the reaction vessel is discharged from another port provided the other end. Chemical synthesis is made to proceed by sequentially changing the solution that is passed through the reaction vessel.
However, some of the carriers may not readily react, depending on their position in the reaction vessel. That is, even though the solution passes through the reaction vessel, the solution may not reach those carriers located near the side wall surfaces of the reaction vessel, for example. In this case, chemical synthesis at the carrier will not occur sufficiently. As a result, the yield of the obtained target product (nucleic acid) may be lower.
In view of this, it is an object of the present disclosure to reduce the number of carrier that do not readily react in the reaction vessel, and to improve the yield of the target product obtained by chemical synthesis.
The synthesis device disclosed herein comprises a reaction vessel that contains a large number of carriers and to which a solution is supplied, and a gas supply means for stirring the solution and the carriers by supplying a gas to the reaction vessel.
With this synthesis device, there are fewer carriers that do not readily react depending on their location in the reaction vessel, which makes it possible to improve the yield of the target product obtained by chemical synthesis.
The conventional approach has been to fill up the reaction vessel with carriers and pass the solutions through the reaction vessel.
However, with the present disclosure disclosed herein, a gas is supplied into the reaction vessel.
In view of this, the reaction vessel preferably has a vessel upper surface located higher than an upper surface of a layer of the carriers and the solution in a state in which the carriers and a required amount of solution are in place.
With this configuration, the carriers and the required amount of solution are held in the reaction vessel, and a space is formed in the upper part of the reaction vessel. Therefore, when the gas is supplied into the reaction vessel, the solution and the carriers can flow so as to escape into the space at the top part the vessel, so the solution and the carriers are stirred more briskly.
The carriers move vigorously when the gas is supplied into the reaction vessel, and if some of the carriers cling to the upper surface of the reaction vessel, it is possible that those carriers will not make contact with the solution.
Therefore, preferably, the reaction vessel has an internal filter that is located higher than an upper surface of a layer of the carriers in a state prior to the supply of the gas to the reaction vessel, and is located lower than a height of a liquid surface of the solution containing the gas supplied to the reaction vessel, the internal filter being permeable to the solution and impermeable to the carriers.
When an internal filter is provided at such a position, even if the carriers move and cling to the internal filter, they will be mixed with the solution. This prevents some of the carriers from clinging to the upper surface of the reaction vessel and being unable to make contact with the solution.
Also, preferably, the gas supply means is configured to transport the solution outside the reaction vessel by pushing by the gas through a pipe connected to the reaction vessel, and supply the gas used to transport the solution directly to the reaction vessel to stir the carriers and the solution by the gas when the transport of the solution to the reaction vessel by the gas is complete.
In this case, the gas transporting the solution to the reaction vessel is used to stir the carriers and the solution. If a mechanism is provided for transporting the solution to the reaction vessel by means of a gas, it may not be necessary for a gas supply mechanism for stirring to be separately provided, and this simplifies the configuration of the synthesis device.
Also, preferably, the reaction vessel has at its upper part an inlet through which passes the gas used to discharge the solution to outside of the reaction vessel, and has at its lower part an outlet through which the solution is discharged.
With this configuration, if a gas is supplied from the upper part of the reaction vessel, the used solution will be discharged from the lower part of the reaction vessel.
Also, the present disclosure disclosed herein is a synthesis method in which chemical synthesis is performed by supplying a solution to a reaction vessel containing a large number of carriers, the method including a supply step of supplying a required amount of the solution to the reaction vessel containing the carriers, and a synthesis step of chemically synthesizing, while stirring the solution and the carriers, by supplying a gas to the reaction vessel.
With this synthesis method, there are fewer carriers that do not readily react depending on their location in the reaction vessel, which makes it possible to improve the yield of the target product obtained by chemical synthesis.
According to the present disclosure disclosed herein, there are fewer carriers that do not readily react depending on their location in the reaction vessel, which makes it possible to improve the yield of the target product obtained by chemical synthesis.
The synthesis device 1 comprises an area for providing storage containers (reagent bottles) 2 in a number equal to the number of solutions L used. The solutions L are stored in the respective storage containers 2. In
The synthesis device 1 comprises a tank 4 for storing gas, the introduction pipes 5, the outlet pipes 6, an intermediate container 7, an intermediate pipe 8, the reaction vessel 9, a measuring mechanism 15, and a control device 16. The tank 4 is filled with a gas whose pressure is higher than atmospheric pressure, and in the synthesis device 1 disclosed herein, the tank is filled with argon gas (an inert gas). Although argon gas is used in this embodiment, any other gas may be used as long as it is an inert gas and is dehydrated. As described below, this gas is used to transport the solutions L of the storage containers 2 to the reaction vessel 9, as well as to stir the carriers B and the solution L in the reaction vessel 9.
The introduction pipes 5, whose number is equal to that of the plurality of storage containers 2, branch off from a shared upstream pipe 10. A regulator 11 and a valve 12 are provided to the upstream pipe 10. The upstream pipe 10 is connected to the tank 4, the gas in the tank 4 is supplied to the storage containers 2, and the internal pressure of the storage containers 2 is adjusted by the regulator 11. The internal pressure of the storage containers 2 is increased by the gas, which causes the solution L in the storage containers 2 to be pumped out through the outlet pipes 6. That is, the solution L in each storage container 2 is fed under pressure to the intermediate container 7 through the outlet pipe 6 due to the differential pressure between the storage container 2 and the intermediate container 7.
A valve 14 is provided to each of the outlet pipes 6. By selecting which of the valves 14 is in an open state, a specific solution L from among the solutions L in the plurality of storage containers 2 is selectively sent through an outlet pipe 6 to the intermediate container 7. The selection of the valve 14 to be in an open state is performed by the control device 16.
The intermediate container 7 is used for metering each solution L. The intermediate container 7 is a bottomed cylindrical container in which each solution L can be held. The plurality of outlet pipes 6 are gathered together at the inlet region (opening 7a) of the intermediate container 7. Therefore, the solution L selectively sent through an outlet pipe 6 is introduced into the intermediate container 7 and held in this intermediate container 7.
The measuring mechanism 15 measures the solution L stored in the intermediate container 7. In the measuring mechanism 15, the intermediate container 7 functions as a measuring container. The measuring mechanism 15 has, for example, a measuring device featuring a load cell or the like, and measures the solution L held in the intermediate container 7. The weight obtained by the weighing mechanism 15 is transmitted to the control device 16. The control device 16 controls the opening and closing operation of the valve 14 on the basis of the measurement result to acquire the specified amount of solution L in the intermediate container 7. The specified amount of solution L is sent through the intermediate pipe 8 to the reaction vessel 9. A valve 21 is provided to the intermediate pipe 8, and the valve 21 is in a closed state when weighing is being performed.
The method for supplying the solution L from the intermediate container 7 to the reaction vessel 9 is pressure feeding with a gas, and the gas in the tank 4 is used for this. During this pressure feeding, the valve 21 is in an open state. For this pressure feeding, the synthesis device 1 comprises a sealed container 29 in which the intermediate container 7 is housed. A gas pipe 17 is provided between the sealed container 29 and the tank 4. A second regulator 18 and a valve 19 are provided to the gas pipe 17. The valve 19 shown in
The intermediate container 7 is open in the sealed container 29, and when the gas in the tank 4 is supplied to the sealed container 29, the pressure (internal pressure) of the gas in the sealed container 29 acts on the solution L held in the intermediate container 7, and the solution L in the intermediate container 7 is pressure-fed through the intermediate pipe 8 to the reaction vessel 9 by the differential pressure between the sealed container 29 (intermediate container 7) and the reaction vessel 9. Thus, with the synthesis device 1 disclosed herein, the method of feeding the solution L from the intermediate container 7 to the reaction vessel 9 is pumping under gas pressure, and this pumping is performed by a gas supply means 20. With the configuration shown in
Because of this, a solution L is selectively sent to the intermediate container 7 from one or more of the storage containers 2, and when the measurement is performed in the intermediate container 7, that solution L is sent to the reaction vessel 9. The solution L is used for chemical synthesis in the reaction vessel 9. After this, the solution L is discharged through the discharge pipe 26 to a waste liquid tank 27.
This supply of the solution L to the reaction vessel 9 is repeated for each type of solution L so that a plurality of types of solution L are sequentially supplied to the reaction vessel 9, and chemical synthesis is conducted in the reaction vessel 9. A large number of carriers B are provided in the reaction vessel 9, and bases are bonded one after another from the carriers B to synthesize a nucleic acid.
The intermediate pipe 8 is connected to the lower part of the reaction vessel 9. A pipe 23 branching off from the gas pipe 17 (see
As shown in
Of the reaction space 30, the volume of the space Q2 may be larger than the combined volume of the carriers B and the solution L. With the reaction vessel 9 disclosed herein, the cross-sectional area of the reaction space 30 is constant in the vertical direction, so the height H1 of the space Q2 is greater than the height H2 of the layer between the carriers B and the solution L.
Here, the method for supplying the solution L to the reaction vessel 9 will be described again. As discussed above, the method for sending the solution L to the reaction vessel 9 is pressure feeding by means of the gas in the tank 4 (see
Therefore, the volume of the reaction vessel 9 (reaction space 30) is set large. More specifically, the volume of the reaction vessel 9 (reaction space 30) is set large enough that even if the preset maximum amount of the solution L is supplied to the reaction vessel 9, the height of the liquid surface of the solution L will be at the second height position P2 part of the way up in the vertical direction of the reaction space 30. That is, in a state where the required amount of solution L has been supplied to the reaction vessel 9 containing the carriers B, the volume of the reaction vessel 9 will be greater than the combined volume of the required amount of solution L and the carriers B. To put this yet another way, in a state in which the carriers B and the required amount of the solution L are held, the vessel upper surface 31 of the reaction vessel 9 is located higher than the upper surface S of the layer of the carriers B and the solution L. With the synthesis device 1 disclosed herein, as mentioned above, the vessel upper surface 31 is the lower surface of the upper filter 38.
The volume of the reaction vessel 9 is greater than the volume of the supplied solution L. For example, from the standpoint of stirring efficiency, the volume of the reaction vessel 9 is preferably at least 2.5 times the volume (maximum required amount) of the solution L, and is also preferably no more than 4 times the volume of the solution L so as not to make the reaction vessel 9 any larger than necessary.
The supply of gas for stirring is continued for a specific length of time. As shown in
As described above (see
The supply of gas produces a mixed flow of the solution L and the carriers B, containing the gas, inside the reaction vessel 9. The vessel upper surface 31 is located higher than the height of the upper surface of this mixed flow.
A plurality of carriers B may be present as a clump in the reaction vessel 9. In this case, the carriers B at the center of this clump will be less likely to come into contact with the solution L. However, in the present disclosure disclosed herein, clumping is eliminated by the stirring, that is, the carriers B are dispersed in the solution L, and more of the carriers B come into contact with the solution L.
A modification example of the reaction vessel 9 will now be described.
As discussed above (see
In view of this, as shown in
Since the internal filter 35 is provided at the position described above, the carriers B will not stick to the vessel upper surface 31. Furthermore, even if the carriers B should move and stick to the internal filter 35, they can be mixed with the solution L, and those carriers B will also contribute to the production of the target substance (nucleic acid). Thus, when the reaction vessel 9 has the internal filter 35, it is possible to prevent some of the carriers B from sticking to the vessel upper surface 31 of the reaction vessel 9 and being unable to come into contact with the solution L. When the internal filter 35 is provided, the upper filter 38 may be omitted.
Whether the reaction vessel 9 is as shown in
First, the stirring is halted in the reaction vessel 9. To this end, the supply of gas to the reaction vessel 9 is stopped. The gas valve 19 provided along the gas pipe 17 (see
In order to discharge the solution L in the reaction vessel 9, the gas in the tank 4 is supplied to the reaction vessel 9 from the upper part of the vessel. Therefore, even if some of the carriers B stick to the vessel upper surface 31 (upper filter 38) or some of the carriers B stick to the internal filter 35 (see
As shown in
As described above, the synthesis device 1 in each of the modes described above comprises a reaction vessel 9 and a gas supply means 20 capable of supplying gas to this reaction vessel 9. The reaction vessel 9 contains a large number of carriers B and is supplied with the solution L used for chemical synthesis. The gas supply means 20 stirs the solution L and the carriers B by supplying gas to the reaction vessel 9 during chemical synthesis in the reaction vessel 9. With this synthesis device 1, the carriers B are dispersed in the solution L in the reaction vessel 9, allowing the solution L to come into contact with all of the many carriers B. As a result, fewer of the carriers B are unlikely to react than in the past. For example, carriers B that have stuck to the side wall surface of the reaction vessel 9 can move to the center of the reaction vessel 9, or carriers B that have clumped together can be dispersed, allowing more of the carriers B to come into contact with the solution L. As a result, it is possible to improve the yield of the target substance (nucleic acid) obtained by chemical synthesis.
The synthesis method performed by the synthesis device 1 in each of the above-mentioned mode, that is, the synthesis method for performing chemical synthesis by supplying a solution L to the reaction vessel 9 containing a large number of carriers B, includes the following steps.
In the case of the synthesis device 1 disclosed herein, in the supply step S2, the solution L is supplied to the reaction vessel 9 by pressure feeding a gas.
In the synthesis step S3, when the gas is supplied to the reaction vessel 9, the gas supply parameters, such as the pressure and the flow rate of the gas, are controlled by the control device 16. This control is preferably performed by adjusting the supply parameters according to the type of solution L. The gas supply parameters when the solution L is supplied to the reaction vessel 9 in the supply step S2, and the gas supply parameters when the gas is supplied to the reaction vessel 9 for stirring in the synthesis step S3, may be the same or may be different.
Furthermore, the synthesis method disclosed herein (see
In the measuring step S1, the required amount of solution L is measured. In the discharge step S4, as described above, if gas is supplied from the upper part (upper port 41) of the reaction vessel 9, the used solution L is discharged as waste liquid from the lower part (lower port 42) of the reaction vessel 9.
In the determination step S5, if it is determined that the synthesis is not complete, the processing proceeds to the measurement step S1, and the next measurement of the solution L is performed. After this, the supply step S2, the synthesis step S3, and the discharge step S4 are repeated until all the synthesis is complete.
The synthesis method disclosed herein is not a method in which the solution L is circulated by using the reaction vessel 9 as one of the flow paths, and is instead a method in which a mixed flow of the solution L and the carriers B is produced inside the reaction vessel 9 by the gas supplied to the reaction vessel 9. It is therefore possible to reduce the amount of solution L used, and the chemical synthesis also takes less time.
With the synthesis method disclosed herein, gas is supplied for stirring to the reaction vessel 9 containing the carriers B and the solution L. Therefore, a flow of the solution L, the carriers B, and the gas is produced in the reaction vessel 9, and the volume of the contents of the reaction vessel 9 is greater than that before the supply of gas.
In view of this, in the supply step S2 (see
Consequently, the carriers B and the required amount of solution L are held in the lower part of the reaction vessel 9, and the space Q2 is formed in the upper part of the reaction vessel 9. Therefore, in the subsequent synthesis step S3, when gas is supplied to the reaction vessel 9 (see
If the reaction vessel 9 were filled with the carriers B and the solution L, then when gas was supplied to the reaction vessel 9 from below for the purpose of stirring, the solution L would end up flowing out of the pipe 23 connected to the upper part of the reaction vessel 9. However, as described above, the solution L can be prevented from flowing out of the pipe 23 by setting the volume of the reaction vessel 9 such that the upper surface 31 of the reaction vessel 9 is higher than the upper surface of the layer of the carriers B and the solution L.
Also, in the case of the synthesis method disclosed herein, in the supply step S2, the solution L measured by the measuring mechanism 15, that is, a solution L that is outside of the reaction vessel 9, is pushed by gas through the intermediate pipe 8 connected to the reaction vessel 9, and conveyed to the reaction vessel 9. Then, when the conveyance of the solution L to the reaction vessel 9 by the gas is complete, in the synthesis step S3, the gas that was used to convey the solution L is supplied directly to the reaction vessel 9, and the carriers B and the solution L are stirred by this gas. The conveyance of the solution L by gas to the reaction vessel 9 and its stirring by the gas are performed by the gas supply means 20. As discussed above, the gas that conveys the solution L to the reaction vessel 9 is used to stir the carriers B and the solution L. If a mechanism for conveying the solution L to the reaction vessel 9 by gas is provided, there will be no need to separately provide a gas supply mechanism for stirring, and the configuration of the synthesis device 1 can be simplified.
With the synthesis device 1, the gas used to pressure-feed the solution L from the storage container 2 to the measuring mechanism 15, the gas used to pressure-feed the solution L from the measuring mechanism 15 to the reaction vessel 9, and the gas used to stir the carriers B and the solution L in the reaction vessel 9 are all supplied from the same tank 4. However, although not depicted in the drawings, the configuration may instead be such that these gases are supplied from different tanks (gas sources). For example, a gas used for pressure-feeding and a gas used for stirring may be supplied from different tanks (gas sources). Also, with the above-mentioned synthesis device 1, the flow path for supplying the solution L to the reaction vessel 9 and the flow path for supplying the gas for stirring are the same (both use the intermediate pipe 8), but the flow path for supplying the solution L to the reaction vessel 9 and the flow path for supplying the gas for stirring to the reaction vessel 9 may be different. Also, the reaction vessel 9 may have a shape other than what is shown in the drawings.
In the above disclosure, in the reaction vessel 9, a gas is supplied from the lower part of the reaction vessel 9 for stirring. However, although not depicted in the drawings, an internal pipe may be provided so as to extend from the upper part of the reaction vessel 9 into the layer of the carriers B and the solution L, and gas may be supplied through this internal pipe into the layer for stirring. Here again, chemical synthesis can be performed while stirring the solution L and the carriers B.
The embodiments disclosed herein are merely examples in all respects, and are not intended to be restrictive. The technical scope of the present invention is not limited to the above embodiments, and the technical scope encompasses all modifications within a scope equivalent to the configuration described in the claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2019-228421 | Dec 2019 | JP | national |
This application is a U.S. National stage of International Application No. PCT/JP2020/034738 filed on Sep. 14, 2020. This application claims priority to Japanese Patent Application No. 2019-228421 filed on Dec. 18, 2019 with Japan Patent Office. The entire disclosure of Japanese Patent Application No. 2019-228421 is hereby incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2020/034738 | 9/14/2020 | WO |
