Automated manufacturing method and automated manufacturing system for synthetic products

Abstract

The object of the present invention is to provide a synthetic product manufacturing system that allows operations from the synthesis process to the cleavage process to proceed continuously in the production of a synthetic product. [Solution] The above problem has been solved by a synthetic product manufacturing system comprising a channel for passing a synthesis process liquid for synthesizing a synthetic product on a solid support (synthesis process liquid channel), and a column containing the solid support placed on the synthesis process liquid channel, wherein the system includes a channel for passing an excision liquid for excising the synthetic product synthesized on the solid support from the solid support (excision liquid channel), a container containing the excision liquid, and a pump for delivering the excision liquid, wherein the synthesis process liquid channel and the excision liquid channel have a shared channel section, the column containing the solid support is shared within the channel of the shared channel section, and the container containing the excision liquid is arranged to be connectable to the excision liquid channel.

Description

Technical Field

[0001] The present invention relates to a method and a system for producing compounds such as oligonucleotides and peptides.

Background Art

[0002] In the production of compounds such as oligonucleotides and peptides, in a synthesis column filled with a solid-phase synthesis support, after chemically synthesizing the compound bound to the solid-phase synthesis support, the solid-phase synthesis support is manually taken out from the synthesis column and mixed with a cleavage reagent to cleave the compound from the solid-phase synthesis support, and a cleavage operation has been performed. However, in that method, since the operator's intervention is required for the switching operation from the synthesis step to the cleavage step, the freedom of the production schedule is restricted, the total production time becomes long, and there is a problem that the labor cost for production increases. In order to solve that problem, a technique for continuously advancing the operation from the synthesis step to the cleavage step has been demanded, but such a production method and an automatic production system have not been known so far.

[0003] In Chinese Patent Application Publication No. 115382481 (Patent Document 1) and Chinese Patent Application Publication No. 118045559 (Patent Document 2), it is described that after chemically synthesizing the compound, the synthesis column is manually transferred to an automatic cutting device to perform a cutting operation. International Publication No. 2024 / 138152 (Patent Document 3) describes a co-system of cutting, deprotection, ultrafiltration, and diafiltration. International Publication No. 2020 / 261854 (Patent Document 4) describes a cutting device and a cutting method for cutting out the compound synthesized on the solid-phase support from the solid-phase support. However, none of the documents disclose or suggest a technique for continuously advancing the operation from the synthesis step to the cleavage step.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

[0005] The object of the present invention is to provide a synthetic product manufacturing system that allows operations from the synthesis process to the cleavage process to proceed continuously in the production of a synthetic product. [Means for solving the problem]

[0006] To solve the aforementioned problems, the inventors conducted extensive research and found that by having the synthesis liquid channel and the cleavage liquid channel share a column containing a solid support within the channel, it is possible to create a synthetic product manufacturing system that allows operations from the synthesis process to the cleavage process to proceed continuously. Further investigations led to the completion of the present invention.

[0007] In other words, in one embodiment, the present invention relates to the following. [1] A synthesis process liquid channel for passing a synthesis process liquid for synthesizing a compound on a solid support, and A synthesis manufacturing system comprising a column containing the solid support above the liquid flow path of the synthesis process, A channel for passing an excision liquid to excise the compound synthesized on the solid support from the solid support, A container containing the aforementioned excision liquid, and A liquid pump for transporting the aforementioned excised fluid. Includes, The synthesis process liquid channel and the extraction liquid channel have a shared channel section, and the column containing the solid phase support is shared within the channel of the shared channel section. The container containing the excision liquid is arranged to be connectable to the excision liquid channel. A manufacturing system for the aforementioned compound. [2] The compound manufacturing system according to [1], wherein the container containing the excision liquid is arranged so that gases volatilized from the excision liquid do not mix into the synthesis process liquid flow path before the excision liquid is supplied to the excision liquid flow path.

[0008] [3] The synthetic product manufacturing system according to [1] or [2], wherein the excision liquid circulates through the excision liquid channel. [4] The synthesis process solution comprises an acid or basic reagent, and the excision solution comprises a neutralizing component that can react with the acid or basic reagent, according to any one of [1] to [3]. [5] The synthesis production system according to [4], wherein the neutralizing component is volatile at room temperature and pressure. [6] The synthesis production system according to [4] or [5], characterized in that the boiling point of the neutralizing component is 10 to 90°C.

[0009] [7] The synthetic product manufacturing system according to any one of [1] to [6], wherein the container containing the excised liquid is connected to the excised liquid flow path via a valve so that gas volatile from the excised liquid does not enter the synthesis process liquid flow path. [8] The compound manufacturing system according to [7], wherein the gas concentration of the neutralizing component in the excision liquid channel is 50% or less of the gas concentration of the neutralizing component between the container containing the excision liquid and the valve. [9] The synthesis production system according to either [7] or [8], wherein the gas concentration of the neutralizing component in the excision fluid channel is 0 to 15,000 ppm.

[10] The compound manufacturing system according to any one of [7] to [9], wherein the valve is selected from the group consisting of rotary valves, ball valves, diaphragm valves, butterfly valves, choke valves, gate valves, globe valves, knife valves, needle valves, pinch valves, piston valves, plug valves, solenoid valves, spool valves, gate valves, and solenoid valves.

[0010]

[11] A compound manufacturing system according to any one of [1] to

[10] , comprising a liquid for sealing the volatile gas in a channel between the container containing the excision liquid and the synthesis process liquid channel, so that the gas volatile from the excision liquid does not mix into the synthesis process liquid channel.

[12] The synthesis product manufacturing system according to

[11] , wherein in one or more flow path sections from each end where the synthesis process liquid flow path and the extraction liquid flow path begin or end sharing a flow path to the container containing the extraction liquid, the gas concentration of the neutralizing component in the flow path section between the end and the liquid is 50% or less of the gas concentration of the neutralizing component in the flow path section between the container containing the extraction liquid and the liquid.

[13] The synthesis production system according to

[11] or

[12] , wherein in one or more flow path sections from each end where the synthesis process liquid flow path and the extraction liquid flow path begin or end sharing a flow path to the container containing the extraction liquid, the gas concentration of the neutralizing component in the flow path section between the end and the liquid is 0 to 15,000 ppm.

[14] The synthesis manufacturing system according to any one of

[11] to

[13] , wherein the liquid is present in the extraction liquid channel.

[15] The synthesis product manufacturing system according to any one of

[11] to

[14] , wherein in one or more of the flow path sections from each end where the synthesis process liquid flow path and the excision liquid flow path begin or end sharing a flow path to the container containing the excision liquid, the liquid is present on the flow path section, blocking the flow path section, to a total length of 0.6 mm or more in the direction of the flow path section.

[16] In one or more of the flow path sections from each end where the shared flow path between the synthesis process liquid flow path and the cut-out liquid flow path starts or ends to the cut-out liquid-containing container, the liquid exists in a container connected on the flow path section, the synthesis product manufacturing system according to any one of

[11] to

[15] .

[17] The liquid contains water, methanol, acetonitrile, or a mixture thereof, the synthesis product manufacturing system according to any one of

[11] to

[16] .

[0011]

[18] The synthesis product is any one of a compound containing an oligonucleotide, a compound containing a peptide, or a compound containing both an oligonucleotide and a peptide, the synthesis product manufacturing system according to any one of [l] to

[17] .

[19] The protecting group bonded to the synthesis product is removed by the cut-out liquid, the synthesis product manufacturing system according to any one of [1] to

[18] .

[20] Before supplying the cut-out liquid to the cut-out liquid flow path, injecting gas into the cut-out liquid flow path, the synthesis product manufacturing system according to any one of [1] to

[19] .

[21] The cut-out liquid contains ammonia, and the concentration of ammonia in the liquid in the cut-out liquid flow path after supplying the cut-out liquid to the cut-out liquid flow path is 15% to 30%, the synthesis product manufacturing system according to any one of [1] to

[20] .

[22] A part of the cut-out liquid can be sampled from the cut-out liquid flow path, the synthesis product manufacturing system according to any one of [1] to

[21] .

[0012]

[23] The number of flow paths in the shared flow path section between the synthesis process liquid flow path and the cut-out liquid flow path is the same as or more than the number of flow paths in the non-shared cut-out liquid flow path section excluding the shared flow path section from the cut-out liquid flow path, the synthesis product manufacturing system according to any one of [1] to

[22] .

[24] The number of cut-out liquid flow paths is plural, the synthesis product manufacturing system according to any one of [1] to

[23] .

[25] The number of channels in the shared channel section of the synthesis process liquid flow path and the cut-out liquid flow path is the same as or greater than the number of channels in the non-shared synthesis process liquid flow path section excluding the shared channel section from the synthesis process liquid flow path. The composition manufacturing system according to any one of [1] to

[24] .

[26] The number of the synthesis process liquid flow paths is plural. The composition manufacturing system according to any one of [1] to

[25] .

[27] The number of channels in the shared channel section of the synthesis process liquid flow path and the cut-out liquid flow path is plural, and two channels selected from the channels in the plurality of shared channel sections are independent of each other. The composition manufacturing system according to any one of [1] to

[26] , which is arranged such that the synthesis process liquid can be passed through the channels of one shared channel section and the cut-out liquid can be passed through the channels of the other shared channel section simultaneously.

[0013]

[28] A method for manufacturing a composition on a solid phase carrier using a column enclosing the solid phase carrier, comprising the following steps: Step (1): Synthesizing a composition on a solid phase carrier by a synthesis reaction of a solid phase synthesis method, including passing a synthesis process liquid through a synthesis process liquid flow path. Step (2): After the composition is synthesized on the solid phase carrier, stopping the passage of the synthesis process liquid. Step (3): Cutting out the composition from the solid phase carrier by a cut-out reaction of a solid phase synthesis method, including passing a cut-out liquid through a cut-out liquid flow path. including Here, the synthesis process liquid flow path and the cut-out liquid flow path have a shared channel section that shares a channel, and a column enclosing the solid phase carrier is shared in the channel of the shared channel section. The cut-out liquid is supplied from a cut-out liquid-containing container arranged to be connectable to the cut-out liquid flow path. The method for manufacturing the composition.

[29] The method according to

[28] , wherein the cut-out liquid is supplied from a cut-out liquid-containing container arranged such that gas volatilized from the cut-out liquid does not mix into the synthesis process liquid flow path before the passage of the cut-out liquid.

[0014]

[30] The method according to

[28] or

[29] , wherein the number of flow paths in the shared flow path section between the synthesis process liquid flow path and the extraction liquid flow path is multiple.

[31] The method according to

[30] , wherein two channels selected from the channels of the plurality of shared channel sections are independent of each other, and the synthesis process liquid is passed through the channel of one of the shared channel sections and the extraction liquid is passed through the channel of the other shared channel section at the same time.

[32] Two channels selected from the channels of the plurality of shared channel sections are independent of each other, In the flow path of one of the shared flow path sections, after steps (1) and (2) are completed, In the flow path of one of the shared flow path sections, step (3) is performed, and independently thereafter, in the flow path of the other shared flow path section, step (1) is performed. The method described in

[30] or

[31] , including the method described in

[30] or

[31] .

[33] Two channels selected from the channels of the plurality of shared channel sections are independent of each other, In the flow path of one of the shared flow path sections, after steps (1) and (2) are completed, In the flow path of one of the shared flow path sections, step (3) is performed, and at the same time, in the flow path of the other shared flow path section, step (1) is performed. A method that includes any one of

[30] to

[32] .

[34] A compound manufacturing system according to any one of [1] to

[27] , comprising a compound extraction system according to any one of [A] to [V] below.

[35] A compound manufacturing system according to any one of [1] to

[27] , comprising the configuration described in any one of [i] to [viii] below.

[0015] In one embodiment, the present invention relates to the following. [A] A synthetic compound cleavage system comprising a cleavage fluid channel through which a cleavage fluid circulates, for cleaving a synthetic compound from a solid-phase support by passing a cleavage fluid through a column containing a solid-phase support, The synthetic excision system comprises, in this order, a liquid delivery pump, a column containing a solid support, and a defoaming unit on the excision liquid flow path, and a heating unit downstream of the defoaming unit and upstream of the column containing the solid support. [B] The synthetic excision system according to [A], wherein the defoaming unit defoams from the excised liquid by a method comprising at least one selected from the group consisting of cooling defoaming, static defoaming, filter defoaming, stirring defoaming, ultrasonic defoaming, reduced pressure defoaming, pressurized defoaming, shaking defoaming, and heating defoaming. [C] The synthetic product extraction system according to [A] or [B], wherein the degassing unit has the function of degassing bubbles in the extraction liquid and cooling the gas that was present in the bubbles or the gas released from the bubbles by degassing. [D] The compound extraction system according to any one of [A] to [C], wherein the degassing unit has at least one cooling function selected from the group consisting of natural cooling, cooling using a cooling device, cooling using a heat exchanger, constant temperature bath cooling, oven cooling, jacket cooling, and coil cooling. [E] A compound excision system according to any one of [A] to [D], wherein a degassing unit degasssing the excision liquid by a method including cooling degassing.

[0016] [F] A compound excision system according to any one of [B] to [E], wherein the cooling defoaming method includes at least one cooling method selected from the group consisting of natural cooling, cooling using a cooling device, and cooling using a heat exchanger. [G] A compound excision system according to any one of [A] to [F], wherein the degassing unit degasss the excised liquid by a method including static degassing. [H] The compound extraction system according to any one of [A] to [G], wherein a heating unit is provided downstream of the liquid delivery pump and upstream of the column containing the solid support. [I] The synthetic excision system according to any one of [A] to [H], wherein the degassing unit is equipped with a storage section capable of storing a portion of the excised liquid circulating in the excised liquid channel. [J] The compound extraction system according to [I], wherein the storage section is a container and includes a gas portion in which gas generated by degassing is collected.

[0017] [K] The compound extraction system according to [J], wherein the temperature of the gas portion inside the container is below the boiling point of the extraction solution. [L] The compound extraction system according to [J] or [K], wherein the temperature of the gas portion inside the container is 20-38°C. [M] The compound excision system according to any one of [A] to [L], wherein the percentage of bubbles in the excised liquid in the section downstream of the degassing unit and upstream of the liquid delivery pump is smaller than the percentage of bubbles in the excised liquid in the section downstream of the degassing unit and upstream of the degassing unit. [N] A compound excision system according to any one of [A] to [M], wherein the bubble rate in the excised liquid in the section downstream of the degassing unit and upstream of the liquid delivery pump is 0 to 90% of the bubble rate in the excised liquid in the section downstream of the column and upstream of the degassing unit.

[0018] [O] The compound excision system according to any one of [A] to [N], wherein the percentage of bubbles in the excised liquid in the section downstream of the liquid delivery pump and upstream of the column is smaller than the percentage of bubbles in the excised liquid in the section downstream of the column and upstream of the degassing unit. [P] The compound extraction system according to any one of [A] to [O], wherein the liquid transfer pump is at least one selected from the group consisting of a plunger pump, a diaphragm pump, a bellows pump, a tube pump, a gear pump, a rotary pump, and a screw pump. [Q] A synthetic compound extraction system according to any one of [A] to [P], wherein the extraction solution is a liquid containing ammonia. [R] A synthetic excision system according to any one of [A] to [Q], wherein deprotection is performed by an excision solution.

[0019] [S] A synthetic compound excision system according to any one of [A] to [R], wherein the excision solution is heated to 20-65°C by a heating unit. [T] A synthetic compound excision system according to any one of [A] to [S], wherein the excision solution is heated to 50-65°C by a heating unit. [U] A compound cleavage system according to any one of [A] to [T], wherein the compound cleaved from the solid support is a compound containing an oligonucleotide, a compound containing a peptide, or a compound containing both an oligonucleotide and a peptide. [V] A compound cleavage system according to any one of [A] to [U], wherein the compound cleaved from the solid support is a compound containing an oligonucleotide.

[0020] [W] A method for producing a compound, comprising a cutting step of passing a cutting solution through a column containing a solid support to cut out a compound from the solid support, This includes circulating the excised fluid through the excised fluid channel. The manufacturing method wherein the excision fluid channel comprises, in this order, a liquid delivery pump, a column containing a solid support, and a degassing unit, and a heating unit is provided downstream of the degassing unit and upstream of the column containing the solid support. A compound manufactured by the manufacturing method described in [X] [W]. A method for producing a synthetic product, using a synthetic product cleavage system described in any one of [Y], [A], to [V]. A compound extraction system according to one of [A] to [V], for use in a compound manufacturing system according to one of [Z] [1] to

[27] . [AA] A compound extraction system according to any one of [A] to [V] below, for use in a compound manufacturing system according to any one of [i] to [viii] below.

[0021] Furthermore, in one embodiment, the present invention relates to the following. [i] A synthesis process liquid channel for passing a synthesis process liquid for synthesizing a compound on a solid support, and A synthesis manufacturing system comprising a column containing the solid support above the liquid flow path of the synthesis process, A channel for passing an excision liquid to excise the compound synthesized on the solid support from the solid support, A synthesis process liquid flow path control unit having a function to control the delivery of the synthesis process liquid, and Includes a cut-out fluid flow path control unit that has a function to control the delivery of the cut-out fluid, The synthesis process liquid channel and the extraction liquid channel share the column containing the solid support within the channel. It includes a trigger device that generates a cutting process start command to initiate the cutting process after the synthesis process is completed, The trigger device, Contact fluid detector, A communication fluid channel that sends communication fluid from a supply port controlled by the synthesis process fluid channel control unit to a communication fluid detector, and A signaling device that sends a command to the dispensing fluid flow path control unit to start the dispensing process after the communication fluid detector detects the flow of communication fluid. A synthesis manufacturing system including the above.

[0022] [ii] The compound manufacturing system according to [i], wherein the communication fluid detector is a time-of-flight type flow meter. [iii] The compound manufacturing system according to [i] or [ii], wherein the liquid supply of the connecting liquid is carried out by a double-plunger pump in which the phases of each plunger are synchronized. [iv] The synthesis manufacturing system according to any one of [i] to [iii], wherein the delivery of the communication fluid is pulsating. [v] The synthesis production system according to any one of [i] to [iv], wherein the liquid transfer of the connecting liquid is at a flow rate of 0.1 mL / min to 1 L / min. [vi] The synthesis production system according to any one of [i] to [v], wherein the communication fluid contains air bubbles. [vii] The synthesis manufacturing system according to any one of [i] to [vi], wherein, before the start of the delivery of the communication fluid, the communication fluid channel is pre-filled with the same or a different liquid as the communication fluid. [viii] The synthesis production system according to any one of [i] to [vii], wherein the communication fluid channel is pre-filled with an organic solvent before the start of the communication fluid delivery.

[0023] [ix] A method for producing a compound on a solid support using a column containing a solid support, comprising the following steps: (1) Synthesizing a compound on a solid support by a synthesis reaction of a solid-phase synthesis method, which includes passing a synthesis process liquid through a synthesis process liquid channel. (2) After the compound has been synthesized on the solid support, stop the flow of the synthesis process liquid. (3) Eclecting the product from the solid support by an cleavage reaction of a solid-phase synthesis method, which includes passing the cleavage liquid through the cleavage liquid channel. Includes, Here, the synthesis process liquid channel and the extraction liquid channel share the column containing the solid support within the channel. After the completion of (1) and (2) above, the trigger device sends a command to start the cutting process to the cutting fluid flow path control unit to start (3), Includes, The trigger device, Contact fluid detector, A communication fluid channel that sends communication fluid from a supply port controlled by the synthesis process fluid channel control unit to a communication fluid detector, and A signaling device that sends a command to the dispensing fluid flow path control unit to start the dispensing process after the communication fluid detector detects the flow of communication fluid. A method for producing the aforementioned compound, including the following. [x] A method for producing a compound on a solid support using a column containing a solid support, wherein the compound production system is described in any one of [i] to [viii]. A compound manufacturing system according to any one of [i] to [viii], further comprising the configuration described in any one of [1] to

[27] . A compound manufacturing system according to any one of [i] to [viii], comprising a compound extraction system according to any one of [A] to [V]. [Effects of the Invention]

[0024] According to the present invention, in the production of a synthetic product, operations from the synthesis process to the extraction process can be carried out continuously. As a result, it becomes possible to automate operations from the synthesis process to the extraction process, achieving a reduction in manufacturing time and labor costs through the reduction of manual work.

[0025] According to the present invention, in one embodiment, the operations from the synthesis process to the excision process can be carried out continuously in the production of a compound. As a result, the operations from the synthesis process to the excision process can be automated, and manual work is reduced, thereby shortening the production time and reducing labor costs. The excision process can be started immediately after the completion of the synthesis process, or it can be started after a predetermined amount of time has elapsed since the completion of the synthesis process. The start time of the excision process can also be delayed so that the excision process is completed at a desirable time. By adjusting the start time of the excision process in this way, the work after the excision process can be carried out on an efficient schedule. Furthermore, such adjustment of the start time of the excision process is also advantageous from the viewpoint of ensuring the stability of the compound, as it avoids leaving the compound unnecessarily for a long time after the completion of the excision process. [Brief explanation of the drawing]

[0026] [Figure 1] Figure 1 is a schematic diagram showing the configuration of the synthetic product manufacturing system in Comparative Example 1. [Figure 2] Figure 2 is a schematic diagram showing the configuration of the compound manufacturing systems of Examples 1 and 2. In these systems, a container containing the excision liquid is connected to the excision liquid flow path via an excision liquid valve. [Figure 3] Figure 3 shows the method of connecting the container containing the excised liquid in comparative connection example 1. [Figure 4] Figure 4 shows the connection methods for containers containing the excised fluid in connection examples 1 to 4. [Figure 5] Figure 5 shows the connection methods for containers containing the excised liquid in connection examples 5 to 8. [Figure 6]Figure 6 is a schematic diagram showing an example of the configuration of the synthetic product manufacturing system of the present invention. In this system, a liquid layer is provided at any position in the flow path between the container containing the excision liquid and the flow path switching valve. [Figure 7] Figure 7 is a schematic diagram showing an example of the configuration of the synthetic product manufacturing system of the present invention. In this system, a liquid layer is provided at any position in the flow path between the container containing the extraction liquid and the flow path switching valve. [Figure 8] Figure 8 is a schematic diagram showing an example of the configuration of the synthetic product manufacturing system of the present invention. In this system, a container containing the extraction liquid is connected to the extraction liquid flow path via an extraction liquid valve, and a liquid layer is provided at any position in the flow path between the container containing the extraction liquid, the extraction liquid valve, and the flow path switching valve. [Figure 9] Figure 9 is a schematic diagram showing an example of the configuration of the synthetic product manufacturing system of the present invention. In this system, a container containing the extraction liquid is connected to the extraction liquid flow path via an extraction liquid valve, and a liquid layer is provided at any position in the flow path between the container containing the extraction liquid, the extraction liquid valve, and the flow path switching valve. [Figure 10] Figure 10 is a schematic diagram showing an example of the configuration of the synthetic product manufacturing system of the present invention. In this system, a container containing the extraction liquid is connected to the extraction liquid flow path via an extraction liquid valve, and a liquid layer is provided at any position in the flow path between the container containing the extraction liquid, the extraction liquid valve, and the flow path switching valve.

[0027] [Figure 11] Figure 11 is a schematic diagram showing an example of the configuration of the synthetic product manufacturing system of the present invention in a configuration in which the excision liquid does not circulate through the excision liquid channel. In this system, a container containing the excision liquid is connected to the excision liquid channel via an excision liquid valve. [Figure 12] Figure 12 is a schematic diagram showing an example of the configuration of the synthetic product manufacturing system of the present invention in a configuration in which the excised liquid does not circulate through the excised liquid channel. In this system, a liquid layer is provided at any position in the channel between the container containing the excised liquid and the channel switching valve. [Figure 13]Figure 13 is a schematic diagram showing an example of the configuration of the synthetic product manufacturing system of the present invention in a configuration in which the excision liquid does not circulate through the excision liquid channel. In this system, a container containing the excision liquid is connected to the excision liquid channel via an excision liquid valve, and a liquid layer is provided at any position in the channel between the container containing the excision liquid, the excision liquid valve, and the channel switching valve. [Figure 14] Figure 14 is a schematic diagram showing an example of the configuration of the synthetic product manufacturing system of the present invention in a configuration in which the excision liquid does not circulate through the excision liquid channel. In this system, a container containing the excision liquid is connected to the excision liquid channel via an excision liquid valve, and a liquid layer is provided at any position in the channel between the container containing the excision liquid, the excision liquid valve, and the channel switching valve. [Figure 15] Figure 15 is a schematic diagram showing an example of the configuration of the synthetic product manufacturing system of the present invention in which the excised liquid circulates through an excised liquid channel. In this system, a container containing the excised liquid is connected to the excised liquid channel via an excised liquid valve, and a gas inlet is provided at any position in the excised liquid channel. [Figure 16] Figure 16 is a schematic diagram showing an example of the configuration of the synthetic product manufacturing system of the present invention in a configuration in which the excised liquid does not circulate through the excised liquid channel. In this system, a container containing the excised liquid is connected to the excised liquid channel via an excised liquid valve, and a gas inlet is provided at any position in the excised liquid channel.

[0028] [Figure 17] Figure 17 is a schematic diagram showing the configuration of the synthetic product extraction system in Comparative Example 2. Figure 17(A) is a schematic diagram of the connection state before the extraction liquid is supplied to the circulating extraction liquid channel. Figure 17(B) is a schematic diagram of the state after the extraction liquid has been supplied to the circulating extraction liquid channel and is being circulated. [Figure 18] Figure 18 is a schematic diagram showing the configuration of the synthetic excision system in Example 3. Figure 18(A) is a schematic diagram of the connection state before supplying the excision liquid to the circulating excision liquid channel. Figure 18(B) is a schematic diagram of the state after supplying the excision liquid to the circulating excision liquid channel and circulating the excision liquid. [Figure 19]Figure 19 is a schematic diagram showing the temperature of the gaseous portion inside the degassing unit measured by installing a thermocouple in the degassing unit. Figure 19(A) is a schematic diagram of the connection state before supplying the excised liquid to the circulating excised liquid channel. Figure 19(B) is a schematic diagram of the state after supplying the excised liquid to the circulating excised liquid channel and circulating the excised liquid. [Figure 20] Figure 20 is a schematic diagram of the degassing unit being actively cooled (cooled using a cooling device) using an ice-filled container. Figure 20(A) is a schematic diagram of the connection state before supplying the excised fluid to the circulating excised fluid channel. Figure 20(B) is a schematic diagram of the state after supplying the excised fluid to the circulating excised fluid channel and circulating the excised fluid.

[0029] [Figure 21] Figure 21 is a schematic diagram showing an example of a configuration with a changed placement of the heating unit. Figure 21(A) is a schematic diagram of the connection state before supplying the excised fluid to the circulating excised fluid channel. Figure 21(B) is a schematic diagram of the state after supplying the excised fluid to the circulating excised fluid channel and circulating the excised fluid. [Figure 22] Figure 22 is a schematic diagram showing an example of a configuration with a changed placement of the heating unit. Figure 22(A) is a schematic diagram of the connection state before supplying the excised fluid to the circulating excised fluid channel. Figure 22(B) is a schematic diagram of the state after supplying the excised fluid to the circulating excised fluid channel and circulating the excised fluid. [Figure 23] Figure 23 is a schematic diagram showing an example of a configuration not connected to the synthesis process fluid flow path. Figure 23(A) is a schematic diagram of the connection state before supplying the cutoff fluid to the circulating cutoff fluid flow path. Figure 23(B) is a schematic diagram of the state after supplying the cutoff fluid to the circulating cutoff fluid flow path and circulating the cutoff fluid. [Figure 24] Figure 24 is a schematic diagram showing an example of a configuration (connected to the synthesis process liquid flow path) when using the various degassing units described herein. Figure 24(A) is a schematic diagram of the connection state before supplying the excised liquid to the circulating excised liquid flow path. Figure 24(B) is a schematic diagram of the state after supplying the excised liquid to the circulating excised liquid flow path and circulating the excised liquid. [Figure 25]Figure 25 is a schematic diagram showing an example of a configuration (not connected to the synthesis process liquid flow path) when using the various degassing units described herein. Figure 25(A) is a schematic diagram of the connection state before supplying the excised liquid to the circulating excised liquid flow path. Figure 25(B) is a schematic diagram of the state after supplying the excised liquid to the circulating excised liquid flow path and circulating the excised liquid.

[0030] [Figure 26] Figure 26 is a schematic diagram showing one embodiment of the present invention, in which the communication fluid channel is directly connected to the communication fluid supply port of the synthesis process fluid channel control unit. When a communication fluid detector connected to the communication fluid channel detects the supply of communication fluid, it communicates a signal to the extraction fluid channel control unit (wireless or wired communication), and this signal becomes the command to start the extraction process. [Figure 27] Figure 27 is a schematic diagram showing one embodiment of the present invention, in which the connecting fluid channel is directly connected to the synthesis process fluid / connecting fluid supply port of the synthesis process fluid channel control unit. A channel switching valve 3 is located in the middle of the connecting fluid channel, and the connecting fluid channel shares a portion of the piping with the synthesis process fluid channel (the section from the synthesis process fluid / connecting fluid supply port of the synthesis process fluid channel control unit to the channel switching valve 3). When a connecting fluid detector connected to the connecting fluid channel detects the supply of connecting fluid, it communicates a signal to the extraction fluid channel control unit (wireless or wired communication), and this signal becomes the command to start the extraction process. [Figure 28] Figure 28 shows the results of measuring the flow rate of the connecting liquid using a connecting liquid detector in the synthetic product manufacturing system of the present invention. The dotted line shows the results for Example A, and the dotted arrow indicates the time when the liquid flow was detected. The solid line shows the results for Example B, and the solid arrow indicates the time when the liquid flow was detected. [Figure 29] Figure 29 shows the results of Example C, in which liquid delivery was detected using a UV spectrophotometer in a synthetic product manufacturing system. The solid arrows indicate the time when liquid delivery was detected. [Figure 30] Figure 30 shows the results of Example D, in which liquid flow was detected using an elliptical gear-type microflow meter in a synthetic product manufacturing system. The solid arrows indicate the time when liquid flow was detected. [Figure 31]Figure 31 is a schematic diagram showing one embodiment of the present invention in which the excised liquid circulates through an excised liquid channel. In this embodiment, an excised liquid sampling mechanism is provided in the synthetic product manufacturing system of the present invention. In this system, an excised liquid sampling container is connected to the excised liquid channel via a channel switching valve 4. A liquid transfer pump 2, connected to a container containing an excision reaction stopping solution, is also connected to the excised liquid channel via a channel switching valve 5, and the channel switching valve 5 is provided with a gas inlet.

[0031] [Figure 32] Figure 32 is a schematic diagram showing one embodiment of the present invention, which includes one non-shared synthesis process liquid channel section obtained by removing the shared channel section from the synthesis process liquid channel, three shared channel sections between the synthesis process liquid channel and the extraction liquid channel, and three non-shared extraction liquid channel sections obtained by removing the shared channel sections from the extraction liquid channel. [Figure 33] Figure 33 is a schematic diagram illustrating one aspect of the present invention. This figure shows an example in which, in one aspect of Figure 32, each of the flow path switching valves 1 and 2 is configured as a valve complex formed by combining multiple valves (circles within the area enclosed by the dashed line in the figure represent one valve). [Figure 34] Figure 34 is a schematic diagram showing one embodiment of the present invention, which includes two non-shared synthesis process liquid flow path sections obtained by removing the shared flow path section from the synthesis process liquid flow path, four shared flow path sections between the synthesis process liquid flow path and the extraction liquid flow path, and four non-shared extraction liquid flow path sections obtained by removing the shared flow path section from the extraction liquid flow path. [Modes for carrying out the invention]

[0032] In one aspect, the present invention relates to a system for producing synthetic products. In the present invention, the compound is synthesized on a solid-phase support by a solid-phase synthesis reaction via a linker that can be cleaved by a cutting solution. In the present invention, the synthetic product is one of the following: a compound containing an oligonucleotide, a compound containing a peptide, or a compound containing both an oligonucleotide and a peptide. In the present invention, the solid-phase support is, for example, porous resin beads containing polystyrene, porous resin beads containing polyethylene glycol, or porous beads containing glass, and preferably porous resin beads containing polystyrene. In the present invention, the synthesis step refers to the step of synthesizing a compound on a solid-phase support, and in the synthesis step, the compound is synthesized on the solid-phase support by a solid-phase synthesis reaction, which includes passing the synthesis process liquid through the synthesis process liquid channel. More specifically, starting from a linker on the surface of the solid-phase support, the solid-phase synthesis reaction is repeated on the solid-phase support to synthesize a compound of the desired length. In the synthesis reaction, the reaction conditions such as reaction temperature and reagents are not particularly limited, and the reaction conditions for general solid-phase synthesis reactions can be used. In the present invention, the cleavage step refers to the step of cleaving a synthesized product from a solid-phase support, and in the cleavage step, the product is cleaved from the solid-phase support by a cleavage reaction of the solid-phase synthesis method, which includes passing a cleavage liquid through a cleavage liquid channel. More specifically, the cleavage liquid acts on a cleavable part such as a linker, and the product is cleaved from the solid-phase support. The reaction conditions such as reaction temperature and reagents are not particularly limited, and the reaction conditions for a general cleavage reaction of a solid-phase synthesis method can be used.

[0033] In the synthesis process of the present invention, compounds containing oligonucleotides are synthesized using known solid-phase synthesis methods, such as the so-called phosphoramidite method, for example, (a) A step of removing (deprotecting) a protecting group from a protected nucleoside that is directly or indirectly supported on a solid support and has a protecting group bonded to a hydroxyl group, thiol group, or amino group at the 3' or 5' position (deprotection step), (b) A coupling step in which a nucleoside phosphoramidite is coupled (coupled) to the hydroxyl group, thiol group, or amino group at the 3' or 5' position of a nucleoside directly or indirectly supported on a solid support from which a protecting group has been removed, in the presence of an activator. (c) A step of sulfiding or oxidizing the bond formed in step (b) (sulfiding step or oxidation step), (d) A step of capping the unbound hydroxyl group, thiol group, or amino group at the 3' or 5' position of the nucleoside directly or indirectly supported on a solid support (capping step), It is synthesized by a method that includes [a specific component].

[0034] Examples of synthesis solutions used in the synthesis process of compounds containing oligonucleotides in the present invention include nucleoside phosphoramidite solution, 4,5-dicyanoimidazole (DCI) solution, DCA (dichloroacetic acid) solution in toluene, bis(phenylacetyl) disulfide solution in pyridine, lutidine or pyridine solution in acetonitrile, mixed solution of N-methylimidazole and acetic anhydride, diethylamine solution in acetonitrile, and TBA (tert-butylamine) solution in acetonitrile. In the present invention, the synthesis solution used in the synthesis step of a compound containing an oligonucleotide includes an acidic reagent (such as dichloroacetic acid). In the present invention, the linker that can be cut by the cutting solution is, when the compound is a compound containing an oligonucleotide, for example, a universal linker, a nucleoside linker, or a special linker, and preferably a universal linker. In the present invention, the cleavage solution is not particularly limited as long as it is a commonly used cleavage solution when the synthesized product is a compound containing an oligonucleotide, but preferably, the cleavage solution contains a neutralizing component that can react with the synthesis process solution, which is an acidic reagent. In the present invention, when the compound is a compound containing oligonucleotides, the excision solution is, for example, aqueous ammonia, methylamine solution, aqueous ammonia / methylamine mixed solution, diethylamine solution, or triethylamine / hydrogen trifluoride solution, and the neutralizing components are ammonia, methylamine, diethylamine, triethylamine, or hydrogen trifluoride.

[0035] In the synthesis process of the present invention, the peptide-containing compound is synthesized using a known solid-phase synthesis method, for example, (a) Step of immobilizing initial amino acids on a carrier. (b) Deprotection step, which removes the protecting group at the N-terminus of the previous amino acid before attaching the next amino acid. (c) A condensation step in which the following amino acid is added together with a condensing agent and reacted with the carboxyl group of the previous amino acid to form a peptide bond. It is synthesized by a method that includes [a specific component].

[0036] In the present invention, examples of synthesis solution used in the synthesis process of a compound containing a peptide include a piperidine-DMF (N,N-dimethylformamide) mixed solution, an amino acid solution, an anhydride-pyridine-DMF mixed solution, DMF, dichloromethane, isopropanol, and the like. In the present invention, the synthesis solution used in the synthesis step of a compound containing a peptide contains a basic reagent (such as piperidine). In the present invention, when the synthetic product is a compound containing a peptide, the linker that can be cleaved by the cutting solution is, for example, a one-linker, a linkamide linker, a 2-chlorotrityl linker, a sas-linker, a peptide-amide linker, a 4-hydroxymethylbenzoic acid linker, a Sever linker, or an Elman linker, and preferably a one-linker or a linkamide linker.

[0037] In the present invention, the cleavage solution is not particularly limited as long as it is a commonly used cleavage solution when the synthesized product is a compound containing a peptide, but preferably, the cleavage solution contains a neutralizing component that can react with the basic reagent of the synthesis process solution. In the present invention, when the synthetic product is a compound containing peptides, the excision solution is, for example, a mixed solution of trifluoroacetic acid, a mixed solution of hydrogen fluoride, or a mixed solution of trifluoromethanesulfonic acid, and the neutralizing component is, for example, trifluoroacetic acid, anhydrous hydrogen fluoride, or trifluoromethanesulfonic acid.

[0038] In the present invention, the synthetic product manufacturing system is A channel through which a synthesis process liquid for synthesizing a compound on a solid support is passed (synthesis process liquid channel), A channel (cleavage liquid channel) through which a cleavage liquid is passed for cleaving the synthesized product from the solid support, A container containing the excision fluid, and A liquid pump for transporting the aforementioned excised fluid. Includes.

[0039] In the present invention, the synthetic product manufacturing system includes a column (solid-phase support-encapsulated column) on the synthesis process liquid channel, the synthesis process liquid channel and the extraction liquid channel have a shared channel section, and the column with the solid-phase support-encapsulated column is shared within the channel of the shared channel section. For example, as shown in Figures 2 and 6-16, a flow path switching valve 1 may be connected to the end (on the inlet side of the solid-phase support-encapsulated column) where the synthesis process liquid flow path and the excision liquid flow path begin to share a flow path, and a flow path switching valve 2 may be connected to the end (on the outlet side of the solid-phase support-encapsulated column) where the synthesis process liquid flow path and the excision liquid flow path end to sharing a flow path. In this case, the section [flow path switching valve 1 → solid-phase support-encapsulated column → flow path switching valve 2] is part of the synthesis process liquid flow path and part of the excision liquid flow path.

[0040] For example, operations to continuously carry out operations from the synthesis process to the extraction process include flow path switching operations by controlling flow path switching valves 1 and 2. During the synthesis process, the ports on the synthesis process liquid flow path control side of flow path switching valves 1 and / or 2 (flow path switching valves 1 and 2 if the synthesis process flow path is circulating, or flow path switching valve 1 or 2 if the synthesis process flow path is non-circulating) are opened, the ports on the solid-phase carrier-embedded column side of flow path switching valves 1 and 2 are also opened, and the ports on the piping side leading to the container containing the extraction liquid (the side of the piping leading to the container containing the extraction liquid via the extraction liquid flow path control, extraction liquid valve, and / or liquid layer) of flow path switching valves 1 and / or 2 (flow path switching valves 1 and 2 if the extraction liquid flow path is circulating, or flow path switching valve 1 if it is non-circulating) are closed, allowing the synthesis process liquid to flow through the section [flow path switching valve 1 → solid-phase carrier-embedded column → flow path switching valve 2]. Then, during the excision process after the completion of the synthesis process, the ports on the synthesis process liquid flow control side of flow path switching valves 1 and 2 are closed, and the ports on the solid-phase support-encapsulated column side of flow path switching valves 1 and 2 are opened. The ports on the piping side of flow path switching valves 1 and / or 2 (flow path switching valves 1 and 2 if the excision liquid flow path is circulating, flow path switching valve 1 if the excision liquid flow path is non-circulating) toward the container containing the excision liquid (piping toward the container containing the excision liquid via the excision liquid flow control, excision liquid valve, and / or liquid layer) are kept open. Furthermore, if an excision liquid valve is interposed between flow path switching valves 1 and / or 2 and the container containing the excision liquid, the ports of the excision liquid valve that need to be opened to allow the excision liquid supplied from the container to pass through the excision liquid valve and be sent to flow path switching valves 1 and / or 2 are kept open. This allows the flow path to be switched so that the excision liquid flows through the section [flow path switching valve 1 → solid-phase support-encapsulated column → flow path switching valve 2].

[0041] In the present invention, the synthetic product manufacturing system further includes a synthesis process liquid flow path control unit, which is not limited to the above but includes, for example, a liquid delivery pump for delivering the synthesis process liquid to the synthesis process liquid flow path, a synthesis process liquid switching mechanism for switching the synthesis process liquid to be delivered, and a liquid temperature control device for controlling the temperature of the synthesis process liquid. The synthesis process flow path that delivers the synthesis process liquid from the synthesis process liquid flow path control unit to the solid phase support-encapsulated column may be either a circulating flow path or a non-circulating flow path, but is preferably a circulating flow path. In the present invention, the synthetic product manufacturing system further includes an excision fluid flow path control unit, which is not limited to the above but includes, for example, a liquid temperature control device for controlling the temperature of the excision fluid. A liquid delivery pump for delivering the excision fluid may be provided as part of the excision fluid flow path control unit. In the present invention, the container containing the excision fluid is arranged to be connectable to the excision fluid channel. In the present invention, the container holding the cutting liquid is positioned so that gases volatilized from the cutting liquid do not mix into the synthesis process liquid flow path before the cutting liquid is supplied to the cutting liquid flow path. In a synthetic product manufacturing system, by positioning containers containing the extraction liquid to prevent gases volatilized from the extraction liquid from mixing into the synthesis process liquid flow path, the precipitation of salts in the flow path within the system can be avoided, thereby preventing blockage of the flow path and poor liquid delivery.

[0042] In one embodiment of the present invention, the container holding the excised liquid includes an internal pressure adjustment section. Examples of the internal pressure adjustment section include, but are not limited to, a vent valve, a check valve, and a relief valve. These internal pressure adjustment sections may be provided in the excised liquid flow path. In one embodiment of the present invention, the solid-phase support-encapsulated column includes a column temperature control unit. The column temperature control unit is not limited to these, but examples include a column oven, an electric heater, and a jacket-type temperature control device. In one embodiment of the present invention, the synthetic product manufacturing system includes an excision liquid flow path control unit. The excision liquid flow path control unit is not limited to these, but examples include a liquid temperature control device that controls the temperature of the excision liquid. The temperature of the excision liquid flow path may be controlled by the liquid temperature control device. In one embodiment of the present invention, the excision fluid channel includes a sensor. Examples of sensors, though not limited to these, include thermocouples, thermometers, flow meters, pressure gauges, and spectrophotometers. The thermocouple may be included in the column.

[0043] In the present invention, the excision liquid may or may not circulate through the excision liquid channel. When the excision liquid channel is circulating, the synthesis production system may be configured as shown in Figures 2, 6-10, and 15, for example, and when the excision liquid channel is not circulating, it may be configured as shown in Figures 11-14, and 16, for example. In the present invention, at least one of the synthesis process solutions contains an acid or a basic reagent, and the cutting solution contains a neutralizing component that can react with the acid or basic reagent. In this invention, the neutralizing component is volatile at room temperature and pressure (25°C, 1 atm). In the present invention, the boiling point of the neutralizing component is 10 to 90°C, for example, 10°C to 80°C, 10°C to 70°C, 10°C to 60°C, 10°C to 50°C, 10°C to 40°C, 10°C to 30°C, 10°C to 20°C, 20°C to 80°C, 20°C to 70°C, 20°C to 60°C, 20°C to 50°C, 20°C to 40°C, 20°C to 30°C, 30°C to 80°C, 30°C to 70°C, 30°C to 60°C, 30°C to 50°C, and 30°C to 40°C.

[0044] In one embodiment of the present invention, the container containing the cutting liquid is connected to the cutting liquid channel via a valve so that gases volatilized from the cutting liquid do not mix into the synthesis process liquid channel. The valve connected to the container of excision liquid (excision liquid valve) may be installed at any position between the end (the inlet or outlet side of the solid-phase support-encapsulated column) where the synthesis process liquid flow path and the excision liquid flow path begin or end sharing the flow path, and the container of excision liquid. For example, as shown in Figures 2, 8-10, and 15, it may be installed between the flow path switching valve 2 and the excision liquid flow path control unit, or, as shown in Figures 11, 13, 14, and 16, it may be installed in the order of flow path switching valve 1 ~ excision liquid flow path control unit ~ excision liquid valve ~ container of excision liquid.

[0045] In one embodiment of the present invention, a defoaming unit is provided in the excision liquid flow path. In this case, as shown in Figure 22, the defoaming unit, excision liquid valve, heating unit, liquid delivery pump, and solid phase carrier-encapsulated column may be arranged in that order. In order to efficiently aspirate the cutoff liquid from the container containing the cutoff liquid, it is preferable to place the liquid delivery pump after the cutoff liquid valve. For this reason, it is preferable to arrange the components in the order of degassing unit ~ cutoff liquid valve ~ liquid delivery pump ~ heating unit ~ solid support-encapsulated column, as shown in Figure 18, or in the order of degassing unit ~ heating unit ~ cutoff liquid valve ~ liquid delivery pump ~ solid support-encapsulated column, as shown in Figure 21. Considering the preferred arrangement of the heating unit mentioned above, the optimal arrangement is as shown in Figure 18: degassing unit ~ excision valve ~ liquid transfer pump ~ heating unit ~ solid support-encapsulated column. With this arrangement, the excision liquid can be efficiently aspirated from the container containing the excision liquid, and the heated excision liquid can be efficiently supplied to the solid support-encapsulated column. In one embodiment, the compound extraction system can be used in a compound manufacturing system that allows operations from the synthesis process to the extraction process to proceed continuously. In particular, the compound extraction systems shown in Figures 18-22 and 24 can constitute a part of the compound manufacturing system shown in Figure 2. In one embodiment, the synthetic compound extraction system of the present invention can be used not as a synthetic compound manufacturing system that allows operations from the synthesis process to the extraction process to proceed continuously, but as an independent synthetic compound extraction system. In this case, first, a solid-phase synthesis reaction is carried out in a column using any synthesis apparatus and method to obtain a column containing a synthetic compound bound to a solid-phase synthesis support. Then, this column can be incorporated into the synthetic compound extraction system of the present invention to extract the synthetic compound from the solid-phase synthesis support. That is, the synthetic compound extraction system in Figures 18-22 and 24 can be an independent synthetic compound extraction system that does not include a synthesis process liquid flow path, and is not a part of the synthetic compound manufacturing system in Figure 2. Examples of the configuration in this case are shown in Figures 23 and 25.

[0046] In the present invention, the valve is selected from the group consisting of, but is not limited to, rotary valves, ball valves, diaphragm valves, butterfly valves, choke valves, gate valves, globe valves, knife valves, needle valves, pinch valves, piston valves, plug valves, solenoid valves, spool valves, gate valves, and solenoid valves. In the present invention, a container containing the extraction liquid is connected to the extraction liquid flow path via an extraction liquid valve, and with the extraction liquid container side port of the extraction liquid valve closed, the gas concentration of the neutralizing component in the extraction liquid flow path is 50% or less of the gas concentration of the neutralizing component between the extraction liquid container and the valve, for example, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less. In the present invention, a container containing the extraction solution is connected to the extraction solution flow path via an extraction solution valve, and with the extraction solution container side port of the extraction solution valve closed, the gas concentration of the neutralizing component in the extraction solution flow path is 0 to 15000 ppm, for example, 0 to 14000 ppm, 0 to 13000 ppm, 0 to 12000 ppm, 0 to 11000 ppm, 0 to 10000 ppm, 0 to 9000 ppm. The ppm ranges are: pm, 0-8000 ppm, 0-7000 ppm, 0-6000 ppm, 0-5000 ppm, 0-4000 ppm, 0-3000 ppm, 0-2000 ppm, 0-1000 ppm, 0-900 ppm, 0-800 ppm, 0-700 ppm, 0-600 ppm, 0-500 ppm, 0-400 ppm, 0-300 ppm, 0-200 ppm, and 0-100 ppm.

[0047] In one embodiment of the present invention, a liquid for sealing the volatile gas is included in the flow path between the container containing the cutting liquid and the synthesis process liquid flow path, so that the gas volatile from the cutting liquid in the container containing the cutting liquid does not mix into the synthesis process liquid flow path. In one embodiment of the present invention, the liquid for sealing the volatile gas is present in the excision fluid channel. In another embodiment, the liquid is present in the excision fluid channel in an amount sufficient to block the channel. In one embodiment of the present invention, the liquid for sealing the volatile gas may be provided in a flow path between the end (on the inlet or outlet side of the solid-phase carrier-encapsulated column) where the synthesis process liquid flow path and the cleavage liquid flow path begin or end sharing of the flow path, and the container containing the cleavage liquid, without the synthesis process liquid flow path valve being included in the flow path, for example, in a flow path between the end and the cleavage liquid flow path control unit, or in a flow path between the cleavage liquid flow path control unit and the container containing the cleavage liquid. In one embodiment of the present invention, the liquid for sealing the volatile gas may be provided in the flow path between the end (on the inlet or outlet side of the solid-phase carrier-encapsulated column) where the synthetic process liquid flow path and the cleavage liquid flow path begin or end sharing of the flow path, and the cleavage liquid flow path control unit, or in the flow path between the cleavage liquid flow path control unit and the cleavage liquid valve, provided in the flow path between the end and the cleavage liquid flow path control unit and the cleavage liquid valve, provided in the flow path between the end and the cleavage liquid flow path control unit and the cleavage liquid valve, provided in the flow path between the flow path switching valve 2 and the cleavage liquid valve, provided in the flow path between the cleavage liquid flow path control unit and the cleavage liquid valve, provided in the flow path between the cleavage liquid flow path control unit and the cleavage liquid valve, provided in the flow path between the cleavage liquid flow path control unit and the cleavage liquid valve, provided in the flow path between the end and the cleavage liquid flow path control unit and the cleavage liquid valve, provided in the flow path between the end and the cleavage liquid flow path control unit and the cleavage liquid valve, provided in the synthetic process synthesizer production system includes a cleavage liquid valve in the flow path. In one embodiment of the present invention, the liquid for sealing the volatile gas may be located outside the dispensing fluid flow path, for example, in the flow path between the dispensing fluid valve and the container containing the dispensing fluid, as shown in Figure 10.

[0048] In the present invention, the liquid for sealing the volatile gas includes water, methanol, acetonitrile, or a mixture thereof. In the present invention, in one or more channel sections of the channel from each end (on the inlet or outlet side of the solid-phase support-encapsulated column) where the synthesis process liquid channel and the excision liquid channel begin or end sharing a channel, to the container containing the excision liquid, the gas concentration of the neutralizing component in the channel section between the end and the liquid is 50% or less, for example, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less, of the gas concentration of the neutralizing component between the container containing the excision liquid and the liquid. In the present invention, in one or more channel sections from each end (on the inlet or outlet side of the solid-phase support-encapsulated column) where the synthesis process liquid channel and the excision liquid channel begin or end sharing the channel, to the container containing the excision liquid, the gas concentration of the neutralizing component in the channel section between the end and the liquid is 0 to 15000 ppm, for example, 0 to 14000 ppm, 0 to 13000 ppm, 0 to 12000 ppm, 0 to 11000 ppm. The ranges are 0-10000ppm, 0-9000ppm, 0-8000ppm, 0-7000ppm, 0-6000ppm, 0-5000ppm, 0-4000ppm, 0-3000ppm, 0-2000ppm, 0-1000ppm, 0-900ppm, 0-800ppm, 0-700ppm, 0-600ppm, 0-500ppm, 0-400ppm, 0-300ppm, 0-200ppm, and 0-100ppm.

[0049] In one embodiment of the present invention, the liquid for sealing the volatile gas is present in one or more channel sections from each end (on the inlet or outlet side of the solid-phase support-encapsulated column) where the synthesis process liquid channel and the excision liquid channel begin or end sharing the channels, to the container containing the excision liquid, blocking the channel section, and in a total length of 0.6 mm or more in the direction of the channel section, for example, 0.7 mm or more, 0.8 mm or more, 0.9 mm or more, 1.0 mm or more, 1.1 mm or more, 1.2 mm or more, 1.3 mm or more, 1.4 mm or more, 1.5 mm or more, 1.6 mm or more, 1.7 mm or more, 1.8 mm or more, 1.9 mm or more, 2.0 mm or more, 2.5 mm or more, and 3.0 mm or more.

[0050] In one embodiment of the present invention, the liquid for sealing the volatile gas is present in a container connected to the flow path in one or more of the flow path sections from each end (on the inlet or outlet side of the solid-phase support-encapsulated column) where the synthesis process liquid flow path and the excision liquid flow path begin or end sharing the flow path to the container containing the excision liquid. In one embodiment of the present invention, the method for installing the liquid for sealing the volatile gas at the various liquid installation locations (inside the piping or in the container) described above is not particularly limited, but for example, it can be installed by injecting the liquid from any inlet (valve, etc.) inside or outside the liquid extraction flow path. Alternatively, for example, the liquid can be installed at the liquid installation location by injecting the liquid as a flow path cleaning liquid from any inlet (valve, etc.) inside or outside the liquid extraction flow path to clean the flow path, and then discharging the cleaning liquid from any outlet (valve, etc.) inside or outside the liquid extraction flow path, leaving a portion of the cleaning liquid at the liquid installation location. In one embodiment of the present invention, protecting groups bonded to the compound are removed (deprotected) by a cleavage solution. In the present invention, examples of protecting groups bonded to the synthetic product include benzoyl groups, phenoxyacetyl groups, acetyl groups, isobutyryl groups, and dimethylformamidine groups.

[0051] In one embodiment of the present invention, a gas is injected into the excision fluid channel before the excision fluid is delivered to the excision fluid channel. The gas is not particularly limited as long as it does not substantially affect the excision fluid, but examples include air and nitrogen. In the present invention, the location from which the injected gas is injected is not particularly limited, as long as the injected gas reaches the excised liquid channel. The gas can be injected from any position inside or outside the excised liquid channel, and the gas inlet can be installed at any position inside or outside the excised liquid channel, but preferably the gas inlet can be installed inside the excised liquid channel.

[0052] The compound manufacturing system may be configured as shown in Figure 15, for example, when the excision fluid channel is circulating, or as shown in Figure 16, for example, when the excision fluid channel is not circulating. As shown in Figures 15 and 16, for example, gas may be injected from the channel switching valve 1 or 2 or the excision fluid valve. The injected gas can be discharged, for example, from the waste liquid port in the excision fluid channel. In the present invention, before injecting gas into the excision fluid channel, a washing solution may be injected to remove the synthesis process fluid from the channel shared by the synthesis process fluid channel and the excision fluid channel. By injecting gas into the cutting fluid channel before the cutting fluid is delivered to the cutting fluid channel, dilution of the cutting fluid caused by the synthesis process fluid or washing fluid present in the cutting fluid channel is suppressed. For example, water, acetonitrile, methanol, or a mixture thereof can be used as the washing solution. The cleaning solution may be passed through either the synthesis process fluid channel or the extraction fluid channel. For example, the cleaning solution may be delivered as a type of synthesis process fluid via the synthesis process fluid control unit or via the extraction fluid control unit. In the present invention, when the excision fluid is a liquid containing ammonia, the concentration of ammonia in the liquid within the excision fluid channel when the excision fluid is sent to the excision fluid channel is 15% to 30%, for example, 15% to 25% or 15% to 20%.

[0053] In one aspect, the present invention relates to a method for producing a synthetic product. In the present invention, a method for producing a compound on a solid support using a column containing a solid support comprises the following steps: (1) Synthesis of a compound on a solid support by a synthesis reaction using a solid-phase synthesis method, which includes passing the synthesis process liquid through the synthesis process liquid channel. (2) After the compound has been synthesized on the solid support, stop the flow of the synthesis process liquid. (3) The process of cleaving the synthesized product from the solid support by a cleavage reaction of the solid-phase synthesis method, which includes passing the cleavage liquid through the cleavage liquid channel (cleavage step) Includes, Here, the synthesis process liquid channel and the extraction liquid channel have a shared channel section, and the column containing the solid phase support is shared within the channel of the shared channel section. The cutting liquid is supplied from a container containing the cutting liquid, which is connected to the cutting liquid channel and is positioned so that gases volatilized from the cutting liquid do not mix into the synthesis process liquid channel before the cutting liquid passes through.

[0054] The compound cleavage system equipped with the defoaming unit of the present invention is capable of cleaving compounds bound to a solid-phase synthesis support. For example, it can be used in a compound manufacturing system in which operations from the synthesis process to the cleavage process can be carried out continuously (Figures 2, 18-22, 24). In particular, it can be used in a compound manufacturing system developed by the present inventors. Alternatively, for example, the compound cleavage system of the present invention can be used as an independent compound cleavage system, rather than in the above-mentioned compound manufacturing system in which operations from the synthesis process to the cleavage process can be carried out continuously. In that case, first, a solid-phase synthesis reaction is carried out in a column using any synthesis apparatus and synthesis method to obtain a column containing a compound bound to a solid-phase synthesis support. Then, that column can be incorporated into the compound cleavage system of the present invention to cleave the compound from the solid-phase synthesis support (Figures 23, 25).

[0055] In one embodiment, the present invention relates to a synthetic excision system equipped with an excision fluid channel through which an excision fluid is circulated for excising a synthetic product from a solid-phase support by passing an excision fluid through a column containing a solid-phase support. In one embodiment, the synthetic product extraction system of the present invention comprises, in this order, a liquid delivery pump, a column containing a solid support, and a defoaming unit on the extraction liquid flow path, with a heating unit provided downstream of the defoaming unit and upstream of the column containing the solid support. By installing a degassing unit on the flow path of a circulating excision fluid channel that includes a synthesis column, it is possible to suppress the incorporation of air bubbles into the liquid delivery pump on the flow path, and the heated excision fluid can be passed through the synthesis column on the flow path, allowing the excision reaction of the synthesized product to proceed smoothly. In the present invention, the compound is synthesized on a solid-phase support by a solid-phase synthesis reaction via a linker that can be cleaved by a cutting solution. In the present invention, the synthetic product is a compound containing an oligonucleotide, a compound containing a peptide, or a compound containing both an oligonucleotide and a peptide.

[0056] Examples of liquid transfer pumps that can be used in the compound dispensing system of the present invention include plunger pumps, diaphragm pumps, bellows pumps, tube pumps, gear pumps, rotary pumps, and screw pumps. In the present invention, the solid-phase support is, for example, porous resin beads containing polystyrene, porous resin beads containing polyethylene glycol, or porous beads containing glass, and preferably porous resin beads containing polystyrene. In this invention, the solid phase support is used sealed in a column. The column may be installed in a column oven to provide a temperature control function. The column temperature may be measured by a temperature meter attached to the column oven.

[0057] In the present invention, the shape of the column is not particularly limited, but it is preferably cylindrical. The size of the column is not particularly limited, but in one embodiment, the inner diameter of the internal cross-section of the column (or the major axis if the cross-section is not circular) is 0.5 cm to 100 cm. In this invention, the inner diameter of the pipes used in each flow path is not particularly limited, but in one embodiment, the inner diameter is 1 mm to 200 mm. In this invention, the inner diameter (or major axis) of the column's internal cross-section is larger than the inner diameter of the piping connected to the column. The ratio of the inner diameter (or major axis) of the column's internal cross-section to the inner diameter of the column piping is not particularly limited, but in one embodiment it is 5 to 50. The larger the ratio of the inner diameter (major axis) of the column's internal cross-section to the inner diameter of the column piping, the more likely air bubbles are to be generated when the excised liquid is passed through the column, thus making this invention highly useful.

[0058] In the present invention, the defoaming unit defoams the cut liquid by a method comprising at least one selected from the group consisting of cooling defoaming, static defoaming, filter defoaming, stirring defoaming, ultrasonic defoaming, reduced pressure defoaming, pressurized defoaming, shaking defoaming, and heating defoaming. It is preferable that the defoaming unit defoams the cut liquid by a method including cooling defoaming. When a defoaming unit defoams from the excised liquid using a method that includes cooling defoaming, the method and function of cooling are not particularly limited, as long as the excised liquid can be cooled. In one embodiment, the defoaming unit can cool the excised liquid by at least one of the following cooling functions: natural cooling by heat exchange with a naturally present medium in the surroundings (gas, liquid, solid, etc.); cooling using a cooling device that applies ice or a refrigerant directly or indirectly; cooling using a heat exchanger; constant temperature bath cooling, in which the container or piping is immersed in a constant temperature bath containing cooling water, refrigerant, or beads; oven cooling, in which the container or piping is placed in a temperature-controlled oven room for cooling; jacket cooling, in which cooling water or a refrigerant is circulated through a cooling jacket provided on the outside of the container or piping; and coil cooling, in which cooling water or a refrigerant is circulated through a cooling coil provided inside the container or piping. Cooling may also be performed on the container and / or piping.

[0059] Cooling degassing is, in one embodiment, a degassing method that includes at least one cooling method selected from the group consisting of natural cooling, cooling using a cooling device, and cooling using a heat exchanger. In one embodiment, the degassing unit has the function of degassing air bubbles in the excised liquid and cooling the gas that was present in the air bubbles or the gas released from the air bubbles by degassing. The degassing unit degasssed the excised liquid by a method including static degassing, according to one embodiment. The degassing unit may include a storage section capable of storing a portion of the excised liquid circulating through the excised liquid channel. The storage section may be a container. The material of the container is not particularly limited as long as it is heat-conductive, such as glass, metal, or plastic, but glass is preferred. The shape and size of the container are not particularly limited. Such storage sections and containers are suitable for degassing by cooling degassing and / or static degassing.

[0060] If the storage portion is a container, it may contain a gas portion in which the gas generated by degassing is collected. The temperature of the gas portion in the container is preferably below the boiling point of the excised liquid. Typically, the temperature of the gas portion in the container is 20 to 38°C, preferably 25 to 38°C, and more preferably 30 to 38°C. Such temperature conditions are suitable for degassing by cooling degassing and / or static degassing. The temperature of the gaseous portion inside the degassing unit can be measured, for example, by installing a thermocouple in the degassing unit.

[0061] The defoaming unit has a defoaming means for defoaming air bubbles present in the excised liquid in the circulating excision channel. Defoaming by the defoaming means includes defoaming by cooling, defoaming by standing, defoaming by filtration, defoaming by stirring, defoaming by ultrasound, defoaming by reduced pressure, defoaming by pressurization, defoaming by shaking, and defoaming by heating, with standing defoaming being preferred. Standing defoaming is a method of defoaming in which air bubbles mixed in the liquid rise due to buoyancy, reach the liquid surface, and then burst and disappear.

[0062] The degassing unit may have the function of degassing bubbles in the cutting solution and cooling the gas that was present in the bubbles or the gas released from the bubbles by degassing. This is because such cooling is expected to redissolve the gas that was present in the bubbles or the gas released from the bubbles by degassing into the cutting solution, thereby preventing a decrease in the concentration of the cutting reagent in the cutting solution. Such a cooling function can be at least one selected from the group consisting of natural cooling, cooling using a cooling device, cooling using a heat exchanger, constant temperature bath cooling, oven cooling, jacket cooling, and coil cooling.

[0063] In the present invention, the heating unit is provided to heat the excision liquid so that the excision liquid circulating through the degassing unit is supplied to the column containing the solid support at a temperature suitable for excision. The heating unit is typically located downstream of the degassing unit and upstream of the column containing the solid-phase support. The heating unit may also be located downstream of the degassing unit and upstream of the liquid delivery pump. In particular, as shown in Figure 21, it may be located downstream of the degassing unit and upstream of the cutoff valve that supplies the cutoff liquid, or as shown in Figure 22, it may be located downstream of the cutoff valve that supplies the cutoff liquid and upstream of the liquid delivery pump. From the viewpoint of efficiently supplying the heated cutoff liquid to the column containing the solid-phase support, it is more preferable to position the heating unit downstream of the liquid delivery pump and upstream of the column containing the solid-phase support, as shown in Figure 18.

[0064] The ideal temperature for cutting is typically 50–65°C, preferably 52.5–65°C, and more preferably 55–65°C. To ensure that the excision solution is supplied to the column at a temperature suitable for excision, the excision solution is typically heated by a heating unit to 20-65°C, preferably to 30-65°C, 40-65°C, or 45-65°C, and more preferably to 50-65°C or 55-65°C.

[0065] In the synthetic extracting system of the present invention, bubbles are generated in the extracting liquid when it passes through the column after being heated by the heating unit while circulating in the extracting liquid channel. However, when the extracting liquid then passes through the defoaming unit, the bubbles are removed from the extracting liquid. This situation can be expressed by the bubble ratio, which is the proportion of bubbles in the extracting liquid. The bubble ratio is the ratio of the total length in the direction of piping (total section length B) of the sections in any given section of piping (let's call the section length in the direction of piping A) in which bubbles exist. Without distinguishing between where bubbles exist in the pipe cross-section (e.g., the center or the periphery) or how many bubbles exist in the same cross-section, if even one bubble exists at any position in the cross-section, that cross-section is considered a section with bubbles. The total section length B is determined by calculating the length in the direction of piping for each section in the section with bubbles and summing the lengths of these sections. A and B are determined by measuring the length of the corresponding section in a still image of the piping to be evaluated using a ruler. The bubble rate is calculated using the formula (total section length B / section length A) × 100 (%). In other words, in one embodiment of the synthetic excision system of the present invention, the bubble rate of the excised liquid circulating in the excised liquid flow path is smaller in the section downstream of the defoaming unit and upstream of the liquid delivery pump than in the section downstream of the column and upstream of the defoaming unit. The bubble rate of the excised liquid in the section downstream of the defoaming unit and upstream of the liquid delivery pump is 0 to 90% of the bubble rate of the excised liquid in the section downstream of the column and upstream of the defoaming unit, preferably 0 to 80%, more preferably 0 to 70%, more preferably 0 to 60%, more preferably 0 to 50%, more preferably 0 to 40%, more preferably 0 to 30%, more preferably 0 to 20%, more preferably 0 to 10%, more preferably 0 to 5%, and even more preferably 0 to 1%. In one embodiment, the synthetic extracting system of the present invention has a system in which, in the extracting liquid circulating in the extracting liquid channel, the bubble rate of the extracting liquid in the section downstream of the liquid delivery pump and upstream of the column is smaller than the bubble rate of the extracting liquid in the section downstream of the column and upstream of the degassing unit.

[0066] In one embodiment, the present invention provides a method for producing a synthetic product, comprising a cutting step of passing a cutting solution through a column containing a solid-phase support to cut out a synthetic product from the solid-phase support, This includes circulating the excised fluid through the excised fluid channel. The present invention relates to a manufacturing method wherein the excision fluid channel comprises, in this order, a liquid delivery pump, a column containing a solid support, and a degassing unit, and a heating unit is provided downstream of the degassing unit and upstream of the column containing the solid support. In one embodiment, the present invention relates to a compound produced by the manufacturing method of the present invention. In one embodiment, the present invention relates to a method for producing a synthetic product using the synthetic product production system of the present invention.

[0067] In one embodiment, the synthetic product manufacturing system of the present invention includes a trigger device that generates a cutting process start command to initiate a cutting process after the completion of the synthesis process. The trigger device of the present invention includes a communication fluid detector, a communication fluid channel that delivers the communication fluid from a delivery port controlled by the synthesis process fluid channel control unit to the communication fluid detector, and a signaling device that sends a cutting process start command to the cutting fluid channel control unit after the communication fluid detector detects the delivery of the communication fluid.

[0068] The liaison fluid of the present invention is not particularly limited as long as it is a liquid that can be delivered by the control of the synthesis process liquid flow path control unit. The liaison fluid may be a liquid used in the synthesis process, or a liquid not used in the synthesis process. The type of liaison fluid may be an organic solvent or an inorganic solvent, but preferably a solvent containing an organic solvent. The liaison fluid may be an aqueous solvent or a non-aqueous solvent, but a non-aqueous solvent is preferred if the reaction in the synthesis process includes a water-reactive reaction. Preferably, the liaison fluid is an organic solvent with a water content of 30% v / v or less, more preferably an organic solvent with a water content of 20% v / v or less, more preferably an organic solvent with a water content of 10% v / v or less, and even more preferably an organic solvent with a water content of 1% v / v or less.

[0069] The liaison fluid of the present invention can be delivered from a delivery port controlled by the synthesis process fluid flow path control unit to a liaison fluid detector via a liaison fluid flow path. In one embodiment, the liaison fluid of the present invention may be programmed in the synthesizer to be delivered to the liaison fluid flow path after the completion of the synthesis process, or, if a cleaning solution is delivered to clean the flow path after the completion of the synthesis process, the synthesizer may be programmed to be delivered to the liaison fluid flow path after the delivery of the cleaning solution is completed. The container containing the liaison fluid may be directly or indirectly connected to the synthesis process fluid flow path control unit. The container containing the liaison fluid is connected to a delivery port controlled by the synthesis process fluid flow path control unit. The configuration in which the container containing the liaison fluid is indirectly connected to the synthesis process fluid flow path control unit includes cases where the container containing the liaison fluid is connected to any position within the flow path of the synthesis machine. When a synthesis process fluid is used as the liaison fluid, the container containing the synthesis process fluid corresponds to the container containing the liaison fluid.

[0070] The liaison fluid channel of the present invention is a channel from a fluid delivery port controlled by a synthesis process fluid channel control unit to a liaison fluid detector (see, for example, Figure 26). In some embodiments of the present invention, the liaison fluid channel of the present invention may pass through a portion of the synthesis process fluid channel (see, for example, Figure 27). After the completion of the synthesis process, the synthesis process fluid channel control unit sends the liaison fluid from a container directly or indirectly connected to the synthesis process fluid channel control unit into the liaison fluid channel. The liaison fluid channel is connected to a liaison fluid detector, and the liaison fluid reaches the liaison fluid detector via the liaison fluid channel. After reaching the liaison fluid detector, the liaison fluid may be discharged as waste liquid after passing through the liaison fluid detector. The communication fluid channel may be pre-filled with the same or a different liquid as the communication fluid before the start of the communication fluid delivery. In one embodiment, the communication fluid channel may be pre-filled with an organic solvent before the start of the communication fluid delivery.

[0071] There are no particular restrictions on the material, shape, or inner diameter of the piping for the communication fluid channel, but it is sufficient to deliver a liquid volume necessary to signal the completion of the synthesis process. Therefore, the inner diameter is 0.1 to 15 mm, preferably 0.2 to 5 mm, more preferably 0.3 to 3 mm, and even more preferably 0.5 to 1 mm. The flow rate of the communication fluid in the communication fluid channel is 0.1 mL / min to 1 L / min, preferably 0.5 to 10 mL / min, and even more preferably 0.5 to 1 mL / min.

[0072] The communication fluid detector of the present invention detects the communication fluid delivered through the communication fluid channel and transmits the detection of the communication fluid to a signaling device that sends a command to start the cutting process to the cutting fluid channel control unit. Any flow meter can be used as the communication fluid detector. Regarding generating some kind of signal based on information detected by a flow meter, there is prior art such as U.S. Patent Application Publication No. 2013 / 0116941 (Patent Document 5) and Japanese Patent Publication No. 2015-509250 (Patent Document 6), but both aimed to accurately measure the flow rate and give some kind of instruction based on the result. In contrast, the flow meter used in the present invention is not intended to accurately measure the flow rate, but is used as a device for detecting the completion of the synthesis process. As the communication fluid detector of the present invention, for example, a flow meter, an electrical conductivity meter, a UV spectrophotometer, a pH meter, a pressure gauge, a thermometer, a vibration sensor, etc., can be used. A micro-flow meter is preferred as the flow meter. Examples of micro-flow meters include time-of-flight type micro-flow meters and micro-flow meters that measure flow velocity by rotating an elliptical gear. The communication fluid detector of the present invention is preferably a time-of-flight type micro-flow meter. By passing the communication fluid from the synthesizer through the communication fluid channel when synthesis is complete and detecting the flow velocity obtained by the time-of-flight type micro-flow meter, stable information transmission becomes possible. Because micro-flow meters have high responsiveness, the amount of liquid used as the communication fluid can be sufficiently saved and malfunctions can be prevented. Since the time-of-flight type micro-flow meter is a tube-type micro-flow meter without a stagnant part, air bubbles contained in the liquid do not accumulate, and stable measurement is possible.

[0073] The liquid delivery pump in the synthesis process fluid flow path control unit is not particularly limited, as long as it can reliably deliver the synthesis process fluid and the connecting fluid. From the viewpoint of enabling stable liquid delivery, the liquid delivery pump is preferably a plunger pump, more preferably a double plunger pump, and even more preferably a double plunger pump in which the phases of each plunger are aligned. Normally, pulsation occurs during the delivery of the connecting fluid, and this pulsation makes it easy for bubbles to form in the connecting fluid. Bubbles are especially likely to form when an organic solvent is used as the connecting fluid. However, in this invention, the purpose is to communicate information that the synthesis process has been completed from the synthesis process fluid flow path control unit to the extraction fluid flow path control unit, and there is no need to measure the flow rate precisely. Therefore, even if pulsation occurs during the delivery of the connecting fluid or if bubbles form in the connecting fluid, the purpose can still be achieved.

[0074] In particular, in one embodiment, the present invention is realized by delivering a connecting liquid after the completion of the synthesis process and detecting the delivery of the liquid. However, in particular, synthesizers such as nucleic acid synthesizers use a liquid delivery system with a double-plunger pump in which the phases of each plunger are synchronized, resulting in large pulsations and a tendency for bubbles (cavitation) to be generated when organic solvents are passed through, making it difficult to stably detect the delivery of the liquid. However, according to one embodiment of the present invention, the problems of pulsation and bubbles can be solved, the delivery of the liquid can be stably detected, and a stable start trigger can be provided.

[0075] The trigger device of the present invention includes a signaling device that sends a command to start the cutting process to the cutting fluid flow path control unit after the communication fluid detector detects the delivery of the communication fluid. After the communication fluid detector detects the arrival of the communication fluid, the output device of the signaling device attached to the communication fluid detector transmits a signal indicating the arrival of the communication fluid. The transmitted signal is promptly communicated to the cutting fluid flow path control unit by wired or wireless communication means. That is, a signal input device or communication device attached to the cutting fluid flow path control unit receives the transmitted signal indicating the arrival of the communication fluid. This received signal becomes a command to start the cutting process, and the cutting process is started. Specifically, the signal output device or communication device of the cutting fluid flow path control unit transmits a signal indicating the command to start the cutting process. Each component unit involved in the cutting process (including a control PC, control microcomputer, liquid delivery pump, liquid temperature control device, valves, etc.) receives the command to start the cutting process by the signal input device or communication device attached to each component unit. The cutting process is started in accordance with this command to start the cutting process.

[0076] The communication method of the present invention is particularly effective in older synthesizers that cannot utilize electrical signal communication means such as TTL (Transistor-Transistor Logic). Even older synthesizers have the basic function of transporting liquid, so a communication liquid, a communication liquid channel, and a communication liquid detector can be used as communication means in such synthesizers. That is, by using a trigger device including a communication liquid detector, even in synthesizers without TTL communication functionality, information that the synthesis process has been completed can be transmitted from the synthesis process control system to the extraction process control system promptly after the synthesis process is completed, or at a suitable time according to the production schedule, allowing the extraction process to be started promptly, or at a suitable time according to the production schedule.

[0077] In the present invention, the synthesis process liquid channel may be a single channel or a plurality of independent channels. In the present invention, the extraction liquid channel may be a single channel or a plurality of independent channels. One or more synthesis process liquid channels and one or more extraction liquid channels have a shared channel section in which some channels are shared, and the channels in the shared channel section may be a single channel or a plurality of channels.

[0078] In one embodiment of the present invention, the number of channels in the shared channel section between the synthesis process liquid channel and the extraction liquid channel may be equal to or greater than the number of channels in the non-shared extraction liquid channel section obtained by subtracting the shared channel section from the extraction liquid channel. Here, the number of channels in the shared channel section between the synthesis process liquid channel and the extraction liquid channel may be multiple. In one embodiment of the present invention, the number of channels in the shared channel section between the synthesis process liquid channel and the extraction liquid channel may be equal to or greater than the number of channels in the non-shared synthesis process liquid channel section obtained by subtracting the shared channel section from the synthesis process liquid channel. Here, the number of channels in the shared channel section between the synthesis process liquid channel and the extraction liquid channel may be multiple.

[0079] In one embodiment of the present invention, the number of channels in the shared channel section between the synthesis process liquid channel and the extraction liquid channel may be multiple. In one embodiment of the present invention, two channels selected from the channels of the multiple shared channel sections may be independent of each other and arranged so that the synthesis process liquid can be passed through the channel of one shared channel section and the extraction liquid can be passed through the channel of the other shared channel section simultaneously.

[0080] In one embodiment, the present invention provides a method for producing a compound on a solid support using a column containing a solid support, comprising the following steps: (1) Synthesizing a compound on a solid support by a synthesis reaction of a solid-phase synthesis method, which includes passing a synthesis process liquid through a synthesis process liquid channel. (2) After the compound has been synthesized on the solid support, stop the flow of the synthesis process liquid. (3) Eclecting the product from the solid support by an cleavage reaction of a solid-phase synthesis method, which includes passing the cleavage liquid through the cleavage liquid channel. Includes, Here, the synthesis process liquid channel and the extraction liquid channel have a shared channel section, and the column containing the solid phase support is shared within the channel of the shared channel section. After the completion of (1) and (2) above, the trigger device sends a command to start the cutting process to the cutting fluid flow path control unit to start (3), Includes, The trigger device, Contact fluid detector, A communication fluid channel that sends communication fluid from a supply port controlled by the synthesis process fluid channel control unit to a communication fluid detector, and A signaling device that sends a command to the dispensing fluid flow path control unit to start the dispensing process after the communication fluid detector detects the flow of communication fluid. The present invention relates to a method for producing the aforementioned compound, which includes [the specified compound]. Furthermore, in one embodiment, the present invention relates to a method for producing a compound on a solid phase support using a column containing a solid phase support, wherein the method for producing the compound uses the compound production system of the present invention.

[0081] In one embodiment, the synthetic product manufacturing system of the present invention may include a cleavage liquid sampling mechanism on the cleavage liquid channel, which samples a portion of the cleavage liquid flowing through the cleavage liquid channel at any time during the progress of the cleavage reaction (and deprotection reaction), preferably sampling only a small amount of the cleavage liquid circulating in the cleavage liquid channel. The cleavage liquid sampled by the cleavage liquid sampling mechanism can be evaluated in any way appropriate to the type of synthetic product, protecting group, and cleavage liquid to determine the cleavage status from the solid support and the progress of the deprotection reaction that releases the protecting group from the synthetic product. The cleavage liquid sampling mechanism is useful as a method for confirming the completion of the cleavage reaction (and deprotection reaction) and for determining the appropriate time required for the cleavage reaction (and deprotection reaction). The number of samplings may be one or multiple times. The sampled cleavage liquid may be evaluated as is, or it may be evaluated after stopping the cleavage reaction by mixing it with a cleavage reaction stopping solution to stop the cleavage reaction in the sampled cleavage liquid.

[0082] The cutoff liquid sampling mechanism may be configured such that a cutoff liquid sampling container is connected to the cutoff liquid flow path via a flow path switching valve. As a mechanism for mixing the sampled cutoff liquid with the cutoff reaction stop solution, the cutoff reaction stop solution may be supplied to the cutoff liquid sampling container via a flow path switching valve on the cutoff liquid flow path or via the cutoff liquid flow path. The mixing order within the cutoff liquid sampling container may be such that the sampled cutoff liquid is added first, followed by the cutoff reaction stop solution, or in the reverse order, or added to the container almost simultaneously. After supplying the cutoff reaction stop solution to the cutoff liquid sampling container, any gas such as air or nitrogen can be flowed through the cutoff liquid flow path to remove any remaining cutoff reaction stop solution in the cutoff liquid flow path, thereby avoiding or suppressing its influence on the cutoff reaction (and deprotection reaction) in the cutoff liquid flow path after sampling.

[0083] In one embodiment, the excision fluid sampling mechanism of the present invention includes an excision fluid sampling container, a flow path switching valve 4, a container containing an excision reaction stopping solution, a liquid delivery pump 2, a flow path switching valve 5, and a gas inlet, as shown in Figure 31. When sampling the excised fluid, first, the fluid delivery pump of the excised fluid flow control unit is stopped to halt the circulation of the excised fluid. Then, the flow path switching valve 4 is switched to a predetermined port, and the fluid delivery pump of the excised fluid flow control unit is restarted, allowing the excised fluid to be sampled into the excised fluid sampling container. After a predetermined time has elapsed, the fluid delivery pump of the excised fluid flow control unit is stopped again. Then, the flow path switching valve 5 is switched to a predetermined port, and the fluid delivery pump 2 is used to deliver the excised reaction stopping solution, which is contained in a container of excised reaction stopping solution, to the excised fluid sampling container, thereby stopping the excised reaction of the sampled excised fluid. Then, gas is injected into the excised fluid sampling container from the gas inlet provided on the flow path switching valve 5, pushing the remaining excised fluid in the piping connecting the flow path switching valve 4 and the excised fluid sampling container into the excised fluid sampling container. Alternatively, the excised reaction stopping solution may be placed in the excised fluid sampling container, and only the gas from the container of excised reaction stopping solution may be delivered using the fluid delivery pump 2. Finally, the ports of the flow path switching valve 4 and the flow path switching valve 5 are returned to their state before the circulation and supply of the excised fluid was stopped, and the supply pump of the excised fluid flow path control unit is restarted, thereby restarting the circulation and supply of the excised fluid.

[0084] In the present invention, valves are not limited to these, but examples include rotary valves, ball valves, diaphragm valves, butterfly valves, choke valves, gate valves, globe valves, knife valves, needle valves, pinch valves, piston valves, plug valves, solenoid valves, spool valves, gate valves, and solenoid valves. In the present invention, the liquid transfer pump is not limited to these, but examples include plunger pumps, diaphragm pumps, bellows pumps, tube pumps, gear pumps, rotary pumps, screw pumps, and the like. In the present invention, the cleavage reaction stopping solution is not limited to these, but for example, water, acetonitrile, methanol, or a mixture thereof can be used.

[0085] In one embodiment of the present invention, the gas injected from the gas inlet is not particularly limited as long as it does not substantially affect the excised liquid, but examples include air and nitrogen. In the present invention, the sampled excised solution can be evaluated by the following methods. Methods for evaluating the excision status from the solid support are not limited to these, but include, for example, measuring the absorbance (260 nm) of the sampled excised solution using a UV-Vis spectrophotometer. Methods for evaluating the deprotection status of protecting groups from the synthetic product are not limited to these, but include, for example, quantitative measurement of deprotected protecting groups contained in the sampled excised solution using high-performance liquid chromatography (LC / MS). Oligonucleotides contained in the sampled excised solution may be measured by high-performance liquid chromatography (HPLC) and evaluated by the ratio of peak areas of the target product to impurities.

[0086] In the method of the present invention, the synthesis process liquid channel may be a single channel or a plurality of independent channels. In the method of the present invention, the extraction liquid channel may be a single channel or a plurality of independent channels. One or more synthesis process liquid channels and one or more extraction liquid channels have a shared channel section in which some of the channels are shared, and the channels in the shared channel section may be a single channel or a plurality of channels. In the method of the present invention, two flow paths selected from the flow paths of the plurality of shared flow path sections are independent of each other, and the method may include simultaneously passing the synthesis process liquid through the flow path of one shared flow path section and passing the extraction liquid through the flow path of the other shared flow path section.

[0087] In one embodiment, the method of the present invention is such that two flow paths selected from the flow paths of the plurality of shared flow path sections are independent of each other. In the flow path of one of the shared flow path sections, after steps (1) and (2) are completed, In the flow path of one of the shared flow path sections, step (3) is performed, and independently thereafter, in the flow path of the other shared flow path section, step (1) is performed. It may include. In one embodiment, the method of the present invention is such that two flow paths selected from the flow paths of the plurality of shared flow path sections are independent of each other. In the flow path of one of the shared flow path sections, after steps (1) and (2) are completed, In the flow path of one of the shared flow path sections, step (3) is performed, and at the same time, in the flow path of the other shared flow path section, step (1) is performed. It may include.

[0088] In one embodiment, the present invention can also be applied to a case where, for example, there is one section obtained by excluding the shared channel section from the synthesis process liquid channel (non-shared synthesis process liquid channel section), and there are two shared channel sections. When "section 1" and "section 2" are selected as the two shared channel sections selected from the plurality of shared channel sections, first, steps (1) and (2) are carried out in "section 1". Next, step (3) is carried out in "section 1" and simultaneously, steps (1) and (2) are carried out in "section 2". After that, step (3) is carried out in "section 2". By using it in this way, steps (1), (2), and (3) can be carried out in overlapping time in "section 1" and "section 2", respectively, thereby improving manufacturing efficiency.

[0089] In one embodiment, the present invention can also be applied to a case where, as shown in Figure 32, there is one section obtained by excluding the shared channel section from the synthesis process liquid channel (non-shared synthesis process liquid channel section), and there are three shared channel sections. In this case, two of the shared channel sections selected from the plurality of shared channel sections can be a combination of "section 1" and "section 2", and a combination of "section 2" and "section 3". In this case, steps (1), (2), and (3) can be carried out in each of "section 1", "section 2", and "section 3" with temporal overlap. That is, first, steps (1) and (2) can be carried out in "section 1". Next, step (3) can be carried out in "section 1", and at the same time, steps (1) and (2) can be carried out in "section 2". Next, step (3) can be carried out in "section 2", and at the same time, steps (1) and (2) can be carried out in "section 3". Next, step (3) can be carried out in "section 3".

[0090] In the embodiment shown in Figure 32, the number of shared channel sections (3) in which the synthesis process fluid channel and the excision fluid channel share the column within the channel is equal to the number of non-shared excision fluid channel sections (3) obtained by subtracting the shared channel sections from the excision fluid channel. In this embodiment, the number of shared channel sections (3) in which the synthesis process fluid channel and the excision fluid channel share the column within the channel is greater than the number of non-shared synthesis process fluid channel sections (1) obtained by subtracting the shared channel sections from the synthesis process fluid channel. In this embodiment, the shared channel sections in which the synthesis process fluid channel and the excision fluid channel share the column within the channel include a plurality (3) of the shared channel sections. In this embodiment, the channel switching valves 1 and 2 are valves that have the function of switching the channel without direct contact between the synthesis process fluid and the excision fluid, and may be configured as a single valve or as a valve complex combining multiple valves. In this embodiment, the flow path switching valves (composite valves) 1 and 2 are arranged so that two of the shared flow path sections selected from a plurality (three) of the shared flow path sections can simultaneously pass the synthesis process liquid into one of the shared flow path sections and pass the extraction liquid into the other of the shared flow path sections.

[0091] In one embodiment, the present invention can also be applied to a case where, as shown in Figure 34, there are two sections obtained by excluding the shared channel sections from the synthesis process liquid flow path, and there are four shared channel sections. In this case, two combinations of "section 1" and "section 2" and "section 3" and "section 4" can be selected as the two shared channel sections to be carried out from the plurality of shared channel sections, and processes (1), (2), and (3) can be carried out in a time-overlapping manner in the combination of "section 1" and "section 2," and processes (1), (2), and (3) can be carried out in a time-overlapping manner in the combination of "section 3" and "section 4," either simultaneously or with a time delay.

[0092] In the embodiment shown in Figure 34, the number of shared channel sections (4) in which the synthesis process fluid channel and the excision fluid channel share the column within the channel is equal to the number of non-shared excision fluid channel sections (4) obtained by subtracting the shared channel sections from the excision fluid channel. In this embodiment, the number of shared channel sections (4) in which the synthesis process fluid channel and the excision fluid channel share the column within the channel is greater than the number of non-shared synthesis process fluid channel sections (2) obtained by subtracting the shared channel sections from the synthesis process fluid channel. In this embodiment, the shared channel sections in which the synthesis process fluid channel and the excision fluid channel share the column within the channel include a plurality (4) of the shared channel sections. In this embodiment, the channel switching valves 1 and 2 are valves that have the function of switching the channel without direct contact between the synthesis process fluid and the excision fluid, and may be configured as a single valve or as a valve complex combining multiple valves. In this embodiment, two of the shared flow channel sections selected from a plurality (four) of the shared flow channel sections are arranged so that the synthesis process liquid can be passed through one of the shared flow channel sections and the extraction liquid can be passed through the other shared flow channel section at the same time.

[0093] The present invention is not limited to the examples shown in Figures 32-34, and can be applied similarly even when the number of sections obtained by excluding the shared flow path sections from the synthesis process liquid flow path, or the number of shared flow path sections, is different from those described above. With the structure shown in Figures 32-34, or a method using this structure, after the synthesis process using the first column containing the solid phase support is completed, the first column can be moved to the excision process, and at the same time, the synthesis process using the second column can be carried out in parallel, thereby improving manufacturing efficiency.

[0094] Figures 32-34 are configuration diagrams showing an example of a synthetic product manufacturing system having the multiple shared flow path sections, but the number of solid-phase support-encapsulated columns, the number of excision liquid flow path control units, and the number of synthesis process liquid flow path control units are arbitrary. The flow path switching valve is not particularly limited as long as it is a valve that has the function of switching the flow path without direct contact between the synthesis process liquid and the excision liquid, and may be configured as a single valve or as a valve complex combining multiple valves. The valve used for the flow path switching valve, whether as a single valve or as a valve used to constitute a valve complex, may be a valve selected from the group consisting of rotary valves, ball valves, diaphragm valves, butterfly valves, choke valves, gate valves, globe valves, knife valves, needle valves, pinch valves, piston valves, plug valves, solenoid valves, spool valves, gate valves, and solenoid valves. [Examples]

[0095] I. Investigation of salt precipitation in the synthesis system 1. Configuration of the synthetic product manufacturing system Figure 1 shows the configuration of the compound manufacturing system in Comparative Example 1, and Figure 2 shows the configuration of the example compound manufacturing systems in Examples 1 and 2. Flow path switching valves 1 and 2 (both rotary 6-way valves (From Co., Ltd., model number VA-21-618 (wetted parts material: PEEK, ceramic))) are connected to the inlet and outlet sides of the solid-phase support-embedded column, respectively. Flow path switching valves 1 and 2 are connected to the synthesis process fluid flow path and the excision fluid flow path, respectively. The section [flow path switching valve 1 → solid-phase support-embedded column → flow path switching valve 2] is part of both the synthesis process fluid flow path and the excision fluid flow path. By switching between flow path switching valves 1 and 2, the section [flow path switching valve 1 → solid-phase support-embedded column → flow path switching valve 2] is supplied with the synthesis process fluid during the synthesis process and with the excision fluid during the excision process. A synthesis process fluid flow path control unit (including a synthesis process fluid switching mechanism, a fluid temperature control device, a fluid delivery pump, etc.) is placed in the synthesis process fluid flow path, and multiple types of synthesis process fluids used in the synthesis process are connected to the synthesis process fluid flow path control unit. In the synthesis process, multiple types of synthesis process fluids are appropriately delivered to the synthesis process fluid flow path via the synthesis process fluid flow path control unit.

[0096] A fluid extraction channel control unit (including a fluid temperature control device and a fluid delivery pump) is located in the fluid extraction channel. In Comparative Example 1, the container containing the excision solution (containing 38 mL of 28-30% ammonia water as the excision solution) was installed in the middle of the flow path between the flow path switching valve 2 and the excision solution flow path control unit. In Examples 1 and 2, the container containing the excision solution was installed at a position branched off from the excision solution flow path via an excision solution valve placed on the excision solution flow path between the flow path switching valve 2 and the excision solution flow path control unit.

[0097] The liquid extraction valves used in each embodiment are as shown in Table 1, and consisted of a rotary 6-way valve (From Co., Ltd., model number VA-21-618 (wetted parts material: PEEK, ceramic)) and a T-port 3-way valve (AS ONE Corporation, model number 016.802.5.2, material: body / PTFE (polytetrafluoroethylene), handle / PP (polypropylene)).

[0098] 2. Evaluation method for salt precipitation In Comparative Example 1 and Examples 1 and 2, DNA oligonucleotide synthesis (synthesis step) was performed by supplying the required type of synthesis solution to the solid support-encapsulated column at the required timing via the synthesis solution channel, and the presence or absence of salt deposition on the channel switching valve was evaluated after the completion of the synthesis step. In Comparative Example 1, the flow path switching valves 1 and 2 were set to an open state with the synthesis process liquid flow path control side port and the solid phase carrier-encapsulated column side port open, and the port connected to the excision liquid flow path control unit or the container containing the excision liquid closed (standby state). In Examples 1 and 2, the flow path switching valves 1 and 2 were set with the synthesis process liquid flow path control side port and the solid phase carrier-encased column side port open, and the port on the piping side connected to the cut-out liquid flow path control or cut-out liquid valve closed. Furthermore, the cut-out liquid valve was set with the port on the piping side connected to flow path switching valve 1 and the port on the piping side connected to flow path switching valve 2 open, and the port on the container side containing the cut-out liquid closed (standby state).

[0099] DNA oligonucleotide synthesis was performed as follows. Porous resin beads (NittoPhase® HL UnyLinker350) were placed in a solid-supported column (volume 12.6 ml) to a synthesis scale (total reaction sites of beads) of 480 μmol. This column was then set in an AKTAoligopilotplus100 synthesizer (Cytiva), and nucleoside phosphoramidite and 4,5-dicyanoimidazole (DCI) as an activator were added to carry out a coupling reaction (condensation time: 5 minutes). All activators were dissolved in acetonitrile to prepare a 0.7 M solution. Other synthetic reagents used included 3% DCA (dichloroacetic acid) in toluene as a deprotecting agent, 0.2 M xanthan hydride in pyridine as a sulfurizing agent, a mixed solution of lutidine or pyridine, N-methylimidazole, and acetic anhydride in acetonitrile as a capping agent, and 20% TBA (tert-butylamine) in acetonitrile as an amine wash reaction solution. A 24-mer DNA oligonucleotide (5'-TCGACGTATTGACGTATTGACGTA-3', with phosphite esters (nucleoside-to-nucleoside bonds) and the 3' terminal phosphate group all sulfurized (SEQ ID NO: 1)) was synthesized, and the terminal DMTr protecting group was removed. The total time required for the above synthesis steps was approximately 20 hours.

[0100] (Comparative Example 1) After the synthesis of DNA oligonucleotides was complete, the flow path was switched from the synthesis process solution flow path to the excision solution flow path by switching flow path switching valves 1 and 2 (flow path switching valves 1 and 2 were switched to a state where the ports on the synthesis process solution flow path control side were closed, and the ports on the piping side connected to the excision solution flow path control or the container of excision solution, and the solid support-encapsulated column side were opened), and the supply of excision solution was started. After confirming whether the supply of excision solution had started, the supply was stopped, the piping connecting the container of excision solution and flow path switching valve 2 was removed, and the condition of the flow path was visually observed. (Examples 1 and 2) After the synthesis of DNA oligonucleotides was completed, the piping connecting the excision solution valve and the flow path switching valve 2 was removed, and the flow path was visually observed. Then, the removed piping was put back in place, and the flow path switching valves 1 and 2 were switched to switch from the synthesis process solution flow path to the excision solution flow path (flow path switching valves 1 and 2 were switched to a state where the synthesis process solution flow path control side port was closed, and the port on the piping side connected to the excision solution flow path control unit or the excision solution valve, and the port on the solid support-encapsulated column side were opened). The excision solution valve was then opened to the port on the excision solution container side, the port on the piping side connected to flow path switching valve 1, and the port on the piping side connected to flow path switching valve 2, and the supply of excision solution to the excision solution flow path was started.

[0101] 3. Evaluation results of salt precipitation Table 1 shows the evaluation results of salt precipitation in the synthetic product manufacturing system. (Comparative Example 1) After the synthesis of DNA oligonucleotides was completed, the flow path was switched from the synthesis process solution channel to the cleavage solution channel, and when the supply of the cleavage solution was started, the channel became blocked, resulting in a failure to deliver the solution. Upon removing the piping connecting the container of cleavage solution and the channel switching valve 2 and visually inspecting it, salt precipitation was confirmed. (Examples 1 and 2) In both cases, whether the excision fluid valve was a rotary 6-way valve or a T-port 3-way valve, after the completion of DNA oligonucleotide synthesis, a portion of the piping connecting the excision fluid valve and the flow path switching valve 2 was removed and visually inspected, and no salt precipitation was observed. Furthermore, when the removed piping was returned, the flow path was switched, and the excision fluid supply was started, the supply of excision fluid could be started without any blockage of the flow path. From the above results, it was found that even if a container of cutting liquid is connected in advance to transition to the cutting process after the completion of the synthesis process, if the container of cutting liquid is placed in a position that is part of the cutting liquid flow path, salt precipitation occurs and liquid delivery problems occur. Furthermore, it was found that salt precipitation can be avoided if the container of cutting liquid is placed in a position branched off from the cutting liquid flow path via a cutting liquid valve (installed on the cutting liquid flow path in the section from flow path switching valve 1 to cutting liquid flow path control unit to flow path switching valve 2).

[0102] [Table 1]

[0103] II. Elucidation of the Mechanism of Salt Deposition <Materials and Methods> As a synthetic reagent containing acid, a few drops of 3% DCA in toluene, a deprotecting agent, were added to a container. A few drops of 28-30% aqueous ammonia were then added, and the presence of a precipitate was visually confirmed. Furthermore, after drawing up and expelling 28-30% ammonia water with a dropper, the air (containing ammonia gas) inside the dropper was blown into the previously added 3% DCA in toluene, and the presence of precipitates was visually confirmed. <Result> The results are shown in Table 2. When a few drops of 28-30% ammonia water were added, no precipitate was observed. It is presumed that even if precipitates (neutralized salts) form in the presence of a solvent such as water, they dissolve immediately. However, when air (containing ammonia gas) from a dropper was blown onto the solution, precipitates were observed. From the above, it was found that salt precipitates due to a reaction between the acid (DCA) in the synthesis reagent and the basic reagent gas (ammonia gas) volatilized from the extraction solution. In other words, it was found that controlling the gas concentration of ammonia (neutralizing component) volatilized from the extraction solution container is important to avoid salt precipitation.

[0104] [Table 2]

[0105] III. Evaluation of the gas concentration of ammonia gas (neutralizing component gas) volatilizing from the excision liquid container in the piping. We investigated methods for connecting a container containing the cutting fluid (containing 38 mL of 28-30% ammonia water as the cutting fluid) to the cutting fluid channel. Figure 3 shows the connection method for the cutting fluid container in comparative connection example 1, Figure 4 shows the connection methods for the cutting fluid container in connection examples 1-4, and Figure 5 shows the connection methods for the cutting fluid container in connection examples 5-8. In comparative connection example 1, the container containing the cut-out liquid was connected to the piping (piping directly above the container containing the cut-out liquid) without using either the cut-out liquid valve or the liquid layer (corresponding to the connection method in comparative example 1), and the opposite end of the piping directly above the container containing the cut-out liquid was sealed with a plug. In connection examples 1 to 4, the container containing the cut-out liquid was connected to the piping corresponding to the cut-out liquid flow path (piping equivalent to the cut-out liquid flow path) via the cut-out liquid valve (connection example 1 corresponds to the connection method in Example 1, and connection example 4 corresponds to the connection method in Example 2). In connection examples 5 to 8, the container containing the cut-out liquid was connected to the piping equivalent to the cut-out liquid flow path via the cut-out liquid valve and the liquid layer, and the opposite end of the piping equivalent to the cut-out liquid flow path was sealed with a plug. As a method for forming the liquid layer, the piping equivalent to the cut-out liquid flow path was removed from the cut-out liquid valve, liquid was injected into the piping using a microsyringe to form a liquid layer inside the piping, and then the piping was reconnected to the cut-out liquid valve. At that time, the volume of the injected liquid was read using the scale of a microsyringe, and the thickness of the liquid layer (length in the flow direction of the channel) was calculated considering the inner diameter of the pipe through which the liquid was injected. In both the comparative connection example and the connection example, the ammonia gas concentration in the piping directly above the container containing the cutting solution or in the piping corresponding to the cutting solution flow path was evaluated after 20 hours (a time equivalent to the synthesis time of DNA oligonucleotides) had elapsed since connecting the container containing the cutting solution.

[0106] (Comparison and connection example 1) After 20 hours with a container containing the extraction solution (containing 38 mL of 28-30% ammonia water as the extraction solution) connected to the piping directly above the container, the piping directly above the container (the piping between the plug and the pipe connector in Figure 3, with an inner diameter of 1 mm) was removed and connected to an ammonia gas detection tube (Gastec, 3L / 3La / 3M) and a gas sampler (Gastec, GV-100S) to aspirate the gas from the piping. The detection result [ppm] shown by the detection tube was read visually, and the ammonia gas concentration [ppm] in the piping directly above the container containing the extraction solution was evaluated using the following formula (1), which takes into account the volume [mL] of the portion of the piping removed for measurement and the amount of air [mL] aspirated by the gas sampler. Ammonia gas concentration [ppm] in the piping directly above the container containing the excised fluid = Detection result shown by the detector tube [ppm] × (Amount of air drawn in by the gas sampler [mL] ÷ Volume of a portion of the piping removed for measurement (Note) [mL]) ... (1) Note: This refers to the piping between the plug and the pipe connector in Figure 3.

[0107] (Connection examples 1-4) In connection examples 1-4, a container containing the extraction solution (containing 38 mL of 28-30% ammonia water as the extraction solution) was connected to a pipe equivalent to the extraction solution flow path (1 mm inner diameter) via an extraction solution valve (the extraction solution valve was configured with the port on the pipe equivalent to the extraction solution flow path side open, and all other ports, including the port on the container side containing the extraction solution, closed). After 20 hours, the pipe equivalent to the extraction solution flow path (the pipe between the stopper and the extraction solution valve in Figure 4) was removed, and the gas in the pipe was aspirated by connecting it to an ammonia gas detection tube (Gastec, 3L / 3La / 3M) and a gas sampler (Gastec, GV-100S).

[0108] (Connection examples 5-8) In connection examples 5-8, a container containing the extraction solution (containing 38 mL of 28-30% ammonia water as the extraction solution) was connected to a pipe equivalent to the extraction solution flow path (1 mm inner diameter) via an extraction solution valve and a liquid layer (the extraction solution valve was set to open the port on the pipe equivalent to the extraction solution flow path side, and closed all other ports, including the port on the container containing the extraction solution). After 20 hours, the pipe equivalent to the extraction solution flow path (the pipe between the stopper and the extraction solution valve in Figure 5 (however, the section between the liquid layer and the stopper)) was removed, and the gas in the pipe was aspirated by connecting it to a detection tube for ammonia gas (Gastec, 3L / 3La / 3M) and a gas sampler (Gastec, GV-100S).

[0109] The fluid extraction valves used in each connection example are listed in Table 3, and include a rotary 6-way valve (From Co., Ltd., model number VA-21-618 (wetted parts material: PEEK, ceramic)), a diaphragm 3-way valve (Takasago Electric Industry Co., Ltd., model number MTV-3-14UKGH-17-R0, wetted parts material: PTFE, FFKM, PEEK), a ball 3-way valve (Fujikin Co., Ltd., model number PUBVT-95-3.2-V, material: body / stainless steel (SCS14), socket / ball / stem / stainless steel (SUS316), O-ring / fluororubber, seat / gland packing / G-PTFE), and a T-port 3-way valve (AS ONE Corporation, model number 016.802.5.2, material: body / PTFE (polytetrafluoroethylene), handle / PP (polypropylene)).

[0110] In each connection example, after aspirating the gas from the piping, the detection result [ppm] indicated by the detection tube was read visually, and the ammonia gas concentration [ppm] in the pipe equivalent to the liquid extraction channel was evaluated using the following equation (2), which takes into account the volume [mL] of the portion of the piping removed for measurement (in connection examples 1 to 4, this refers to the piping equivalent to the liquid extraction channel (the piping between the stopper and the liquid extraction valve in Figure 4), and in connection examples 5 to 8, this refers to the piping equivalent to the liquid extraction channel (the piping between the stopper and the liquid extraction valve in Figure 5 (however, the section between the liquid layer and the stopper))) and the amount of air aspirated by the gas sampler [mL]. Ammonia gas concentration [ppm] in the piping corresponding to the excised fluid flow path = Detection result shown by the detector tube [ppm] × (Amount of air drawn in by the gas sampler [mL] ÷ Volume of a portion of the piping removed for measurement (Note) [mL]) ... (2) Note: In connection examples 1-4, the piping equivalent to the liquid extraction channel refers to the piping between the stopper and the liquid extraction valve in Figure 4, while in connection examples 5-8, the piping equivalent to the liquid extraction channel refers to the piping between the stopper and the liquid extraction valve in Figure 5 (however, this refers to the section between the liquid layer and the stopper).

[0111] The results of the ammonia gas concentration measurements are shown in Table 3. (Comparison and connection example 1) The ammonia gas concentration directly above the container holding the extraction solution was evaluated and found to be 34783 ppm. Comparing this with the results of Comparative Example 1, it is considered that when the ammonia gas concentration in the extraction solution channel is at the 34783 ppm level, a neutralization reaction occurs in the channel between the acid-containing synthesis process solution and the ammonia gas volatilized from the container holding the extraction solution (a neutralization reaction near the region where the synthesis process solution channel and the extraction solution channel share a channel), and a neutralized salt precipitates.

[0112] (Connection examples 1 and 4) When the ammonia gas concentration in the piping equivalent to the extraction fluid flow path was evaluated, it was 261 ppm when the extraction fluid valve was a rotary 6-way valve (Connection Example 1) (corresponding to 0.8% when Comparative Connection Example 1 (concentration directly above the ammonia water bottle) is set to 100%), and 14783 ppm when the extraction fluid valve was a T-port valve (Connection Example 4) (corresponding to 42.5% when Comparative Connection Example 1 (concentration directly above the ammonia water bottle) is set to 100%). Comparing these results with those of Comparative Example 1, Comparative Connection Example 1, and Examples 1 and 2, it is considered that the inflow of ammonia gas volatilized from the container containing the extraction fluid was suppressed by the valve, thereby suppressing the precipitation of neutralization salts due to the neutralization reaction between the acid-containing synthesis process liquid and the ammonia gas volatilized from the container containing the extraction fluid. From these results, it is considered that salt precipitation can be suppressed by limiting the inflow of ammonia gas to a gas concentration of 42.5% or less relative to the concentration directly above the container containing the extraction fluid (Comparative Connection Example 1).

[0113] (Connection examples 2 and 3) When the ammonia gas concentration in the piping equivalent to the extraction fluid flow path was evaluated, it was found to be 0 ppm in connection example 2 where the extraction fluid valve was a diaphragm valve (0% compared to comparison connection example 1 (concentration directly above the container containing the extraction fluid)) and 87 ppm in connection example 3 where the extraction fluid valve was a ball valve (0.3% compared to comparison connection example 1 (concentration directly above the container containing the extraction fluid)). Since the inflow of ammonia gas was suppressed to 42.5% or less, it is considered that salt precipitation can be suppressed even with these valves.

[0114] (Connection examples 5-8) Experiments were conducted using water layers of varying thicknesses as examples of liquid layers, and the ammonia gas concentration in the piping equivalent to the liquid extraction channel was evaluated. It was confirmed that the gas concentration could be reduced as the thickness of the liquid layer increased. Connection example 5 (T-port valve + 0.6 mm thick liquid layer) achieved a gas concentration of 261 ppm, which was 0.8% of comparative connection example 1 (no valve), and 1.8% of connection example 4 (T-port valve only, no liquid layer). From this, it can be inferred that while using a valve and a liquid layer together can further reduce the gas concentration, simply providing a liquid layer without using a valve can also significantly reduce the gas concentration.

[0115] [Table 3]

[0116] From the evaluation results above, it was found that even if a container of cutting solution is connected in advance to transition to the cutting process after the completion of the synthesis process, if the container of cutting solution is placed in a position that is part of the cutting solution flow path, salt precipitation occurs and liquid delivery problems occur. However, if the container of cutting solution is placed in a position branched off from the cutting solution flow path via a cutting solution valve (installed on the cutting solution flow path between flow path switching valve 1, cutting solution flow path control unit, and flow path switching valve 2), salt precipitation can be avoided. Furthermore, it was found that the mechanism of salt precipitation is that the acid (DCA) in the synthesis process reagent and the basic reagent gas (ammonia gas) volatilized from the cutting solution react to form a neutralization reaction, causing salt to precipitate. From the above, it was found that controlling the gas concentration of ammonia (neutralizing component) volatilized from the cutting solution container is important to avoid salt precipitation. Furthermore, it was considered that when the concentration of basic reagent gas (ammonia gas) in the extraction solution flow channel piping was at the 34783 ppm level, a neutralization reaction would occur in the flow channel between the acid-containing synthesis process solution and the ammonia gas volatilized from the container holding the extraction solution, resulting in the precipitation of a neutralized salt. It was considered that the precipitation of neutralized salt could be avoided if the concentration of basic reagent gas (ammonia gas) in the extraction solution flow channel piping was suppressed to 14783 ppm or less. It was considered that salt precipitation could be suppressed by suppressing the concentration of basic reagent gas (ammonia gas) to 42.5% or less of the concentration directly above the container holding the extraction solution.

[0117] It was found that by installing a container of cut-off liquid at a location branched off from the cut-off liquid flow path via a cut-off liquid valve (installed on the cut-off liquid flow path between flow path switching valve 1, cut-off liquid flow path control unit, and flow path switching valve 2), the concentration of basic reagent gas (ammonia gas) in the cut-off liquid flow path piping can be reduced to a level that avoids salt precipitation. Similar effects can be achieved not only by using a cut-off liquid valve but also by installing a liquid layer (0.6 mm or more) in the piping, and it was found that a better effect is achieved by combining a cut-off liquid valve and a liquid layer. The liquid layer can be installed by injecting the liquid into the cut-off liquid flow path from any position (valve, etc.) inside or outside the cut-off liquid flow path, or by injecting the liquid as a cleaning solution into the cut-off liquid flow path from any position (valve, etc.) inside or outside the cut-off liquid flow path to clean the flow path, and then discharging the cleaning solution from any position (valve, etc.) inside or outside the cut-off liquid flow path, leaving a portion of the cleaning solution in the cut-off liquid flow path. Adopting a method that involves cleaning the excision fluid channel and leaving a liquid layer within it has the advantage of easily installing the liquid layer. Based on the above findings, it is clear that the automated manufacturing system for the compound of the present invention can suppress salt precipitation not only with the system shown in Figure 2, but also with the systems shown in Figures 6 to 16.

[0118] IV. Effect of aqueous ammonia concentration on the rate of the cleavage and deprotection reaction (C&D reaction) <Materials and Methods> (1) Synthesis of DNA oligonucleotides Porous resin beads (NittoPhase® HL UnyLinker350) were placed in a synthesis column (volume 12.6 ml) to a synthesis scale (total reaction sites of beads) of 480 μmol. This column was then set in an AKTAoligopilotplus100 synthesizer (Cytiva), and nucleoside phosphoramidite and 4,5-dicyanoimidazole (DCI) as an activator were added to carry out a coupling reaction (condensation time: 5 minutes). All activators were dissolved in acetonitrile to prepare a 0.7 M solution. Other synthetic reagents used included 3% DCA in toluene as a deprotecting agent, 0.2 M xanthan hydride in pyridine as a sulfurizing agent, a mixed solution of lutidine or pyridine, N-methylimidazole, and acetic anhydride in acetonitrile as a capping agent, and 20% TBA (tert-butylamine) in acetonitrile as an amine wash reaction solution. A 24-mer DNA oligonucleotide (5'-TCGACGTATTGACGTATTGACGTA-3', with phosphite ester (nucleoside-to-nucleoside bond) and the 3' terminal phosphate group all sulfurized (SEQ ID NO: 1)) was synthesized, and the terminal DMTr protecting group was removed. Porous resin beads to which the DNA oligonucleotide was bound were extracted and dried.

[0119] (2) Excavation and deprotection of synthesized DNA oligonucleotides The synthesized porous resin beads were divided into 0.2g portions. Each portion was immersed in ammonia water of different concentrations, and DNA oligonucleotides were cleaved from the porous resin beads, followed by a deprotection reaction of the base amino group. After 5 hours, the cleavage and deprotection reaction was stopped, and a filtrate containing dissolved DNA oligonucleotides and deprotection components was obtained.

[0120] (3) Evaluation of C&D reaction progress rate by quantitative measurement of deprotective components The deprotection components were quantitatively measured by high-performance liquid chromatography (LC / MS) under the following conditions for oligonucleotide sample filtrates diluted 3750 times. The amount of protecting groups was set to 100% assuming successful synthesis reactions at all reaction sites on the porous resin beads, and the quantitative result of the deprotection components relative to this amount was evaluated as the C&D reaction progress rate. Column: Waters Atlantis T3 Column, 100A, 3μm, 2.1 mm x 150 mm Mobile phase A: 0.1% formic acid aqueous solution Mobile phase B: Acetonitrile Column temperature: 40℃ Detection mode: MS (ESI-Pos.) Detected m / z: Isobutylamide m / z = 88.0757, Benzamide m / z = 122.0600

[0121] <Results and Discussion> The results are shown in Table 4. A tendency was observed for the reaction rate to decrease as the concentration of aqueous ammonia decreased. At this point, the decrease in the reaction rate was small when the aqueous ammonia concentration was up to 22%, but it decreased significantly at 14%. From this, it was found that even when the column is washed and then replaced with the excision solution, the impact on the excision and deprotection reactions can be minimized by controlling the concentration of aqueous ammonia after the excision solution replacement to 15% to 30%. It was found that the decrease in C&D reaction efficiency can be suppressed by controlling the concentration of ammonia water after the extraction fluid replacement to 15% to 30% by injecting gas into the extraction fluid channel to remove residual liquid in the piping before supplying the extraction fluid to the channel.

[0122] [Table 4]

[0123] V. Time course of the dissection and deprotection reaction (C&D reaction) using the dissection fluid sampling mechanism. <Materials and Methods> (1) Synthesis and excision / deprotection of DNA oligonucleotides Porous resin beads (NittoPhase® HL UnyLinker 350) were placed in a synthesis column (volume 12.6 ml) to a synthesis scale (total reaction sites of beads) of 480 μmol. This column was then set in an AKTA® oligopilot plus 100 synthesizer (manufactured by Cytiva), and nucleoside phosphoramidite and 4,5-dicyanoimidazole (DCI) as an activator were added to carry out a coupling reaction (condensation time: 5 minutes). All activators were dissolved in acetonitrile to prepare a 0.7 M solution. Other synthetic reagents used included 3% DCA in toluene as a deprotecting agent, 0.2 M xanthan hydride in pyridine as a sulfurizing agent, a mixed solution of lutidine or pyridine, N-methylimidazole, and acetic anhydride in acetonitrile as a capping agent, and 20% TBA (tert-butylamine) in acetonitrile as an amine wash reaction solution. A 24-mer DNA oligonucleotide (5'-TCGACGTATTGACGTATTGACGTA-3', with phosphite esters (internucleoside bonds) and the 3' terminal phosphate group all sulfurized (SEQ ID NO: 1)) was synthesized, and the terminal DMTr protecting group was removed.

[0124] (2) Excavation and deprotection of synthesized DNA oligonucleotides After the synthesis of DNA oligonucleotides was complete, the flow path was switched from the synthesis fluid channel to the cleavage fluid channel, and the supply of cleavage fluid (containing 38 mL of 28-30% ammonia water as the cleavage fluid) was started. DNA oligonucleotides were cleaved from porous resin beads, and the deprotection reaction of the base amino group was carried out. At 8, 10, 12, and 14 hours, approximately 1 g of circulating ammonia water was collected in bottles using the cleavage fluid sampling mechanism to obtain solutions in which the DNA oligonucleotides and deprotection components were dissolved.

[0125] (3) Evaluation of C&D reaction progress rate Oligonucleotide sample filtrates were prepared to 5OD and measured by high-performance liquid chromatography (HPLC) under the following conditions. The C&D reaction progress rate was evaluated by the ratio (%) of the peak area of ​​the principal component to the sum of the peak areas of the principal component and related impurity components (components in which the protecting group remains without being removed). Column: Waters ACQUITY UPLC Oligonucleotide BEH C18 Column, 130 Å, 1.7 μm, 2.1 mm × 100 mm UV detection: 260nm Mobile phase A: 400mM HFIP / 15mM TEA aqueous solution Mobile phase B: methanol Column temperature: 60℃

[0126] <Results and Discussion> The results are shown in Table 5. At 8 and 10 hours, the C&D reaction progress rate had not reached 100%, indicating that the C&D reaction was incomplete. In contrast, at 12 and 14 hours, the C&D reaction progress rate was 100%, indicating that the C&D reaction was completed at 12 hours. It was considered that using a solution sampling mechanism could be helpful in evaluating the change in the C&D reaction progress rate over time.

[0127] [Table 5]

[0128] VI. Examination of the compound extraction system in the compound manufacturing system 1. Synthesis manufacturing system In testing the present invention, a compound manufacturing system developed by the inventors was used. The outline of the compound manufacturing system is shown in Figure 2 and is configured as follows. Flow path switching valves 1 and 2 (both rotary 6-way valves (From Co., Ltd., model number VA-21-618 (wetted parts material: PEEK, ceramic))) are connected to the inlet and outlet sides of the solid-phase support-embedded column, respectively. Flow path switching valves 1 and 2 are connected to the synthesis process fluid flow path and the excision fluid flow path, respectively. The section [flow path switching valve 1 → solid-phase support-embedded column → flow path switching valve 2] is part of both the synthesis process fluid flow path and the excision fluid flow path. By switching between flow path switching valves 1 and 2, the section [flow path switching valve 1 → solid-phase support-embedded column → flow path switching valve 2] is supplied with the synthesis process fluid during the synthesis process and with the excision fluid during the excision process.

[0129] A synthesis process fluid flow path control unit (including a synthesis process fluid switching mechanism, a fluid temperature control device, a fluid delivery pump, etc.) is placed in the synthesis process fluid flow path, and multiple types of synthesis process fluids used in the synthesis process are connected to the synthesis process fluid flow path control unit. In the synthesis process, multiple types of synthesis process fluids are appropriately delivered to the synthesis process fluid flow path via the synthesis process fluid flow path control unit. A liquid extraction flow control unit (including a liquid temperature control unit and a liquid delivery pump) was placed in the liquid extraction flow path. The containers containing the liquid extraction fluid were installed at a location branched off from the liquid extraction flow path, via a liquid extraction valve located on the liquid extraction flow path between the flow path switching valve 2 and the liquid extraction flow control unit.

[0130] In Comparative Example 2 and Example 3, prior to the circulation of the excision solution in the synthetic product manufacturing system shown in Figure 2, DNA oligonucleotides were synthesized by the following method. At that time, the solid-phase support-encapsulated column was connected to the synthesis process solution flow control unit via flow switch valves 1 and 2 by switching the flow paths of flow switch valves 1 and 2. The synthesis process solution flow control unit supplied the required type of synthesis process solution to the solid-phase support-encapsulated column at the required timing to advance the DNA oligonucleotide synthesis reaction.

[0131] 2. Synthesis of DNA oligonucleotides DNA oligonucleotide synthesis was performed as follows. Porous resin beads (NittoPhase® HL UnyLinker350) were placed in a solid-phase support-mounted column (volume 12.6 ml) so that the synthesis scale (total reaction sites of the beads) was 480 μmol. This column was then set in a synthesis process liquid flow control unit (AKTAoligopilotplus100 synthesizer (manufactured by Cytiva)), and nucleoside phosphoramidite and 4,5-dicyanoimidazole (DCI) as an activator were added to carry out a coupling reaction (condensation time: 5 minutes). All activators were dissolved in acetonitrile and prepared to 0.7 M. Other synthesis reagents used included 3% DCA (dichloroacetic acid) in toluene as a deprotecting agent, 0.2 M xanthan hydride in pyridine as a sulfurizing agent, a mixed solution of lutidine or pyridine, N-methylimidazole, and acetic anhydride in acetonitrile as a capping agent, and 20% TBA (tert-butylamine) in acetonitrile as an amine wash reaction solution. A 24-mer DNA oligonucleotide (5'-TCGACGTATTGACGTATTGACGTA-3', with phosphite esters (internucleoside bonds) and the 3' terminal phosphate group all sulfurized (SEQ ID NO: 1)) was synthesized, and the terminal DMTr protecting group was removed.

[0132] 3. Measurement of the bubble ratio The bubble percentage was measured as follows: For each section of piping (outer diameter 1.6 mm, inner diameter 1.0 mm) to be measured, a 5 cm long section was recorded for 30 seconds or more using a digital camera (CANON PowerShot SX740 HS). Three still images were extracted from the recorded video at approximately 10-second intervals. Using these still images, the length in the direction of the piping in the still image of the section to be measured (section length a) and the sum of the lengths in the direction of the piping in the still image of the section filled with bubbles (total section length b) were measured with a ruler. The value was calculated as (total section length b / section length a) × 100 (%) and evaluated as the bubble rate. After evaluating the bubble rate of each of the three still images, the average value was calculated.

[0133] 4. Comparative Example 2 After synthesizing the DNA oligonucleotides, the fluid delivery route of the flow path switching valve was switched to form a circulating flow path for the excised fluid, as shown in Figure 17(A). Flow path switching valves 1 and 2 and the excised fluid valve were all rotary 6-way valves (From Co., Ltd., model number VA-21-618 (wetted parts material: PEEK, ceramic)), and a container of excised fluid was connected to the excised fluid valve. A double plunger pump (manufactured by Nippon Precision Science Co., Ltd.) was used as the fluid delivery pump, and a circulating constant temperature bath (manufactured by Huber Corporation) was used as the heating unit.

[0134] The excision solution (25% ammonia water, 38 mL) was supplied from the excision solution container to the excision solution channel, and the delivery route of the excision solution valve was switched to circulate the excision solution through the excision solution channel. At this time, the heating unit on the excision solution channel was set to 56°C so that the solution temperature in the solid-phase support-embedded column was 50°C or higher. The inner diameter of the piping in the excision solution channel was 1 mm, the inner diameter of the solid-phase support-embedded column was 20 mm, and the flow rate of the circulating excision solution was 12.6 mL / min. As a result, when the heated excised fluid passed through the solid-phase support-embedded column, bubbles were generated from the column outlet, as shown in Figure 17(B). At this time, the bubble rate of the excised fluid in the section downstream of the column and upstream of the fluid delivery pump was approximately 43% on average and approximately 60% at its maximum (see Table 6). Subsequently, to avoid malfunction of the fluid delivery pump, the circulation and delivery of the excised fluid was stopped before the bubbles reached the pump.

[0135] [Table 6]

[0136] 5. Example 3 (a) Configuration of the compound extraction system After synthesizing DNA oligonucleotides, the fluid delivery route was switched using a flow path switching valve to create a circulating flow path for the excised fluid, as shown in Figure 18(A). The flow path was circulating, consisting of a fluid delivery pump, a heating unit, a solid support-embedded column, a degassing unit, and returning to the fluid delivery pump. A glass container was used as the degassing unit, and it was configured to be cooled by heat dissipation from the container wall (natural cooling).

[0137] The flow path switching valves 1 and 2 and the cut-off fluid valve were all rotary 6-way valves (From Co., Ltd., model number VA-21-618 (wetted parts material: PEEK, ceramic)), and a container of cut-off fluid was connected to the cut-off fluid valve. A double plunger pump (manufactured by Nippon Precision Science Co., Ltd.) was used as the liquid delivery pump, and a circulating constant temperature bath (manufactured by Huber) was used as the heating unit. Cut-off fluid (25% ammonia water, 38 mL) was supplied from the container of cut-off fluid to the cut-off fluid flow path, and the liquid delivery route of the cut-off fluid valve was switched to circulate the cut-off fluid flow path for 24 hours. At this time, the heating unit on the cut-off fluid flow path was set to 56°C so that the solution temperature in the solid support-embedded column was 50°C or higher. In addition, the degassing unit (glass container) was placed in a room temperature environment with the temperature controlled to about 25°C to allow the gas in the degassing unit to cool naturally. The inner diameter of the extraction fluid channel piping was 1 mm, the inner diameter of the solid-phase support-embedded column was 20 mm, and the flow rate of the circulating extraction fluid was 12.6 mL / min. The temperature inside the column was measured using a T-type thermocouple (manufactured by Nippon Thermo Sensor Co., Ltd.).

[0138] The condition of the excised fluid channel was visually inspected 1 hour and 24 hours after the start of circulation. After 24 hours, the excised fluid that had been circulating in the channel was collected and adjusted to 5 OD / mL, and measured by high-performance liquid chromatography (HPLC) under the following conditions. The sum of the peak areas from the detection of the main component up to approximately 10 minutes was taken as 100%, and the peak area (%) of the main component was defined as the synthesis purity (Full-length: area %). Column: Waters ACQUITY UPLC Oligonucleotide BEH C18 Column, 130 Å, 1.7 μm, 2.1 mm × 100 mm UV detection: 260nm Mobile phase A: 400mM HFIP / 15mM TEA aqueous solution Mobile phase B: methanol Column temperature: 60℃

[0139] (b) Evaluation results of bubble suppression One hour after the start of circulation, a visual inspection of the excised liquid flow path revealed that bubbles were being generated vigorously in the excised liquid flow path immediately after the solid-phase support-embedded column (Figure 18(B)). The generated bubbles reached the defoaming unit (glass container), but after floating to the surface of the solution in the container, they burst (static defoaming). It was confirmed that the liquid delivery pump in the circulation path was not sucking up the bubbles, and there were no liquid delivery problems. At this time, the bubble rate in the section downstream of the liquid delivery pump and upstream of the column was 0%. Furthermore, the bubble rate in the section downstream of the column and upstream of the defoaming unit was approximately 55% on average and approximately 62% at its maximum. In addition, the bubble rate in the section downstream of the defoaming unit and upstream of the liquid delivery pump was 0% (see Table 8). In other words, the bubble rate in the excised liquid in the section downstream of the defoaming unit and upstream of the liquid delivery pump was lower than the bubble rate in the section downstream of the column and upstream of the defoaming unit. Furthermore, the bubble rate of the excised liquid in the section downstream of the defoaming unit and upstream of the liquid delivery pump was 0% of the bubble rate of the excised liquid in the section downstream of the column and upstream of the defoaming unit. After 24 hours had elapsed since the start of circulation, a visual inspection of the excised liquid flow path confirmed that solution circulation was continuing without any pump delivery failures or pipe ruptures due to pressure increases in the circulation piping. The final product purity was 86.5%, confirming that a high-purity sample solution was obtained. Furthermore, no impurity components with remaining protecting groups that could not be completely deprotected were detected.

[0140] From these results, it was found that even if bubbles are generated in the flow path, installing a defoaming unit between the solid-phase support-encapsulated column and the liquid delivery pump can prevent pump failure. Furthermore, it was found that cooling the excised liquid and gas in the defoaming unit can prevent situations where the pressure in the excised liquid flow path continues to rise and causes the pipe to rupture.

[0141] [Table 7]

[0142] [Table 8]

[0143] 6. Example 4 Temperature measurement of the gas portion inside the container in the degassing unit containing the cutting and deprotection solution in Example 3. (a) Configuration of the compound extraction system Figure 19(A) shows the configuration of the compound extraction system in Example 4. The apparatus and piping configuration are the same as in Example 3, but in order to measure the temperature of the gas in the container in the degassing unit, a T-type thermocouple (Chino Corporation, YC520-13T) was installed on top of the container, and the temperature was logged using a data logger (Graphtec Corporation, midi LOGGER GL240).

[0144] (b) Method for measuring the gas temperature in the container in the degassing unit An empty column without a solid support was attached to the compound extraction system, and the extracted liquid was circulated while being heated. At this time, as in Example 3, the heating unit was set to 56°C so that the solution temperature in the column was 50°C or higher. In addition, the container in the degassing unit was placed in a room temperature environment with the temperature controlled to approximately 25°C to allow the gas in the container in the degassing unit to cool naturally. After 1 hour from the start of circulation, the condition of the extracted liquid flow path was visually checked, and the gas temperature in the container in the degassing unit was checked using the thermocouple. The temperature inside the column was measured using a T-type thermocouple (manufactured by Nippon Thermo Sensor Co., Ltd.).

[0145] (c) Measurement results of the gas temperature in the container in the degassing unit One hour after the start of circulation, a visual inspection of the excision fluid channel revealed that bubbles were being generated vigorously in the excision fluid channel immediately after the solid-phase support-embedded column (Figure 19(B)). The generated bubbles reached the container in the defoaming unit, but after floating to the surface of the solution in the container, they burst (static defoaming). It was confirmed that the liquid delivery pump in the circulation path was not sucking up the bubbles, and that there were no liquid delivery problems. It was also confirmed that no pipe ruptures occurred due to pressure increases in the circulation path piping. At this time, the temperature of the gas in the container within the degassing unit was approximately 37°C. Considering that the boiling point of 25% aqueous ammonia is approximately 38°C, it is thought that if the temperature of the gas in the container is cooled to below its boiling point (38°C or lower), the ammonia gas released when the bubbles burst will redissolve into the solution, allowing for solution circulation without accumulation in the container.

[0146] [Table 9]

[0147] 7. Example 5: Investigation of the lower limit of cooling temperature (a) Configuration of the compound extraction system Figure 20(A) shows the configuration of the compound extraction system in Example 5. The apparatus and piping configuration are the same as in Example 4, but the container in the degassing unit was actively cooled by placing it in a container filled with ice. Also in this case, when visually checking the state of the cut-out liquid flow path after the start of circulation, bubbles gradually generated in the cut-out liquid flow path immediately after the solid-phase carrier-encapsulated column as the temperature inside the column increased. When the temperature inside the column reached 50 °C, it was confirmed that bubbles were vigorously generated in the cut-out liquid flow path immediately after the solid-phase carrier-encapsulated column (Figure 20(B)). The generated bubbles reached the defoaming unit (glass container), but burst after floating on the liquid surface in the container (static defoaming). It was confirmed that the liquid feed pump in the circulation path did not suck in the bubbles and no liquid feed failure occurred. At this time, the bubble rate in the section downstream of the liquid feed pump and upstream of the column was 0%. Also, the bubble rate in the section downstream of the column and upstream of the defoaming unit was approximately 46% on average and 56% at maximum. Furthermore, the bubble rate in the section downstream of the defoaming unit and upstream of the liquid feed pump was 0% (see Table 11). That is, the bubble rate in the cut-out liquid in the section downstream of the defoaming unit and upstream of the liquid feed pump was lower than the bubble rate in the cut-out liquid in the section downstream of the column and upstream of the defoaming unit. Furthermore, the bubble rate of the cut-out liquid in the section downstream of the defoaming unit and upstream of the liquid feed pump was 0% of the bubble rate of the cut-out liquid in the section downstream of the column and upstream of the defoaming unit. Also, it was confirmed that no pipe breakage due to the pressure increase in the circulation path piping occurred.

[0148] (b) Method for measuring the temperature of the gas in the container in the defoaming unit An empty column without a solid-phase carrier was attached to the compound excision system, and the excision solution was circulated while being heated. At this time, the heating unit was set to 65°C. This was because, referring to other companies' examples of excision and deprotection reactions (Glen Research https: / / www.glenresearch.com / reports / gr20-24, TriLink BioTechnologies https: / / www.trilinkbiotech.com / comparison-of-deprotection-methods-for-the-phthalimidyl-amino-cpgs, Yale University https: / / medicine.yale.edu / keck / oligo / services / protocols / trityl / ), the maximum temperature was 65°C for each company. It was speculated that a temperature above 65°C would have an adverse effect on the excision and deprotection reactions, so it was set to 65°C.

[0149] Temperature measurement was started from the state where the container in the defoaming unit was ice-cooled. The gas temperature in the container in the defoaming unit was confirmed by the thermocouple. Also, the temperature inside the column was measured by a T-type thermocouple (manufactured by Nippon Thermo Sensor Co., Ltd.). As a result, the temperature inside the column and the gas temperature in the container in the defoaming unit gradually increased. Circulation was continued, and the temperature of the gas in the container in the defoaming unit was confirmed when the temperature inside the column exceeded 50°C. Since the lower the temperature inside the column, the longer it takes to complete the excision and deprotection reaction, excessive cooling is also unsuitable for the progress of the reaction. According to the prior literature (Wako Pure Chemical HP https: / / labchem-wako.fujifilm.com / jp / siyaku-blog / 010997.html), it is generally shown that the reaction temperature needs to be 50°C or higher. Therefore, it was determined that the temperature inside the column needs to be maintained at 50°C or higher.

[0150] (c) Temperature measurement results of the gas part in the container in the defoaming unit When the column temperature exceeded 50°C, the temperature of the gas in the container within the degassing unit was approximately 20°C. This indicates that a gas temperature of approximately 20°C or higher is required to ensure both the progress of the cleavage and deprotection reaction and the management of bubbles (cooling effect).

[0151] [Table 10]

[0152] [Table 11]

[0153] In summary, Examples 4 and 5 suggest that cooling the gas temperature in the container within the degassing unit to approximately 20-38°C can prevent liquid delivery failures and pipe ruptures while allowing the extraction and deprotection reactions to proceed.

[0154] VII. Investigation of trigger devices in synthetic product manufacturing systems [Example A] Use of microflow meters in hollow piping The synthesis machine (synthesis process fluid flow path control unit) used an AKTA® oligopilot 100 (manufactured by Cytiva). The extraction fluid flow path control unit interconnected various component units (control PC, control microcontroller, fluid delivery pump, fluid temperature control device, etc.) equipped with signaling devices (signal output device, signal input device, communication device). The extraction fluid flow path control unit has the function of controlling the fluid temperature and fluid delivery of the extraction fluid in the extraction fluid flow path by coordinating each component unit within the extraction fluid flow path control unit with valves on the extraction fluid flow path via the signaling devices. As the connecting fluid detector, an in-line microflow meter (time-of-flight (TOF) method: manufactured by Surpass, NTFD-1 / 4UNF-3-PK) with no stagnation section was used. As the connecting fluid flow path, one end of a 1 mm inner diameter plastic tube was attached to one of the fluid delivery ports of the synthesis machine (synthesis process fluid flow path control unit), and the other end of the tube was connected to the connecting fluid detector. The communication fluid channel was filled with liquid (acetonitrile) from the synthesizer side to every corner of the internal piping of the communication fluid detector. In addition, the signal input device attached to the control microcontroller of the cut-off fluid channel control unit and the signal output device attached to the communication fluid detector were connected by a communication line (Figure 26). The system used consisted of the synthesizer (synthesis process fluid channel control unit), cut-off fluid channel control unit, communication fluid detector, and communication fluid channel connected as described above. Once nucleic acid synthesis was complete, an oxidation solution (manufactured by Fujifilm Wako Pure Chemical Industries), a type of synthesis process liquid, was delivered at a rate of 1 mL / min from the liquid delivery port of the synthesizer through the communication liquid channel to the communication liquid detector. The liquid delivery status was then detected by the communication liquid detector. The detection threshold was set to 0.5 ml / min.

[0155] When the transfer of the connecting liquid (oxidizing solution) from the synthesizer to the connecting liquid channel was initiated, the phenomenon of minute air bubbles being mixed into the transferred connecting liquid was observed. The air bubbles were thought to have been generated by the operation of the plunger of the synthesizer's pump. However, despite the inclusion of such air bubbles, the transfer could be detected 9 seconds after the start of transfer (dotted line and dotted arrow in Figure 28). The fact that the fluid was detected in such a short time—just 9 seconds after the start of fluid delivery—is thought to be because the liquid that was already filled in the fluid channel before delivery began was pushed out as a piston flow. Furthermore, the presence of minute air bubbles did not affect the detection of fluid delivery.

[0156] Furthermore, around 86 seconds in Figure 28, waveform distortion (a peak in flow velocity) occurred. This was thought to be due to the aggregation of small bubbles into larger bubbles, which scattered light. However, it is believed that these large bubbles were discharged without accumulating in the communication fluid channel or the communication fluid detector, and fluid delivery detection was able to be performed without problems even after the waveform distortion. Similarly, when acetonitrile was used instead of the oxidizing solution as the liquid to be delivered, it was possible to detect the delivery in a short amount of time. Based on the above, it was confirmed that the method in Example A is the most preferable method because it is not affected by air bubbles and can detect liquid delivery in a short time of 9 seconds.

[0157] [Example B] Use of a micro-flow meter in hollow piping (in combination with noise reduction processing method) A system was used in which the synthesizer (synthesis process liquid flow path control unit), excision liquid flow path control unit, connecting liquid detector, and connecting liquid flow path were connected in the same manner as in Example A. The difference from Example A is that, in order to counteract noise, a moving average (moving average over the past 10 seconds) was calculated and plotted for the flow rate data acquired by the connecting liquid detector, and the liquid delivery detection level was evaluated when the moving average exceeded 0.5 mL / min. As a result, 27 seconds after starting to supply the connecting liquid (oxidizing solution) from the synthesizer to the connecting liquid channel, the moving average reached the above-mentioned liquid supply detection level, and the liquid supply status was detected (solid line and solid arrow in Figure 28). Although the presence of air bubbles was confirmed in the piping, their effect did not appear in the waveform, and stable detection was possible. Similar results were obtained when the liquid being supplied was replaced with acetonitrile. As described above, the liquid delivery detection time for Method B was longer than that of Method A. However, since the waveform was stable, it was considered a useful method for stable detection as a countermeasure against situations where the effects of bubbles are significant, such as when a large number of large bubbles are generated.

[0158] [Example C] Use of a UV spectrophotometer Except for using an inline UV spectrophotometer (Ocean Optics, Flame) and a flow cell (optical path length 0.1 mm) as the contact fluid detector, the same system as in Example A was constructed and used. Once the synthesis was complete, acetonitrile was passed through the synthesizer's liquid delivery port via the connecting liquid channel to the connecting liquid detector at a flow rate of 1 mL / min. The absorbance (260 nm) was measured by passing the acetonitrile through a flow cell (optical path length 0.1 mm) for inline UV spectrophotometric measurement (Ocean Optics, Flame). As a result, the absorbance was low and the sample was unsuitable.

[0159] Therefore, instead of acetonitrile, an oxidation solution (manufactured by Fujifilm Wako Pure Chemical Industries) was passed through the synthesizer at a flow rate of 1 mL / min. The termination condition was set to detect when the absorbance exceeded 1. As a result, detection was achieved in 127 seconds (Figure 29). The reason it took longer compared to using a flow meter is likely because detection is not possible until the oxidizing solution reaches the flow cell. Furthermore, although the flow cell was washed with acetonitrile, the cleaning performance was poor, and it took 3100 seconds for the absorbance to return to its initial value of 0. The spectrophotometer was deemed unsuitable for detection due to its responsiveness and cleanability.

[0160] [Example D] Use of an elliptical gear type microflow meter Except for using an elliptical gear-type inline microflow meter (HORIBA, LM05ZZT) with a retention section as the contact fluid detector, the same system as in Example A was constructed and used. Once the synthesis was complete, acetonitrile (manufactured by Fujifilm Wako Pure Chemical Industries) was passed through the synthesizer's liquid delivery port via a connecting fluid channel at a rate of 1 mL / min, and measured using an elliptical gear-type in-line microflowmeter with a retention section (manufactured by HORIBA, LM05ZZT). The detection threshold was set at 0.2 ml / min. As a result, at this flow rate, the flow rate could not be measured by the elliptical gear type micro flow meter, and thus the detection threshold was not reached either. As a result of increasing the flow rate to 20 ml / min for testing, detection was achieved in 11 seconds. However, due to large pulsation and the remaining bubbles in the detector, the flow rate value fluctuated greatly under the influence (Figure 30).

[0161] The results of Examples A to D are summarized in Table 12. As a desirable detection method, it is a method with a short response time, measurable regardless of the type of contact liquid, and without the influence of the remaining liquid and bubbles in the contact liquid flow path. The UV spectrophotometer of Example C can be detected if it is a solvent having a UV absorption effect such as an oxidation solution, but it has the characteristic that the type of contact liquid is limited. On the other hand, Example D is unstable due to the influence of bubbles due to the structural characteristics of the gear type and cannot acquire continuous data, so it is unsuitable as a stable trigger. Devices like those in Examples A and B, which are not affected by the type of contact liquid or the remaining liquid and can detect even with bubbles within a response time of several tens of seconds or less, were the most stable detection devices.

[0162]

Table 12

Claims

1. A synthesis process liquid channel for passing a synthesis process liquid for synthesizing a compound on a solid support, and A synthesis manufacturing system comprising a column containing the solid support above the liquid flow path of the synthesis process, A channel for passing an excision liquid to excise the compound synthesized on the solid support from the solid support, A container containing the aforementioned excision liquid, and A liquid pump for transporting the aforementioned excised fluid. Includes, The synthesis process liquid channel and the extraction liquid channel have a shared channel section, and the column containing the solid phase support is shared within the channel of the shared channel section. The container containing the excision fluid is arranged to be connectable to the excision fluid channel, The excised liquid circulates through the excised liquid channel, The aforementioned synthesis solution contains an acid or basic reagent, and the aforementioned extraction solution contains a neutralizing component that can react with the acid or basic reagent. To prevent gases volatile from the extraction liquid from mixing into the synthesis process liquid channel, a liquid for sealing the volatile gases is included in the channel between the container holding the extraction liquid and the synthesis process liquid channel. In one or more of the flow path sections from each end where the synthesis process liquid flow path and the extraction liquid flow path begin or end sharing a flow path to the container containing the extraction liquid, the liquid is present in a container connected to the flow path section. A manufacturing system for the aforementioned compound.

2. The synthesis production system according to claim 1, wherein the neutralizing component is volatile at room temperature and pressure.

3. The synthesis production system according to claim 1, characterized in that the boiling point of the neutralizing component is 10 to 90°C.

4. A synthetic product manufacturing system according to any one of claims 1 to 3, wherein the container containing the extraction liquid is connected to the extraction liquid flow path via a valve so that gas volatile from the extraction liquid does not mix into the synthesis process liquid flow path.

5. The synthetic product manufacturing system according to claim 4, wherein the gas concentration of the neutralizing component in the extraction liquid channel is 50% or less of the gas concentration of the neutralizing component between the container containing the extraction liquid and the valve.

6. The synthetic product manufacturing system according to claim 4, wherein the gas concentration of the neutralizing component in the excision liquid channel is 0 to 15,000 ppm.

7. The compound manufacturing system according to claim 4, wherein the valve is selected from the group consisting of rotary valves, ball valves, diaphragm valves, butterfly valves, choke valves, gate valves, globe valves, knife valves, needle valves, pinch valves, piston valves, plug valves, solenoid valves, spool valves, gate valves, and solenoid valves.

8. The synthesis production system according to claim 1, wherein the liquid comprises water, methanol, acetonitrile, or a mixture thereof.

9. The synthesis production system according to claim 1, wherein the synthesized product is any one of the following: a compound containing an oligonucleotide, a compound containing a peptide, or a compound containing both an oligonucleotide and a peptide.

10. The synthetic product manufacturing system according to claim 1, wherein the protecting group bonded to the synthetic product is removed by the cleavage solution.

11. The compound manufacturing system according to claim 1, wherein gas is injected into the excision fluid channel before the excision fluid is supplied to the excision fluid channel.

12. The compound manufacturing system according to claim 11, wherein the excision liquid contains ammonia, and the concentration of ammonia in the liquid in the excision liquid channel after the supply of the excision liquid to the excision liquid channel is 15% to 30%.

13. The synthetic product manufacturing system according to claim 1, wherein a portion of the excised liquid can be sampled from the excised liquid channel.

14. A method for producing a compound on a solid support using a column containing a solid support, comprising the following steps: Step (1) Synthesis of a compound on a solid support by a synthesis reaction using a solid-phase synthesis method, which includes passing the synthesis process liquid through the synthesis process liquid channel. Step (2) After the compound has been synthesized on the solid support, stop the flow of the synthesis process liquid. Step (3) The synthesis product is cleaved from the solid support by a solid-phase synthesis reaction, which includes passing the cleavage liquid through the cleavage liquid channel. Includes, Here, the synthesis process liquid channel and the extraction liquid channel have a shared channel section, and the column containing the solid phase support is shared within the channel of the shared channel section. The excision fluid is supplied from a container containing the excision fluid, which is arranged to be connectable to the excision fluid channel. The excised fluid circulates through the excised fluid channel, The aforementioned synthesis solution contains an acid or basic reagent, and the aforementioned extraction solution contains a neutralizing component that can react with the acid or basic reagent. To prevent gases volatile from the extraction liquid from mixing into the synthesis process liquid channel, a liquid for sealing the volatile gases is included in the channel between the container holding the extraction liquid and the synthesis process liquid channel. In one or more of the flow path sections from each end where the synthesis process liquid flow path and the extraction liquid flow path begin or end sharing a flow path to the container containing the extraction liquid, the liquid is present in a container connected to the flow path section. A method for producing the aforementioned synthetic product.

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