Peptide production device and peptide production method

Abstract

[Problem] To enable, even when the capacity of a reaction vessel is increased, implementation of multiple steps pertaining to solid state synthesis of a peptide using the same reaction vessel. [Solution] A peptide production device 1 is configured to comprise: a reaction vessel 3 used to elongate a peptide chain binding to a carrier; a stirring device 11 having one or more stirring blades 15, 16 for stirring contents of the reaction vessel 3; a filtering device 25 that allows foreign substances generated during peptide chain elongation to pass so that the foreign substances can be discharged from the reaction vessel 3 and that traps the carrier; a cleaning solvent introduction pipe P3 that is composed of a plurality of pipes connected to the reaction vessel 3 and that introduces, into the reaction vessel 3, a cleaning solvent for cleaning foreign substances; a drying substituent solvent introduction pipe P3 for introducing, into the reaction vessel 3, a drying substituent solvent for drying the carrier to which an elongated peptide chain has bound; an exhaust pipe P5a for vacuum exhausting gas inside the reaction vessel 3; and an inert gas introduction pipe P2 for introducing an inert gas into the reaction vessel 3.

Description

Peptide production apparatus and peptide production method

[0001] The present invention relates to a peptide production apparatus and a peptide production method using solid phase synthesis.

[0002] Various peptides have been developed for use in pharmaceuticals, foods, etc. Known peptide synthesis methods include solid-phase synthesis, liquid-phase synthesis, and tag synthesis. Solid-phase synthesis has the advantage that while a peptide chain bound to a solid support is extended, other low-molecular-weight contaminants (e.g., remaining substrates and by-products) can be easily removed by washing.

[0003] With regard to solid-phase synthesis, for example, a cleavage treatment device is known that includes a drain port, a reaction vessel having a filter that separates the reaction chamber from the drain port and that can support a solid-phase support to which a peptide is bound, a plug member for sealing the drain port, and a pressurizing device for pressurizing the inside of the reaction vessel (see Patent Document 1).

[0004] Japanese Patent Application Publication No. 5-239089

[0005] Conventional processing equipment such as that described in Patent Document 1 is designed to use a reaction vessel with a relatively small capacity (e.g., about several liters) used in research, etc. Therefore, it is difficult to directly apply the configuration of that reaction vessel to an equipment with a relatively large capacity (e.g., about 50 liters to 1000 liters) used in peptide production.

[0006] Furthermore, it is desirable that the apparatus used for solid-phase peptide synthesis be capable of performing multiple steps (e.g., elongation of a peptide chain bound to a support, drying of the support to which the elongated peptide is bound, and cleavage of the elongated peptide from the support) using the same reaction vessel. This eliminates the need to prepare separate apparatus for each step and reduces the workload of the operator when transitioning between steps.

[0007] In view of the above background, an object of the present invention is to provide a peptide production apparatus and a peptide production method that are capable of performing multiple steps related to solid-phase peptide synthesis using the same reaction vessel, even when the capacity of the reaction vessel is increased.

[0008] In order to solve the above-mentioned problems, one aspect of the present invention is a peptide production apparatus comprising: a reaction vessel used for elongating a peptide chain bound to a support; an agitator having one or more agitating blades for agitating the contents of the reaction vessel; a filtration device that allows impurities generated during the peptide chain elongation to pass through the reaction vessel so that they can be discharged from the reaction vessel and captures the support; and a plurality of pipes connected to the reaction vessel, wherein the plurality of pipes include a deprotecting agent solution inlet pipe for introducing into the reaction vessel a deprotecting agent solution for removing a protecting group at the N-terminus of the elongated peptide chain; an amino acid inlet pipe for introducing into the reaction vessel a deprotecting agent solution for removing a protecting group at the N-terminus of the elongated peptide chain; a washing solvent inlet pipe for introducing into the reaction vessel a washing solvent for washing the impurities; a drying replacement solvent inlet pipe for introducing into the reaction vessel a drying replacement solvent for drying the support to which the elongated peptide chain is bound; an exhaust pipe for evacuating the gas in the reaction vessel; and an inert gas inlet pipe for introducing an inert gas into the reaction vessel.

[0009] According to this embodiment, even if the capacity of the reaction vessel is increased, it is possible to carry out multiple steps involved in solid-phase peptide synthesis (including elongation of the peptide chain bound to the support and drying of the support to which the elongated peptide is bound) using the same reaction vessel.

[0010] In the above aspect, the filtration device may be disposed below the one or more stirring blades in the reaction vessel, and the reaction vessel may have a dry carrier outlet through which the carrier, to which the elongated peptide chain has been bound and which has been dried and captured by the filtration device, is discharged by rotation of any of the one or more stirring blades.

[0011] According to this embodiment, the support to which the elongated peptide chain is bound can be easily removed from the reaction vessel after drying using a stirring blade. For example, in a conventional large reaction vessel, the part where the filtration device is attached must be opened in order to remove the dried support, which is a cumbersome procedure. However, this embodiment reduces the burden on the operator of such removal work.

[0012] In the above aspect, the plurality of pipes may further include a cleavage solution inlet pipe for introducing a cleavage solution that cleaves the elongated peptide chain from the support into the reaction vessel, and a peptide outlet pipe for discharging a liquid containing the elongated peptide chain that has been cleaved from the support in the reaction vessel.

[0013] According to this embodiment, more steps involved in solid-phase peptide synthesis (including extension of the peptide chain bound to the support, drying of the support to which the extended peptide is bound, discharge of the dried support, and cleavage and recovery of the extended peptide from the support) can be performed using the same reaction vessel.

[0014] In the above aspect, the plurality of pipes may each be provided with a valve, and may further include a controller that controls the operation of the corresponding valve to sequentially carry out the following: introduction of the deprotecting agent solution, introduction of the amino acid solution, introduction of the drying replacement solvent from the drying replacement solvent inlet pipe to the reaction vessel, evacuation of the gas in the reaction vessel via the exhaust pipe, introduction of the inert gas from the inert gas inlet pipe to the reaction vessel, and introduction of the cleaving solution from the cleaving solution inlet pipe to the reaction vessel.

[0015] According to this embodiment, multiple steps related to solid phase synthesis of a peptide and cleavage of the peptide from the support after synthesis can be easily carried out using the same reaction vessel.

[0016] In the above aspect, the one or more stirring blades may include a first stirring blade arranged at a position corresponding to the dry carrier outlet in the height direction of the reaction vessel, for discharging the carrier to which the elongated peptide chains are bound that has been captured by the filtration device from the dry carrier outlet, and a second stirring blade arranged above and spaced from the first stirring blade.

[0017] According to this aspect, the first and second stirring blades can appropriately discharge the carrier to which the elongated peptide chain is bound from the reaction vessel and stir the contents of the reaction vessel. Note that the stirring device may be provided with additional stirring blades in addition to the first and second stirring blades (i.e., three or more stirring blades).

[0018] In the above aspect, the stirring device may further include an elevator device that changes the vertical position of the one or more stirring blades within the reaction vessel.

[0019] According to this aspect, the contents can be stably stirred regardless of the amount of the contents in the reaction vessel (i.e., the level of the liquid surface).

[0020] In the above aspect, at least one of the stirring blades may include a heat transfer medium passage through which a heat transfer medium for adjusting the temperature of the contents in the reaction vessel flows.

[0021] According to this embodiment, it is possible to effectively suppress temperature changes of the contents due to exothermic reactions in the reaction vessel (e.g., reactions of cleaving elongated peptides from the carrier), etc. For example, the reaction vessel may be provided with a jacket for adjusting the temperature. This allows heat exchange between the jacket and the stirring blades, so that temperature changes of the contents can be suppressed more effectively.

[0022] In the above aspect, the stirring device further includes a rotatable main shaft, a drive unit for rotating the main shaft, and an auxiliary shaft that can be detachably attached to the main shaft, and at least one of the stirring blades is preferably fixed to the auxiliary shaft.

[0023] According to this aspect, multiple auxiliary shafts, each equipped with agitator blades having different shapes, are prepared, and an appropriate one is selected from these auxiliary shafts and connected to the main shaft, thereby easily achieving the required agitation performance.

[0024] In the above aspect, the apparatus may further include a connecting member that connects the main shaft and the auxiliary shaft, and the auxiliary shaft may be detachably attached to the main shaft via the connecting member.

[0025] This embodiment increases the degree of freedom in connecting the main shaft and the auxiliary shaft. Furthermore, by using a connecting member, it is possible to avoid complicating the structures of the main shaft and the auxiliary shaft (for example, both shafts can be configured with the same diameter).

[0026] In the above aspect, the peptide production method may include an elongation step of elongating a peptide chain bound to a support in a reaction vessel; a drying step of drying the support to which the elongated peptide chain bound, elongated in the elongation step, is bound in the reaction vessel; a discharging step of discharging the support to which the elongated peptide chain bound and dried in the drying step is bound from the reaction vessel; a cleavage solution introducing step of introducing a cleavage solution that cleaves the elongated peptide chain into the reaction vessel after the discharging step; and a carrier introducing step of introducing the support to which the elongated peptide chain bound, which has been discharged from the reaction vessel in the discharging step, into the reaction vessel after the introducing step.

[0027] According to this embodiment, even if the capacity of the reaction vessel is increased, it is possible to carry out multiple steps involved in solid-phase peptide synthesis (including elongation of the peptide chain bound to the support, drying of the support to which the elongated peptide is bound, and cleavage of the elongated peptide from the support) using the same reaction vessel.

[0028] In the above aspect, the drying step may include a drying replacement solvent introducing step of introducing a drying replacement solvent into the reaction vessel to dry the support to which the elongated peptide chain is bound, and an inert gas aeration step of bubbling an inert gas into the reaction vessel after the drying replacement solvent introducing step to dry the support.

[0029] According to this aspect, the support to which the elongated peptide chain is bound can be appropriately dried in the reaction vessel in which the elongation step has been carried out.

[0030] In the above aspect, the carrier introduction step may include a step of stirring the contents of the reaction vessel using a stirring device having one or more stirring blades including a heat transfer medium passage through which a heat transfer medium for regulating the temperature of the contents of the reaction vessel flows. For example, the reaction vessel may be provided with a jacket for regulating the temperature.

[0031] According to this embodiment, it is possible to effectively suppress a rise in the temperature of the contents due to the exothermic reaction (the reaction of cleaving the elongated peptide from the carrier) in the adding step.

[0032] In the above aspect, the method may include a step of stirring the contents of the reaction vessel using a stirring device, the stirring device further including a rotatable main shaft, a drive unit for rotating the main shaft, and an auxiliary shaft detachably attached to the main shaft, and a plurality of stirring blade assemblies are prepared, each of which has a stirring blade having a different shape attached to a different auxiliary shaft, and one stirring blade assembly is selected from the plurality of stirring blade assemblies. The stirring blade assembly may be selected, for example, based on the stirring performance required for the contents of the reaction vessel (also referred to as required stirring performance; for example, adjustment of the amount, temperature, type of carrier, and diffusion distribution of the contents, or a combination thereof).

[0033] According to this aspect, by selecting an appropriate stirring blade assembly, it is possible to appropriately stir the contents of the reaction vessel.

[0034] According to the above aspects, it is possible to provide a peptide production apparatus and a peptide production method that can perform multiple steps related to solid-phase peptide synthesis using the same reaction vessel, even if the capacity of the reaction vessel is increased.

[0035] 4A and 4B are diagrams showing an example of the structure of the connecting portion of the first shaft 45 and the second shaft 46; FIG. 4B is a diagram showing another embodiment of the connecting portion shown in FIG. 4; FIG. 4B is a diagram showing another embodiment of the connecting portion shown in FIG. 4; FIG. 4B is a diagram showing another embodiment of the connecting portion shown in FIG. 4; FIG. 4B is a diagram showing another embodiment of the connecting portion shown in FIG. 4; FIG. 4B is a diagram showing another embodiment of the connecting structure of the first shaft 45 and the second shaft 46; FIG. 4B is a diagram showing another embodiment of the connecting portion shown in FIG. 1;

[0036] Hereinafter, a peptide production apparatus and a peptide production method according to an embodiment will be described with reference to the drawings.

[0037] First, the configuration of a peptide production apparatus 1 will be described with reference to FIG.

[0038] The peptide production apparatus 1 is an apparatus for synthesizing a desired peptide based on a peptide production method and for performing a cleavage process (separation of the synthesized peptide from the support) after peptide synthesis. The peptide production apparatus 1 includes a reaction vessel 3 used for an elongation reaction of a peptide chain bound to the support. The peptide chain can be elongated, for example, by a known method in which the amino protecting group of an initial molecule bound to the support is repeatedly removed and coupled so that the peptide chain is gradually extended from the C-terminus to the N-terminus. The support can be, for example, a resin support formed by crosslinking a resin such as polystyrene into particles.

[0039] The reaction vessel 3 has a substantially cylindrical vessel body 5 with a closed top, and a vessel bottom 7 detachably attached to the lower end of the vessel body 5 so as to close the lower opening of the vessel body 5. In the reaction vessel 3, a sealed internal space is formed by the vessel body 5 and the vessel bottom 7. Note that the structure of the reaction vessel 3 is not limited to that shown in FIG. 1 . For example, an automatic attachment / detachment device for attaching and detaching the vessel bottom 7 to and from the vessel body 5 may be attached to the vessel bottom 7.

[0040] A temperature-regulating jacket 9 is provided on the outer wall of the vessel body 5 to regulate the temperature of the contents (e.g., solutions used in solid-phase peptide synthesis and cleavage treatment, and solutions for cleaving elongated (synthesized) peptides from a support) contained within the vessel body 5. In this embodiment, the jacket 9 has a substantially cylindrical shape and is disposed so as to surround the vessel body 5. The jacket 9 is provided with an inlet pipe P11 for introducing a heat transfer medium (e.g., a refrigerant) into a passage (not shown) formed within the body, and an outlet pipe P12 for discharging the heat transfer medium from the passage within the body. The inlet pipe P11 and the outlet pipe P12 are provided with valves V11 and V12, respectively. The introduction temperature of the heat transfer medium can be changed as appropriate depending on the process being performed in the reaction vessel 3. The temperature-regulating jacket 9 is not limited to the vessel body 5, and may be provided on the vessel bottom 7.

[0041] The peptide production apparatus 1 also includes an agitator 11 for agitating the contents of the reaction vessel 3. The agitator 11 has a rotating shaft 13 and first and second agitator blades 15 and 16.

[0042] The rotating shaft 13 extends substantially vertically, and its lower portion is inserted into the reaction vessel 3. Agitating blades 15 and 16 are attached to the lower portion of the rotating shaft 13. In addition, an electric motor 19 (an example of a driving device) is attached to the upper portion of the rotating shaft 13 located outside the reaction vessel 3 as a device for driving the rotating shaft 13 to rotate.

[0043] In the agitator 11, an elevator device 21 for driving the rotary shaft 13 to move up and down is attached below the electric motor 19 above the rotary shaft 13. The elevator device 21 is fixed to a support device (not shown). The elevator device 21 supports the rotating rotary shaft 13 and can move the rotary shaft 13 up and down relative to the reaction vessel 3. A portion of the rotary shaft 13 is configured to be extendable within the elevator device 21. This changes the vertical positions of the first and second agitator blades 15, 16 within the reaction vessel 3. The elevator device 21 can be omitted as appropriate. In the agitator 11, in order to prevent foreign matter from entering the rotary shaft 13 during its elevation movement, an extendable bellows may be installed to surround the rotary shaft 13 between the agitator blade 16 and the elevator device 21.

[0044] The first agitating blade 15 is attached to the lower end of the rotating shaft 13. In addition to stirring the contents of the reaction vessel 3, the first agitating blade 15 is mainly used to discharge the carrier (the dry carrier described later) to which the elongated peptide chain described later is bound. The first agitating blade 15 has a shape suitable for discharging the contents (the dry carrier described later) from the vessel body 5 through an outlet (the dry carrier outlet 31 described later). The second agitating blade 16 is disposed above the first agitating blade 15 at a predetermined distance. The second agitating blade 16 is mainly used to stir the contents of the reaction vessel 3. The blade portion of the first agitating blade 15 and the second agitating blade 16 have different shapes. However, the agitating device 11 may have a single agitating blade (for example, only the first agitating blade 15). The number of blade portions (number of blades) of the first and second agitating blades 15, 16 can be changed as appropriate.

[0045] A heat medium passage 23 through which a heat medium (e.g., a refrigerant) flows is formed inside the first agitating impeller 15 (see FIG. 2). Meanwhile, the rotating shaft 13 is provided with an inlet pipe P21 for introducing the heat medium into the rotating shaft 13 and an outlet pipe P22 for discharging the heat medium from the rotating shaft 13. Valves V21 and V22 are provided on the inlet pipe P21 and the outlet pipe P22, respectively. One end of the heat medium passage 23 of the first agitating impeller 15 is connected to the inlet pipe P21 through the rotating shaft 13. The other end of the heat medium passage 23 is connected to the outlet pipe P22 through the rotating shaft 13.

[0046] 2 shows only a portion of the blade portion of the first agitating impeller 15, the heat transfer medium passages 23 can be similarly provided in other blade portions extending radially from the rotating shaft 13. The second agitating impeller 16 can also be provided with a heat transfer medium passage in the same manner as the first agitating impeller 15.

[0047] The peptide production apparatus 1 includes a filter 25 (an example of a filtration device) attached to the top of the vessel bottom 7 of the reaction vessel 3, and a filter support 26 for supporting the filter 25. The filter 25 is generally disk-shaped. The filter 25 passes a solution containing relatively low-molecular-weight impurities generated during the peptide chain elongation reaction from the vessel body 5 to the vessel bottom 7 so that it can be discharged from the reaction vessel 3. Meanwhile, the filter 25 captures the carrier (including the peptide chain bound thereto). In other words, the filter 25 does not allow the carrier to pass through, but instead retains it in the vessel body 5. The filter 25 can be formed using a polymer material such as Teflon (registered trademark) or polyethylene. The shape and material of the filter 25 are not particularly limited as long as they can perform the required functions. The volume of the space between the lower part of the filter support 26 and the lower wall of the vessel bottom 7 should be set as small as possible.

[0048] The peptide production apparatus 1 also has a plurality of pipes P1-P6 connected to the reaction vessel 3.

[0049] One end of the pipe P1 is connected to the upper wall of the vessel body 5. The pipe P1 is used to exhaust gas from the reaction vessel 3. The pipe P1 is provided with a valve V1 that is opened when the reaction vessel 3 is evacuated. A part of the exhaust pipe P1 functions as a nitrogen introduction pipe P2 (an example of an inert gas introduction pipe). The other end of the pipe P1 may be connected to a vacuum device including a vacuum pump or the like.

[0050] The downstream end of a pipe P2 is connected to the pipe P1 on the upstream side of the valve V1 (more specifically, to the upstream side in the flow of gas discharged from the reaction vessel 3). The nitrogen introduction pipe P2 is used to introduce nitrogen (an example of an inert gas) into the reaction vessel 3 through a part of the exhaust pipe P1. The other end of the pipe P2 is connected to a nitrogen tank or the like. The pipe P2 is provided with a valve V2 that is opened when nitrogen is introduced into the reaction vessel 3. The downstream end of the pipe P2 may be connected directly to the reaction vessel 3 without being connected to the pipe P1.

[0051] One end of pipe P3 (an example of a deprotecting agent dissolving solution introduction pipe) is connected to the upper wall of the vessel body 5. Pipe P3 is used to introduce a carrier swelling solvent into the reaction vessel 3. Pipe P3 is provided with valve V3, which is opened when the carrier swelling solvent is introduced into the reaction vessel 3. The carrier swelling solvent is not particularly limited, but for example, N,N-dimethylformamide (DMF) can be used.

[0052] The pipe P3 is used to introduce a deprotecting agent solution into the reaction vessel 3. The deprotecting agent solution is used for deprotection (i.e., removal of the N-terminal protecting group) in solid-phase peptide synthesis. Examples of the N-terminal protecting group that can be used include a Boc group (tert-butoxycarbonyl group) and an Fmoc group (9-fluorenylmethyloxycarbonyl group). The deprotecting agent solution can be, for example, a DMF (N,N-dimethylformamide) solution in which piperidine is dissolved.

[0053] Furthermore, pipe P3 (an example of a washing solvent introduction pipe) is used to introduce a carrier washing solvent (an example of a washing solvent) such as DMF to wash away impurities produced during the peptide chain elongation reaction.

[0054] The pipe P3 (an example of a drying replacement solvent introduction pipe) is used to introduce into the reaction vessel 3 methanol (an example of a drying replacement solvent) for replacement used when drying the support.

[0055] Furthermore, a pipe P3 (an example of a cleavage solution introduction pipe) is used to introduce a cleavage cocktail solution (an example of a cleavage solution) into the reaction vessel 3. The cleavage cocktail solution is not particularly limited, but for example, when an Fmoc group is used as the N-terminal protecting group, a TFA (Trifluoroacetic Acid) cocktail can be used.

[0056] The other end of pipe P3 is selectively connected to a plurality of tanks that individually store the solvents, etc., flowing through pipe P3 (the above-mentioned carrier swelling solvent, deprotection agent solution, carrier washing solvent, methanol, and cleavage cocktail solution) depending on the type of solvent or the like. Valve V3 is opened when any of the carrier swelling solvent, deprotection agent solution, carrier washing solvent, methanol, and cleavage cocktail solution is flowed through pipe P3. However, pipes and valves for introducing the carrier swelling solvent, deprotection agent solution, carrier washing solvent, methanol, and cleavage cocktail solution into reaction vessel 3 may be provided separately.

[0057] One end of pipe P4 (an example of an amino acid introduction pipe) is connected to the upper wall of the vessel body 5. Pipe P4 is used to introduce an amino acid solution activated for coupling into the reaction vessel 3. Pipe P4 is provided with a valve V4 that is opened when introducing an amino acid into the reaction vessel 3. The other end of pipe P4 is selectively connected to a plurality of tanks that individually store the amino acid solutions to be passed through pipe P4, depending on the type of amino acid solution. In order to avoid contamination by different amino acid solutions in pipe P4, it is necessary to wash away any remaining liquid in the pipe with DMF or the like after introducing the amino acid solution.

[0058] One end of the pipe P5 is connected to the lower wall of the vessel bottom 7. The pipe P5 is used to discharge gas or liquid from the reaction vessel 3. The pipe P5 is provided with a valve V5 that is opened when discharging gas or liquid from the reaction vessel 3.

[0059] The upstream ends of three branch pipes P5a-P5c are connected to the downstream end of the pipe P5. The branch pipe P5a (an example of an exhaust pipe) is used to evacuate the reaction vessel 3 through the pipe P5 during the support drying process. The branch pipe P5a is provided with a valve V5a that is opened when evacuating the reaction vessel 3. The downstream end of the branch pipe P5a is connected to a vacuum device (not shown) including a vacuum pump or the like. The branch pipe P5b is used to drain liquid containing impurities (e.g., unnecessary substances such as used solvent and by-products remaining in the reaction vessel 3) from the reaction vessel 3 through the pipe P5. The branch pipe P5b is provided with a valve V5b that is opened when discharging liquid containing impurities from the reaction vessel 3. The liquid discharge operation from the branch pipe P5b is preferably performed by pressurizing and feeding the liquid using nitrogen gas from the pipe P2. Branch pipe P5c is used to discharge a liquid containing an elongated peptide chain (hereinafter referred to as crude peptide) that has been cleaved from the carrier in reaction vessel 3 through pipe P5. Branch pipe P5c is provided with valve V5c that is opened when discharging a liquid containing crude peptide from reaction vessel 3. The end of branch pipe P5c is connected to a peptide recovery tank (not shown) that stores the recovered crude peptide, and the recovered crude peptide is pressurized and delivered by pressurizing nitrogen gas from pipe P2.

[0060] One end of the pipe P6 penetrates the upper wall of the vessel body 5 and extends into the reaction vessel 3. The pipe P6 is used to introduce a vessel washing solvent for washing the inside of the reaction vessel 3. A spray nozzle 27 is attached to one end of the pipe P6 located inside the reaction vessel 3. The pipe P6 is provided with a valve V6 that is opened when the vessel washing solvent is introduced into the reaction vessel 3.

[0061] In the reaction vessel 3, a dry carrier outlet 31 is formed on the peripheral wall at the bottom of the vessel body 5 for discharging a dried carrier to which an elongated peptide chain is bound (hereinafter referred to as a dry carrier). The lower edge of the dry carrier outlet 31 is located at a height that is roughly the same as the upper surface of the filtration filter 25. The first agitating blade 15 is also located at a position corresponding to the dry carrier outlet 31. In particular, since the first agitating blade 15 contributes to the discharge of the dry carrier from the dry carrier outlet 31, it is preferable that the lower edge of the first agitating blade 15 is located close to the upper surface of the filtration filter 25.

[0062] A drying carrier recovery device 33 is attached to the reaction vessel 3 so as to cover the outside of the drying carrier discharge port 31. The recovery device 33 has a storage case 35 that stores the drying carrier discharged from the drying carrier discharge port 31. The storage case 35 is provided with a shutter (not shown) that can close the drying carrier discharge port 31. A discharge pipe 36 is connected to the bottom of the storage case 35, and the drying carrier in the storage case 35 is discharged to a recovery container (not shown here) through the discharge pipe 36. The discharge pipe 36 may be provided with a valve (e.g., a butterfly valve) that is opened when the drying carrier is recovered. The recovery device 33 is preferably structured so that the drying carrier remaining in the reaction vessel 3 can be manually scraped out.

[0063] The peptide production apparatus 1 also includes a thermometer 37 for measuring the temperature inside the reaction vessel 3. A sensor portion 37A of the thermometer 37 is disposed in a position where the temperature of the contents inside the reaction vessel 3 can be measured in the crude peptide cleavage step (see FIG. 16 ), which will be described later. The reaction vessel 3 has a hand hole 39 in the upper wall of the vessel body 5, which communicates with the internal space of the reaction vessel 3. The hand hole 39 is used to introduce a carrier into the reaction vessel 3 in the carrier swelling step, which will be described later, and to introduce a dried carrier into the reaction vessel 3 in the crude peptide cleavage step. A lid (not shown) that can be opened and closed is attached to the hand hole 39. An operator can also sample the contents inside the reaction vessel 3 through the hand hole 39.

[0064] The peptide production apparatus 1 is also provided with a controller 41. The controller 41 is electrically connected to at least some of the valves V1-V6, V5a-V5c, V11, V12, and V21, V22 provided as automatic valves, and can control the opening and closing of these valves. However, at least some of the valves V1-V6, V5a-V5c, V11, V12, and V21, V22 may be opened and closed by an operator.

[0065] The number, types, and arrangement of the pipes and tubes connected to the reaction vessel 3 are not particularly limited as long as they perform the functions required for solid-phase peptide synthesis and cleavage treatment. The same applies to the number, types, and arrangement of the valves installed in those pipes and tubes.

[0066] Next, modified examples of the stirring device 11 will be described with reference to FIGS.

[0067] In the agitator 11, the rotating shaft 13 may include a first shaft 45 (an example of a main shaft) and a second shaft 46 (an example of an auxiliary shaft) detachably connected to the lower part of the first shaft 45, as shown in Figures 3(A) and 3(B). An electric motor 19 (see Figure 1) is attached to the upper part of the first shaft 45. Agitating blades (here, first and second agitating blades 15, 16) are attached to the second shaft 46. Note that the respective ends of the heat transfer medium passages 23 on the first shaft 45 and the second shaft 46 are configured to be connectable to each other when the first shaft 45 and the second shaft 46 are connected.

[0068] The upper portion of the second shaft 46 is cylindrical with an inner diameter slightly larger than the outer diameter of the first shaft 45 so that the lower portion of the first shaft 45 can fit therein. As shown in FIG. 3A , the lower portion of the first shaft 45 is provided with a plurality of locking protrusions 49 that are retractable from the outer circumferential surface and spaced at predetermined intervals in the circumferential direction. For example, each locking protrusion 49 may be biased radially outward by a spring (not shown). The second shaft 46 also has a plurality of locking holes 51 formed at positions corresponding to the locking protrusions 49 of the first shaft 45. When the second shaft 46 is connected to the first shaft 45, as shown in FIG. 3B , each locking protrusion 49 is fitted into the corresponding locking hole 51. However, the configuration for connecting the first shaft 45 and the second shaft 46 is not limited to the configuration shown in FIGS. 3A and 3B .

[0069] In the agitator 11 configured as described above, for example, a plurality of agitator impeller assemblies are prepared, each of which has a different agitator impeller (or blade portion) attached to a different second shaft 46, and an appropriate one can be selected from the plurality of agitator impeller assemblies (i.e., agitator impellers having different configurations (sizes, shapes, etc.)) and attached to the first shaft 45 depending on the amount of contents (e.g., the level of the liquid surface) in the reaction vessel 3. Furthermore, the selection of such an agitator impeller assembly may also be based on the type of carrier. This allows for more appropriate agitation of the contents in the reaction vessel 3.

[0070] For example, it is possible to use a computer simulation or the like to obtain the liquid level and the type of carrier provided in the reaction vessel 3, and select an appropriate agitator blade assembly based on these detected values ​​(i.e., the amount of content and the type of carrier in the reaction vessel 3). In this case, it is advisable to prepare in advance an information table that associates the amount of content and the type of carrier in the reaction vessel 3 with an appropriate agitator blade assembly. By referring to the information table, it is possible to select one or more agitator blade assemblies based on the amount of content and the type of carrier in the reaction vessel 3.

[0071] As agitator blades having different configurations, for example, blades with different numbers of agitator blades, attachment positions relative to the second shaft 46 (including changes in the length of the second shaft 46), and blade shapes (including sizes) can be prepared.

[0072] The connection portion of the first shaft 45 and the second shaft 46 as shown in FIG. 3 can be configured, for example, as shown in FIGS. 4(A) to 4(C).

[0073] In the example shown in FIG. 4A , two grooves 50A, 50B are formed at the bottom of the first shaft 45, spaced apart in the vertical direction. The grooves 50A, 50B are formed between multiple enlarged diameter portions of the first shaft 45. O-rings 52A, 52B serving as sealing members are housed in the grooves 50A, 50B, respectively. The second shaft 46 is also provided with multiple locking holes 51 corresponding to the multiple locking protrusions 49. O-rings 152C are fitted onto the outer peripheries of the locking protrusions 49 inside the second shaft 46. The O-rings 52A, 52B, 152C prevent liquids and the like from entering through the end (here, the upper end) of the second shaft 46 or the multiple locking holes 51. 2, even when the heat medium passage 23 is provided in the rotary shaft 13, the O-rings 52A, 52B, and 152C prevent the heat medium from leaking out of the rotary shaft 13.

[0074] In the example shown in FIG. 4(B), a seal structure similar to that shown in FIG. 4(A) is employed, but the first shaft 45 has a plurality of locking projections 49A, 49B arranged in two rows spaced apart vertically. The second shaft 46 has a plurality of locking holes 51A corresponding to the upper locking projections 49A and a plurality of locking holes 51B corresponding to the lower locking projections 49B. O-rings 152C fit around the outer peripheries of the locking projections 49A on the inside of the second shaft 46. O-rings 152D fit around the outer peripheries of the locking projections 49B on the inside of the second shaft 46. The O-rings 52A, 52B, 152C, and 152D prevent liquids and other contaminants from entering through the end of the second shaft 46 and the locking holes 51A and 51B. 2, even when the heat medium passage 23 is provided in the rotating shaft 13, the O-rings 52A, 52B, 152C, and 152D prevent the heat medium from leaking out of the rotating shaft 13. In this manner, the rotating shaft 13 may have a plurality of locking protrusions arranged in two rows (or three or more rows).

[0075] In the example shown in FIG. 4(C), the lower portion of the first shaft 45 is cylindrical with an inner diameter slightly larger than the outer diameter of the second shaft 46 so that the upper portion of the second shaft 46 can fit therein. A groove 50A is formed in the second shaft 46 at a location corresponding to the lower end of the first shaft 45. The groove 50A is formed by reducing the diameter of a portion of the second shaft 46. An O-ring 52A serving as a sealing member is accommodated in the groove 50A. In FIG. 4(C), the multiple locking projections 49A, 49B have a configuration generally similar to that shown in FIG. 4(B), but are provided on the second shaft 46. A multiple locking hole 51A, 51B is provided on the first shaft 45. An O-ring 152B fits around the outer periphery of each locking projection 49A inside the first shaft 45. The O-rings 152C are fitted onto the outer peripheries of the respective locking projections 49B on the inside of the first shaft 45, respectively.

[0076] The diameter, number, and arrangement of the O-rings used on the rotating shaft 13 can be changed as appropriate.

[0077] As another embodiment of the connection portion between the first shaft 45 and the second shaft 46 shown in Fig. 4, for example, the structures shown in Fig. 5(A) to (C) are conceivable. Regarding the configurations shown in Fig. 5(A) to (C), matters not specifically mentioned below are the same as those in Fig. 4(A) to (C).

[0078] In the example shown in FIG. 5A, three grooves 50A-50C are formed at intervals in the vertical direction on the lower part of the first shaft 45. The grooves 50A-50C are each formed between multiple enlarged diameter portions of the first shaft 45. Multiple locking protrusions 49 protrude from the middle (groove bottom) of the groove 50C. O-rings 52C and 52D serving as sealing members are housed in the grooves 50C. The O-ring 52C is attached to the outer periphery of the first shaft 45 above each locking protrusion 49. The O-ring 52D is attached to the outer periphery of the first shaft 45 below each locking protrusion 49. A flange portion 60 serving as an enlarged diameter portion is provided below the O-ring 52D. This prevents the O-ring 52D from coming off the first shaft 45. The O-rings 52C and 52D may be configured to fit onto the outer peripheries of the respective locking projections 49 inside the first shaft 45, like the O-ring 152C in FIG. 4A.

[0079] In the example shown in FIG. 5B, three grooves 50A-50C are formed at intervals in the vertical direction on the lower part of the first shaft 45. The grooves 50A-50C are each formed between multiple enlarged diameter portions of the first shaft 45. Multiple locking projections 49A, 49B protrude from the middle (groove bottom) of each groove 50C. O-rings 52C-52F serving as sealing members are housed in the grooves 50C. The O-ring 52C is attached to the outer periphery of the first shaft 45 above each locking projection 49A. The O-ring 52D is attached to the outer periphery of the first shaft 45 below each locking projection 49B. The O-ring 52E is attached to the outer periphery of the first shaft 45 below the O-ring 52D and above each locking projection 49B. Furthermore, O-ring 52F is attached to the outer periphery of first shaft 45 below each locking projection 49B. A flange portion 60 as an enlarged diameter portion is provided on the underside of O-ring 52F. This prevents O-ring 52F from coming off first shaft 45. Note that O-rings 52C, 52D, 52E, and 52F may be configured to fit onto the outer peripheries of each locking projection 49A and 49B inside first shaft 45, like O-ring 152C in FIG. 4A.

[0080] In the example shown in FIG. 5C, an O-ring 52A is housed in the recessed groove 50A. O-rings 52B-52E are attached to the outer periphery of the second shaft 46. The O-rings 52B and 52C are respectively disposed above and below the locking projections 49A. The O-ring 52D is disposed below the O-ring 52C and above the locking projections 49B. The O-ring 52E is disposed above the O-ring 52A and below the locking projections 49B. The O-rings 52B, 52C, 52D, and 52E may be configured to fit onto the outer peripheries of the locking projections 49A and 49B inside the first shaft 45, as in the case of the O-ring 152C in FIG. 4A. 5A and 5B, flanges 60 are provided to reliably prevent O-rings 52D and 52F from coming off first shaft 45. However, depending on the situation, flanges 60 may be omitted. In this case, for example, it may be possible to lengthen the tips of the portions with the locking projections. In other words, it may be possible to ensure a sufficient distance from the respective positions of O-rings 52D and 52F to the end (here, the bottom end) of first shaft 45.

[0081] As another embodiment of the connection portion between the first shaft 45 and the second shaft 46 shown in Fig. 4, for example, the structures shown in Fig. 6(A) to (C) can be considered. Regarding the configurations shown in Fig. 6(A) to (C), matters not specifically mentioned below are the same as those in Fig. 4(A) to (C).

[0082] In the example shown in FIG. 6A, the O-ring 152C (see FIG. 4A) fitted onto the outer periphery of each locking projection 49 is omitted. Meanwhile, four grooves 50A-50D are formed at intervals in the vertical direction in the lower part of the first shaft 45. The grooves 50A-50D are each formed between a plurality of enlarged diameter portions in the first shaft 45. The plurality of locking projections 49 protrude from the middle (groove bottom) of the groove 50C. An O-ring 52C serving as a sealing member is housed in the groove 50D. A flange portion 60 serving as an enlarged diameter portion is provided below the O-ring 52C. This prevents the O-ring 52C from coming off the first shaft 45.

[0083] In the example shown in FIG. 6B, the O-rings 152C, 152D (see FIG. 4B) that fit around the outer peripheries of the locking projections 49A, 49B, respectively, are omitted. Meanwhile, four grooves 50A-50D are formed at intervals in the vertical direction in the lower part of the first shaft 45. The grooves 50A-50D are each formed between multiple enlarged diameter portions of the first shaft 45. The multiple locking projections 49A, 49B protrude from the middle (groove bottom) of the groove 50C. An O-ring 52C serving as a sealing member is housed in the groove 50D. A flange portion 60 serving as an enlarged diameter portion is provided below the O-ring 52C. This prevents the O-ring 52C from coming off the first shaft 45.

[0084] In the example shown in Figure 6(C), an O-ring 52A is housed in the recessed groove 50A. In this example, the O-rings 152C and 152D (see Figure 4(C)) that fit around the outer peripheries of the locking protrusions 49A and 49B, respectively, are omitted. Meanwhile, a recessed groove 50B is further formed in the upper part of the second shaft 46. The recessed groove 50B is formed by reducing the diameter of a portion of the second shaft 46. An O-ring 52B serving as a sealing member is housed in the recessed groove 50B. This prevents the O-ring 52B from coming off the second shaft 46.

[0085] Furthermore, by using a mechanism such as that shown in Figures 6(A)-(C), it is possible to prevent liquid in the main shaft or auxiliary shaft (i.e., liquid that may enter the rotating shaft 13 through the engagement holes 51, 51A, 51B) from mixing with the heat transfer medium.

[0086] As another embodiment of the connection portion between the first shaft 45 and the second shaft 46 shown in Fig. 4, for example, the structures shown in Fig. 7(A) to (C) are conceivable. Regarding the configurations shown in Fig. 7(A) to (C), matters not specifically mentioned below are the same as those in Fig. 4(A) to (C).

[0087] In the example shown in FIG. 7A, the O-ring 152C (see FIG. 4A) fitted onto the outer periphery of each locking projection 49 is omitted. Meanwhile, four grooves 50A-50D are formed at intervals in the vertical direction on the lower part of the first shaft 45. The grooves 50A-50D are each formed between multiple enlarged diameter portions of the first shaft 45. The multiple locking projections 49 protrude from the middle (groove bottom) of the groove 50B. Furthermore, O-rings 52B and 52C are housed in the grooves 50C and 50D, respectively. The O-rings 52B and 52C are attached to the outer periphery of the first shaft 45. A flange portion 60 is provided below the O-ring 52C as an enlarged diameter portion. This prevents the O-ring 52C from coming off the first shaft 45.

[0088] In the example shown in FIG. 7(B), O-rings 152C, 152D (see FIG. 4(B)) fitted onto the outer peripheries of the locking protrusions 49A, 49B, respectively, are omitted. Meanwhile, four grooves 50A-50D are formed at intervals in the vertical direction on the lower part of the first shaft 45. The locking protrusions 49A, 49B protrude from the middle (groove bottom) of the groove 50B. Furthermore, O-rings 52B, 52C are housed in the grooves 50C, 50D, respectively. The O-rings 52B, 52C are attached to the outer periphery of the first shaft 45. A flange portion 60 is provided on the lower side of the O-ring 52C as an enlarged diameter portion. This prevents the O-ring 52C from coming off the first shaft 45.

[0089] In the example shown in FIG. 7C , O-rings 152C and 152D (see FIG. 4C ) that fit around the outer peripheries of the locking protrusions 49A and 49B, respectively, are omitted. The upper portion of the second shaft 46 is cylindrical with an inner diameter slightly larger than the outer diameter of the first shaft 45 so that the lower portion of the first shaft 45 can fit therein. Two grooves 50A and 50B are formed in the lower portion of the first shaft 45, spaced apart in the vertical direction. The grooves 50A and 50B are each formed by reducing the diameter of a portion of the first shaft 45. The groove 50A is located in a portion of the first shaft 45 that corresponds to the upper end of the second shaft 46. The multiple locking protrusions 49A and 49B protrude from the non-reduced portions between the grooves 50A and 50B. O-rings 52A and 52B are housed in the grooves 50A and 50B, respectively. The O-rings 52A and 52B are attached to the outer periphery of the first shaft 45, respectively.

[0090] Furthermore, by using a mechanism such as that shown in Figures 7(A)-(C), it is possible to prevent liquid in the main shaft or auxiliary shaft (i.e., liquid that may enter the rotating shaft 13 through the engagement holes 51, 51A, 51B) from mixing with the heat transfer medium.

[0091] 8A, the first shaft 45 and the second shaft 46 may be indirectly connected via a connecting member 53. The connecting member 53 is generally cylindrical and has coaxial mounting holes 54A and 54B into which the first shaft 45 and the second shaft 46 can be fitted, respectively. The connecting member 53 is provided with grooves 57A-57C that respectively accommodate O-rings 56A-56C arranged on the first shaft 45 side. The connecting member 53 is also provided with grooves 59A-59C that respectively accommodate O-rings 58A-58C arranged on the second shaft 46 side. In the connecting member 53, the uppermost O-ring 56A and the lowermost O-ring 58A have smaller diameters than the other O-rings 56B, 56C, 58B, and 58C.

[0092] Furthermore, in connecting member 53, grooves 62A and 62B are provided on the inside (here, the lower side) of O-ring 56C to accommodate lock rings 61A and 61B arranged on the first shaft 45 side. Furthermore, grooves 64A and 64B are provided on the inside (here, the upper side) of O-ring 58C to accommodate lock rings 63A and 63B arranged on the second shaft 46 side.

[0093] Although not shown in detail, each of the lock rings 61A, 61B, 63A, and 63B includes an annular base and multiple protrusions that protrude diagonally inward from the base. The multiple protrusions resiliently engage with the outer circumferential surface of the first shaft 45 or the second shaft 46. As a result, the multiple protrusions, together with the locking protrusions 49A and 49B, prevent the first shaft 45 or the second shaft 46 from disengaging from the mounting holes 54A and 54B. The lock rings 61A, 61B, 63A, and 63B also prevent rattling of the first shaft 45 and the second shaft 46 within the connecting member 53. The lock rings 61A, 61B, 63A, and 63B may be omitted.

[0094] 8(B) , the ends of the heat medium passages 23 in the first shaft 45 and the second shaft 46 may be configured to be connectable to each other when the first shaft 45 and the second shaft 46 are connected. In this case, the ends of the first shaft 45 and the second shaft 46 are in close contact with each other.

[0095] In this way, it is possible to appropriately change the connection structure of the first shaft 45 and the second shaft 46. The first shaft 45 and the second shaft 46 are not limited to those having a circular or cylindrical cross section, and those having a rectangular or polygonal cross section can also be used.

[0096] As another modification, the agitator 11 may be provided with more agitating blades (here, three agitating blades 15-17), as shown in FIG. 9(A). That is, the agitator 11 may be provided with additional agitating blades in addition to the first and second agitating blades 15 and 16 (i.e., three or more agitating blades). Furthermore, as shown in FIG. 9(B), the outer diameter of the agitating blade 15 may be changed (here, changed to a sawtooth shape). The rotating shaft 13 shown in FIGS. 9(A) and (B) may also be configured to have a first shaft 45 and a second shaft 46, as shown in FIGS. 3(A) and (B). While FIGS. 9(A) and (B) show a modification of the agitating blade, the present invention is not limited thereto, and other modifications of the agitating blades may be selected depending on the purpose and application of the agitation.

[0097] Next, the lifting and lowering operation of the agitator 11 will be described with reference to FIG.

[0098] The amount of the contents (i.e., the height of the liquid surface) in the reaction vessel 3 may vary depending on the required amount of peptide to be synthesized and each process to be performed. Therefore, in the stirring device 11, the lifting device 21 can be used to change the vertical positions of the first and second stirring blades 15, 16 in the reaction vessel 3, thereby enabling more appropriate stirring.

[0099] For example, as shown in Figure 10(A), when the amount of contents is normal (i.e., the liquid level is at the reference position), the rotating shaft 13 is in the initial position. On the other hand, as shown in Figure 10(B), when the amount of contents is greater than normal (i.e., the liquid level is higher than the reference position), the rotating shaft 13 can be raised by the lifting device 21. This also raises the first and second stirring blades 15, 16, making it possible to properly stir the contents in the reaction vessel 3. In this case, when the dried carrier is discharged (carrier drying and carrier discharging process) described below, the rotating shaft 13 can be lowered by the lifting device 21, so that the rotating shaft 13 can be placed in the lowered position again.

[0100] Such a peptide production apparatus 1 is suitable for use in a reaction vessel with a relatively large capacity (e.g., approximately 50 liters to 1000 liters). With the above-described configuration, even when the capacity of the reaction vessel 3 is increased, the peptide production apparatus 1 can perform multiple steps involved in solid-phase peptide synthesis (including at least elongation of the peptide chain bound to the support and drying of the support to which the elongated peptide is bound) using the same reaction vessel 3. In particular, the reaction vessel 3 includes a pipe P3 for introducing a cleavage solution, and further includes pipes P5 and P5c for discharging a solution containing the crude peptide after cleavage, thereby realizing so-called one-pot operation (i.e., the same reaction vessel 3 can be used to perform elongation of the peptide chain bound to the support, drying of the support to which the elongated peptide is bound, and cleavage and recovery of the elongated peptide from the support).

[0101] Next, a peptide production method executed using the peptide production apparatus 1 will be described with reference to FIG. 11 . Furthermore, the main steps in FIG. 11 will be described in detail with reference to FIGS. 12 to 18 . The contents of the reaction vessel 3 are not shown in FIGS. 12 to 18 . In each step described below, valves V1-V6, V5a-V5c, V11, V12, V21, V22, the dried carrier outlet 31, the hand hole 39, and the like in the reaction vessel 3 are assumed to be closed unless otherwise specified. Furthermore, although the activation of the agitator 11 (including the rotation of the agitator blades 15 and 16) will not be described in detail below, the agitator 11 is in an activated state during the reaction and during carrier washing.

[0102] When synthesizing a peptide in the peptide production apparatus 1, first, the filtration filter 25 is attached to the reaction vessel 3 (ST101). The filtration filter 25 can be installed or replaced by removing the vessel bottom 7 from the vessel body 5. When removing the vessel bottom 7, it is possible to install or replace a stirring blade depending on the intended use, for example, based on a prepared information table.

[0103] Next, a carrier swelling step (ST102) is performed. In this step, carriers are introduced into the reaction vessel 3 through the hand hole 39 while evacuating the air with the valve V1 of the pipe P1 open (see FIG. 12(A)). Subsequently, the introduction of nitrogen into the reaction vessel 3 by opening the valve V2 of the pipe P2 and the evacuation of the reaction vessel 3 by opening the valve V1 of the pipe P1 are alternately and repeatedly performed (see FIG. 12(B)).

[0104] When replacement of the gas in the reaction vessel 3 with nitrogen is thereby completed, the valve V3 of the pipe P3 is opened while the valve V1 of the pipe P1 is kept open, thereby introducing the carrier swelling solvent into the reaction vessel 3 (see FIG. 12(C)). After leaving it for a predetermined time, when swelling of the carrier is completed, the valve V2 of the pipe P2 is opened to introduce nitrogen into the reaction vessel 3, while the valve V5 of the pipe P5 and the valve V5b of the branch pipe P5b are opened to discharge the carrier swelling solvent from the reaction vessel 3 (see FIG. 12(D)).

[0105] After the carrier swelling step (ST102) is completed, the deprotection and washing step (ST103) is performed. In this step, while evacuating the reaction vessel 3 with the valve V1 of the pipe P1 open, the valve V3 of the pipe P3 is opened to introduce the deprotection agent solution into the reaction vessel 3 (see FIG. 13(A)). After the deprotection is completed, the valve V2 of the pipe P2 is opened to introduce nitrogen into the reaction vessel 3, while the valve V5 of the pipe P5 and the valve V5b of the branch pipe P5b are opened to discharge the deprotection agent solution and the protecting groups and the like released during the deprotection from the reaction vessel 3 (see FIG. 13(B)).

[0106] After the discharge of the deprotection agent solution is completed, the valve V1 of the pipe P1 is left open to evacuate the air, while the valve V3 of the pipe P3 is opened to introduce the carrier washing solvent into the reaction vessel 3 (see FIG. 13(C)). Furthermore, the valve V2 of the pipe P2 is opened to introduce nitrogen into the reaction vessel 3, while the valve V5 of the pipe P5 and the valve V5b of the branch pipe P5b are opened to discharge the carrier washing solvent (including impurities generated during deprotection) from the reaction vessel 3 (see FIG. 13(D)). Such introduction and discharge of the carrier washing solvent (see FIGS. 13(C) and 13(D)) are alternately repeated until the washing of the carrier is completed.

[0107] After the deprotection and washing step (ST103) is completed, the coupling and washing step (ST104) is performed. In this step, while evacuating the air with the valve V1 of the pipe P1 open, the valve V4 of the pipe P4 is opened to introduce the amino acid solution into the reaction vessel 3 (see FIG. 14(A)). After the coupling is completed, the valve V2 of the pipe P2 is opened to introduce nitrogen into the reaction vessel 3, while the valve V5 of the pipe P5 and the valve V5b of the branch pipe P5b are opened to discharge the excess solution from the reaction vessel 3 (see FIG. 14(B)).

[0108] Thereafter, while evacuating with the valve V1 of the pipe P1 open, the valve V3 of the pipe P3 is opened to introduce the carrier washing solvent into the reaction vessel 3 (see FIG. 14(C)). Furthermore, while introducing nitrogen into the reaction vessel 3 by opening the valve V2 of the pipe P2, the valve V5 of the pipe P5 and the valve V5b of the branch pipe P5b are opened to discharge the carrier washing solvent from the reaction vessel 3 (see FIG. 14(D)). Such introduction and discharge of the carrier washing solvent (see FIGS. 14(C) and 14(D)) are alternately repeated until the washing of the carrier is completed.

[0109] The above-mentioned deprotection and washing step (ST103) and coupling and washing step (ST104) constitute a peptide chain elongation step for elongating the peptide chain bound to the carrier. These steps are repeatedly performed until peptide chain elongation is completed (Yes in ST105) (i.e., until an elongated peptide chain is obtained).

[0110] After peptide chain elongation is complete, the carrier drying and carrier discharge step (ST106) is performed. In this carrier drying and carrier discharge step (an example of a drying step and a discharge step), while evacuating the air with valve V1 on pipe P1 open, valve V3 on pipe P3 is opened to introduce substitution solvent methanol into the reaction vessel 3 (an example of a drying substitution solvent introduction step, see FIG. 15(A)). Furthermore, while nitrogen is introduced into the reaction vessel 3 by opening valve V2 on pipe P2, methanol is discharged from the reaction vessel 3 by opening valve V5 on pipe P5 and valve V5b on branch pipe P5b (see FIG. 15(B)). This introduction and discharge of methanol (see FIGS. 15(A) and 15(B)) is alternately repeated until the solvent in the carrier is replaced with methanol.

[0111] After the solvent replacement with methanol is completed, the valve V2 of the pipe P2 is opened to introduce nitrogen into the reaction vessel 3, while the valve V5 of the pipe P5 and the valve V5a of the branch pipe P5a are opened to expose the carrier to nitrogen gas and evacuate the vessel (an example of an inert gas ventilation process or evacuation process; see FIG. 15(C)). At this time, the valve V11 of the inlet pipe P11 and the valve V12 of the outlet pipe P12 of the jacket 9 are opened to allow a heat transfer medium to flow into the jacket 9. This heat transfer medium is used to heat the reaction vessel 3. The temperature of the heat transfer medium introduced from the inlet pipe P11 is preferably set to at least the boiling point of methanol. At this time, the valve V21 of the inlet pipe P21 of the rotating shaft 13 and the valve V22 of the outlet pipe P22 may be opened to allow the heat transfer medium to further flow through the rotating shaft 13 and the first and second stirring blades 15 and 16. The dried carrier remaining in the reaction vessel 3 may be removed as much as possible from the recovery device 33 using a scraping rod or the like.

[0112] As a result, nitrogen flows from the top to the bottom of the reaction vessel 3, drying the carrier and raising the temperature inside the reaction vessel 3, and residual methanol is removed from the reaction vessel 3. When drying of the carrier with nitrogen is completed, the dried carrier discharge port 31 is opened and the dried carrier is discharged from the discharge pipe 36 of the recovery device 33 (see FIG. 15(D)). At this time, the dried carrier deposited on the upper surface of the filtration filter 25 is easily scraped out from the dried carrier discharge port 31 by rotating the first stirring blade 15. The discharged dried carrier is stored in a recovery container 55.

[0113] After the support drying and support discharge step (ST106) is completed, the crude peptide cleavage step (ST107) is performed as a cleavage process. In this crude peptide cleavage step (an example of a cleavage solution introduction step or support introduction step), the cleavage cocktail solution is introduced into the reaction vessel 3 by opening the valve V3 of the pipe P3 while evacuating the air with the valve V1 of the pipe P1 open (see FIG. 16(A)).

[0114] In this step, the valve V11 on the inlet pipe P11 of the jacket 9 and the valve V12 on the outlet pipe P12 are opened, allowing a heat transfer medium to flow through the jacket 9. This heat transfer medium is used to cool the inside of the reaction vessel 3. Furthermore, the valve V21 on the inlet pipe P21 of the rotating shaft 13 and the valve V22 on the outlet pipe P22 are opened, allowing a heat transfer medium to flow through the rotating shaft 13 and the first and second agitating blades 15, 16 (i.e., the heat transfer medium passage 23). This heat transfer medium is also used to cool the inside of the reaction vessel 3. In this way, in the reaction vessel 3, the contents of the reaction vessel 3 are cooled by the heat transfer medium flowing through the jacket 9 as the first cooling device and the heat transfer medium flowing through the rotating shaft 13 and the first and second agitating blades 16 as the second cooling device, thereby suppressing excessive temperature rise due to the exothermic reaction of cleaving the crude peptide. The heat transfer medium flow path through the first cooling device and the heat transfer medium flow path through the second cooling device may be connected to each other. That is, the first cooling device and the second cooling device may use the same heat medium.

[0115] Thereafter, the dried carrier recovered in the aforementioned carrier drying and carrier discharge step (ST106) is introduced through the hand hole 39 (see FIG. 16(B)). At this time, to prevent the temperature inside the reaction vessel 3 from rising excessively, a balance is considered between the amount of heat generated in the reaction vessel 3 and the amount of heat exchange (cooling capacity) with the heat medium flowing through the jacket 9 and the heat medium flowing through the rotating shaft 13 and the first and second agitating blades 15, 16. The operator can adjust the introduction rate (e.g., the amount of dried carrier introduced at one time and the introduction timing) while monitoring the value detected by the thermometer 37. However, an automatic dried carrier introduction device (e.g., a feeder having a known configuration) may be used to introduce the dried carrier through the hand hole 39. In this case, the automatic dried carrier introduction device can acquire the value detected by the thermometer 37 and control the introduction rate of the dried carrier based on the detected value. Through this process, an elongated peptide chain (crude peptide) separated from the carrier is obtained (i.e., crude peptide is produced).

[0116] When the cleavage cocktail solution is introduced into the reaction vessel 3, some dry support may remain in the reaction vessel 3 and not be completely discharged in the support drying and support discharging step (ST106). Even in this case, the amount of remaining dry support is not large, and therefore does not generally have a significant effect on the amount of heat generated in the reaction vessel 3. However, in the crude peptide cleavage step (ST107), the initial rate of introduction of the dry support may be set lower, taking into account the dry support remaining in the reaction vessel 3, and then the rate of introduction may be increased.

[0117] After the crude peptide cleavage step (ST107) is completed, the crude peptide recovery step (ST108) is then performed. In this step, nitrogen is introduced into the reaction vessel 3 by opening the valve V2 of the pipe P2, while the valve V5 of the pipe P5 and the valve V5c of the branch pipe P5c (an example of a peptide discharge pipe) are opened to send the liquid containing the crude peptide in the reaction vessel 3 under nitrogen pressure (see FIG. 17). However, a liquid sending pump may be provided in the branch pipe P5c. The downstream end of the branch pipe P5c is connected to a peptide recovery tank (not shown), and the crude peptide produced in the reaction vessel 3 is stored in the peptide recovery tank. The crude peptide recovered in the peptide recovery tank (including peptides with incomplete sequences and peptides synthesized by side reactions) can be purified to a desired purity.

[0118] After the crude peptide recovery step (ST108) is completed, the carrier discharge step (ST109) is carried out. In this step, the same steps as the carrier drying and carrier discharge step (ST106) described above (FIG. 15(A)-(D)) are carried out. However, in the carrier discharge step (ST109), the carrier (i.e., the carrier after the peptide chain has been cleaved) is discharged from the discharge pipe 36 of the recovery device 33 in the step corresponding to FIG. 15(D).

[0119] After the carrier discharge step (ST109) is completed, the reaction vessel washing step (ST110) is performed. In this step, the valve V1 of the pipe P1 is opened to evacuate the reaction vessel 3, and the valve V6 of the pipe P6 is opened to introduce a vessel washing solvent into the reaction vessel 3 (see FIG. 18). The vessel washing solvent is sprayed onto the inner surface of the reaction vessel 3 via the spray nozzle 27. At this time, the valve V5 of the pipe P5 and the valve V5b of the branch pipe P5b are opened to discharge the vessel washing solvent from the reaction vessel 3.

[0120] Although the description of the embodiments including specific examples has been completed above, the present invention is not limited to the above-described embodiments and modifications, and can be implemented in a wide variety of modifications. Not all of the components of the peptide production apparatus and peptide production method shown in the above-described embodiments (including each example) are necessarily essential, and at least those skilled in the art can select appropriate components without departing from the scope of the present invention.

[0121] DESCRIPTION OF SYMBOLS 1: Peptide production apparatus 3: Reaction vessel 5: Vessel body 7: Vessel bottom 9: Jacket 11: Stirring device 13: Rotating shaft 15: First stirring blade 15-17: Stirring blade 16: Second stirring blade 19: Electric motor 21: Lifting device 23: Heat transfer medium passage 25: Filtration filter 26: Filter support 27: Spray nozzle 31: Drying carrier outlet 33: Recovery device 35: Storage case 36: Discharge pipe 37: Thermometer 37A: Sensor unit 39: Hand hole 41: Controller 45: First shaft 46: Second shaft 49: Locking protrusion 51: Locking hole 53: Connecting member 55: Recovery vessel P1-P6: Piping P11: Inlet pipe P12 : Outlet pipe P21 : Inlet pipe P22 : Outlet pipe P5a-P5c: Branch pipe V1-V6: Valve V11, V12: Valve V21, V22: Valve

Claims

1. A peptide production apparatus comprising: a reaction vessel used for extending the peptide chain bound to the carrier; A stirring device having one or more stirring blades for stirring the contents of the reaction vessel; a filtration device that allows impurities generated during the elongation of the peptide chain to pass through the reaction vessel so as to be discharged therefrom and captures the carrier; a plurality of pipes connected to the reaction vessel; The reaction vessel has a carrier discharge port for discharging the carrier captured by the filtration device. Peptide manufacturing equipment.

2. The filtration device is disposed below the one or more stirring blades in the reaction vessel, 2. The peptide production apparatus according to claim 1, wherein the reaction vessel discharges the carrier captured by the filtration device from the carrier discharge port by rotation of any one of the one or more stirring blades.

3. The plurality of pipes include a deprotection agent solution introducing tube for introducing a deprotection agent solution for removing a protecting group at the N-terminus of the extended peptide chain into the reaction vessel; an amino acid introduction tube for introducing an activated amino acid solution into the reaction vessel; a washing solvent introduction pipe for introducing a washing solvent for washing the impurities into the reaction vessel; a drying replacement solvent introduction pipe for introducing a drying replacement solvent into the reaction vessel for drying the support to which the elongated peptide chain is bound; an exhaust pipe for evacuating the gas in the reaction vessel; an inert gas introduction pipe for introducing an inert gas into the reaction vessel; a cleavage solution introduction pipe for introducing a cleavage solution for cleaving the extended peptide chain from the support into the reaction vessel; a peptide discharge pipe for discharging a liquid containing the extended peptide chain separated from the carrier in the reaction vessel; Including, The reaction vessel discharges the carrier to which the extended peptide chain is bound and which has been captured by the filtration device and dried, from the carrier discharge port by rotation of any one of the one or more stirring blades. The peptide production apparatus according to claim 2.

4. Each of the plurality of pipes is provided with a valve, 4. The peptide production apparatus according to claim 3, further comprising a controller that sequentially controls operations of the corresponding valves to introduce the deprotection agent solution, the amino acid solution, the drying replacement solvent from the drying replacement solvent inlet pipe to the reaction vessel, evacuating the gas in the reaction vessel via the exhaust pipe, introducing the inert gas from the inert gas inlet pipe to the reaction vessel, and introducing the cleavage solution from the cleavage solution inlet pipe to the reaction vessel.

5. The one or more stirring blades are a first stirring blade that is disposed at a position corresponding to the dry carrier outlet in the height direction of the reaction vessel and that discharges the carrier having the elongated peptide chain bound thereto, which has been captured by the filtration device, from the dry carrier outlet; The peptide production apparatus according to claim 2 , further comprising: a second agitating impeller disposed above the first agitating impeller at a distance.

6. 5. The peptide production apparatus according to claim 3, wherein at least one of the stirring blades includes a heat transfer medium passage through which a heat transfer medium for regulating the temperature of the contents in the reaction vessel flows.

7. A method for producing a peptide, comprising the steps of: an extension step in which extension of the peptide chain bound to the support is carried out in a reaction vessel; a drying step of drying the support to which the extended peptide chains are bonded in the elongation step in the reaction vessel; a discharge step of discharging the support to which the extended peptide chain is bound, which has been dried in the drying step, from the reaction vessel; a cleavage solution introduction step of introducing a cleavage solution for cleaving the extended peptide chain into the reaction vessel after the discharge step; a carrier introduction step of introducing into the reaction vessel, after the cleavage solution introduction step, the carrier to which the extended peptide chain is bonded, which has been discharged from the reaction vessel in the discharge step, into the reaction vessel.

8. The drying step includes: a step of introducing a drying replacement solvent into the reaction vessel to dry the support to which the elongated peptide chain is bound; 8. The method for producing a peptide according to claim 7, further comprising, after the step of introducing the drying replacement solvent, an inert gas aeration step of bubbling an inert gas into the reaction vessel to dry the carrier.

9. 9. The method for producing a peptide according to claim 7 or 8, wherein the carrier introduction step includes a step of stirring the contents of the reaction vessel with a stirring device having one or more stirring blades including a heat transfer medium passage through which a heat transfer medium flows to adjust the temperature of the contents in the reaction vessel.

10. agitating the contents of the reaction vessel with a stirring device; The stirring device further includes a rotatable main shaft, a drive device that rotates the main shaft, and an auxiliary shaft that is detachably attached to the main shaft, A plurality of agitating blade assemblies are prepared, each of which has a different shape and is attached to a different auxiliary shaft; The method for producing a peptide according to claim 7 or 8, wherein one stirring blade assembly is selected from the plurality of stirring blade assemblies.