Continuous flow preparation device for morpholino oligonucleotides

Abstract

The utility model relates to a continuous flow preparation device of morpholino oligonucleotide, and belongs to the field of morpholino oligonucleotide preparation devices. A continuous flow preparation device of morpholino oligonucleotide comprises an automatic synthesizer, a heating device and a constant temperature device, a synthesis column is arranged in the automatic synthesizer, a liquid inlet pipe is connected between an inlet of the synthesis column and the automatic synthesizer, and a liquid outlet pipe is connected between an outlet of the synthesis column and the automatic synthesizer; the heating device covers the surface of the composite column; and the constant temperature device covers the outer wall of the liquid outlet pipe. During continuous flow preparation of morpholino oligonucleotides, the coupling reaction efficiency can be effectively improved, and the impurity content can be controlled.

Description

Technical Field

[0001] This application relates to the field of morpholino oligonucleotide preparation apparatus, and particularly to a continuous flow preparation apparatus for morpholino oligonucleotides. Background Technology

[0002] The synthesis of morpholino oligonucleotides is greatly affected by environmental humidity. Regional and seasonal high humidity can significantly affect product quality and batch-to-batch stability, especially during the scale-up stage, with serious consequences that are difficult for commercial companies to resolve, resulting in persistently high product prices.

[0003] The synthesis of morpholino oligonucleotides often employs traditional batch reactor processes. These processes are cumbersome, inefficient, and have long synthesis cycles. The time required for scale-up production is doubled, further extending the overall synthesis cycle. Cleaning is difficult, and reagent consumption is high. Furthermore, as it is a solid-phase synthesis, there may be scale-up effects. In the early stages of research and development, even small-scale production requires continuous and intensive manual operation, resulting in extremely high R&D labor costs, a high risk of failure, and consequently, extremely high prices for small-scale products.

[0004] Currently, automated synthesis methods have been developed to replace traditional batch reactor processes. Automated synthesis is simple to operate, requires no intensive manual labor, and is low-cost with a short synthesis cycle. However, existing automated synthesis methods produce ammonolysis products with low purity, consume a lot of raw materials, require harsh reaction conditions, and have limited synthesis scale, thus failing to meet actual research and development needs. Utility Model Content

[0005] To address the shortcomings of existing technologies, the purpose of this application is to provide a continuous flow preparation apparatus for morpholine oligonucleotides, thereby improving upon the current automated synthesis methods, which, although having a short synthesis cycle, suffer from low purity of the ammonolysed product, high raw material consumption, harsh reaction conditions, and limited synthesis scale.

[0006] In a first aspect, embodiments of this application provide a continuous flow preparation apparatus for morpholine oligonucleotides, including an automatic synthesizer, a heating device, and a temperature control device. The automatic synthesizer has a synthesis column, with an inlet pipe connected to the inlet of the synthesis column and an outlet pipe connected to the outlet of the synthesis column. The heating device covers the surface of the synthesis column, and the temperature control device covers the outer wall of the outlet pipe.

[0007] In the above-mentioned implementation process, this application can heat the synthesis column to a suitable temperature by coating the surface of the synthesis column with a heating device, so as to improve the coupling reaction efficiency and control the impurity content; at the same time, by coating the outer wall of the liquid outlet tube with a constant temperature device, the circulating reagent during the coupling reaction can be kept at a suitable temperature, thereby effectively improving the coupling reaction efficiency and controlling the impurity content.

[0008] In one possible implementation, both the inlet and outlet pipes are metal pipes.

[0009] This application uses metal pipes for the inlet and outlet pipes, which have better thermal conductivity, allowing the thermostat device wrapped around the outer wall of the outlet pipe to efficiently heat the reagents inside the pipes, ensuring that the reagents circulating in the inlet and outlet pipes are always at a suitable temperature.

[0010] In one possible implementation, the length of the thermostatic device covering the outlet pipe is not less than 1 / 2 of the length of the outlet pipe.

[0011] In one possible implementation, the diameter of the outlet pipe is 0.006-0.05 of the length of the outlet pipe.

[0012] This application, by setting an appropriate length and diameter of the outlet tube, facilitates efficient heating of the reagent inside the outlet tube and ensures that the reagent is always at a suitable temperature.

[0013] This application utilizes a constant temperature device that covers an appropriate length of the outlet tube, which facilitates efficient heating of the reagent inside the outlet tube and ensures that the reagent remains at a suitable temperature.

[0014] In one possible implementation, the volume of the outlet tube does not exceed 80% of the volume of the synthesis column.

[0015] This application, by setting the outlet tube to a suitable volume, facilitates efficient reagent circulation and heating, which is beneficial for efficient coupling reactions.

[0016] In one possible implementation, the height of the heating device covering the surface of the synthesis column is not less than the height of the solid support loaded inside the synthesis column.

[0017] This application, by setting a heating device to cover the synthesis column at a height not less than the height of the solid support loaded inside the synthesis column, can more completely heat the solid support loaded inside the synthesis column, so that the solid support loaded inside the synthesis column reaches a uniform temperature, which is beneficial to uniformly and efficiently improve the coupling reaction efficiency.

[0018] In one possible implementation, both the heating device and the thermostat include a temperature probe.

[0019] This application employs a heating device and a temperature control device with a temperature probe, which can detect the temperature and maintain the required temperature inside the synthesis column and the outlet tube.

[0020] In one possible implementation, the heating device is selected from a heating bag or a heating plate.

[0021] This application uses a heating bag or heating plate as a heating device. The heating bag or heating plate has a certain degree of folding flexibility, which is conducive to tightly covering the surface of the synthesis column, so as to efficiently heat the synthesis column.

[0022] In one possible implementation, the thermostat is selected from an oil bath device.

[0023] This application uses an oil bath device as a temperature control device, which allows the outlet tube to be completely immersed in the oil bath for efficient heating, thereby ensuring that the reagent circulating in the outlet tube is kept at a certain required temperature.

[0024] In one possible implementation, the automated synthesizer is also connected to multiple reagent bottles.

[0025] This application achieves continuous flow preparation of morpholine oligonucleotides by connecting multiple reagent bottles to an automated synthesizer. The reagent bottles are used to load morpholine phosphoramide monomer reagents, activating reagents, deprotecting agents, capping reagents, cleaning reagents, etc. The reagents loaded in each reagent bottle are pumped into the synthesis column by a pump in the automated synthesizer to carry out each step of the reaction. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a schematic diagram of a continuous flow preparation apparatus for morpholine oligonucleotides provided in an embodiment of this application.

[0028] Icons: 1-Automatic synthesizer; 11-Synthesis column; 12-Inlet pipe; 13-Outlet pipe; 2-Heating device; 3-Thermostat; 4-Reagent bottle. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. The components of the embodiments of this application described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0030] Therefore, the following detailed description of the embodiments of this application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments of the application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0031] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0032] In the description of this application, it should be noted that the terms "center," "upper," "lower," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship commonly used when the product is in use. They are used only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application. In addition, the terms "first," "second," and "third," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0033] Furthermore, terms such as "horizontal," "vertical," and "sag" do not imply that components must be absolutely horizontal or suspended, but rather that they can be slightly tilted. For example, "horizontal" simply means that its direction is more horizontal relative to "vertical," and does not mean that the structure must be completely horizontal, but can be slightly tilted.

[0034] In the description of this application, it should also be noted that, unless otherwise expressly specified and limited, the terms "set up," "install," "connect," and "link" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.

[0035] Example

[0036] To address the shortcomings of current automated synthesis methods, which, while offering short synthesis cycles, suffer from low purity of the ammonolysis product, high raw material consumption, stringent reaction conditions, and limited synthesis scale, this application provides a continuous flow preparation apparatus for morpholine oligonucleotides. Figure 1As shown, it includes an automatic synthesizer 1, a heating device 2, and a temperature control device 3. The automatic synthesizer 1 has a synthesis column 11. The inlet of the synthesis column 11 is connected to the automatic synthesizer 1 by a liquid inlet pipe 12, and the outlet of the synthesis column 11 is connected to the automatic synthesizer 1 by a liquid outlet pipe 13. The heating device 2 covers the surface of the synthesis column 11, and the temperature control device 3 covers the outer wall of the liquid outlet pipe 13.

[0037] A heating device 2 is installed on the surface of the synthesis column 11 to heat it to a suitable temperature, thereby improving the coupling reaction efficiency and controlling the impurity content. Simultaneously, a thermostat 3 is installed on the outer wall of the outlet tube 13 to ensure that the circulating reagents during the coupling reaction remain at a suitable temperature, effectively improving the coupling reaction efficiency and controlling the impurity content. Furthermore, placing the thermostat 3 on the outer wall of the outlet tube 13 ensures that during non-circulating steps such as removing the protecting group from the morpholine ring amino group and adding a cap, the reagents are heated only inside the synthesis column, not before the column. This prevents overheating of the reagents during non-circulating steps such as removing the protecting group from the morpholine ring amino group and adding a cap, avoiding the generation of excessive degradation impurities that could affect the reaction results.

[0038] As an example, the automatic synthesizer 1 can be, but is not limited to, Cytiva's Oligopilot Plus 100. Synthesis column 11 can be, but is not limited to, FineLINE 35 Oligo synthesis column 11.

[0039] In some embodiments, both the inlet pipe 12 and the outlet pipe 13 are metal pipes. The use of metal pipes for the inlet pipe 12 and the outlet pipe 13 provides better thermal conductivity, enabling the thermostat 3, which is installed on the outer wall of the outlet pipe 13, to efficiently heat the reagent inside the pipe, thereby ensuring that the reagent circulating in the inlet pipe 12 and the outlet pipe 13 is always at a suitable temperature.

[0040] In some embodiments, the diameter of the outlet tube is 0.006-0.05 times its length. Specifically, the length of the outlet tube 13 is 1-1.5m, and the diameter is 1-5cm. For example, the length of the outlet tube 13 can be, but is not limited to, 1m, 1.1m, 1.2m, 1.3m, 1.4m, or 1.5m. The diameter of the outlet tube 13 can be, but is not limited to, 1cm, 1.5cm, 2cm, 2.5cm, 3cm, 3.5cm, 4cm, 4.5cm, or 5cm. In other embodiments, the length and diameter of the outlet tube 13 can be adjusted as needed. Setting an appropriate length and diameter for the outlet tube 13 facilitates efficient heating of the reagent within the tube and ensures that the reagent remains at a suitable temperature.

[0041] In some embodiments, the length of the thermostat 3 covering the outlet tube 13 is not less than 1 / 2 of the length of the outlet tube 13. Simultaneously, by providing a suitable length for the thermostat 3 to cover the outlet tube 13, a larger portion of the reagent within the outlet tube 13 can be heated, facilitating efficient heating of the reagent within the outlet tube 13 and ensuring that the reagent remains at a suitable temperature.

[0042] In some embodiments, the volume of the outlet tube 13 does not exceed 80% of the volume of the synthesis column 11. Setting the outlet tube 13 to a suitable volume facilitates efficient reagent circulation and heating, which is beneficial for efficient coupling reactions.

[0043] In some embodiments, the height of the heating device 2 covering the surface of the synthesis column 11 is not less than the height of the solid support loaded inside the synthesis column 11. This allows for more complete heating of the solid support loaded inside the synthesis column 11, enabling it to reach a uniform temperature, which is beneficial for uniformly and efficiently improving the coupling reaction efficiency.

[0044] In some embodiments, both the heating device 2 and the temperature control device 3 include temperature probes that can detect temperature and maintain the required temperature inside the synthesis column 11 and the outlet pipe 13.

[0045] In some embodiments, the heating device 2 may be, but is not limited to, a heating bag or a heating sheet. The heating bag or heating sheet has a certain degree of folding flexibility, facilitating a tighter fit onto the surface of the synthesis column 11 for efficient heating of the synthesis column 11. As an example, the temperature of the heating device 2 is controlled between 30-60°C. Specifically, the temperature of the heating device 2 may be controlled at, but is not limited to, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, and 60°C to ensure that the temperature inside the synthesis column 11 is maintained at 30-60°C.

[0046] In some embodiments, the thermostat 3 may be, but is not limited to, an oil bath device. The oil bath device can completely immerse the outlet pipe 13 in the oil bath to efficiently heat the outlet pipe 13, ensuring that the reagent circulating within the outlet pipe 13 is maintained at a certain required temperature. As an example, the temperature of the thermostat 3 is controlled between 30-60°C. Specifically, the temperature of the thermostat 3 may be controlled at, but is not limited to, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, or 60°C, to ensure that the reagent circulating within the outlet pipe 13 is kept at 30-50°C.

[0047] In some embodiments, the automated synthesizer 1 is also connected to multiple reagent bottles 4. The reagent bottles 4 are used to load morpholine phosphoramidite monomer reagents, activating reagents, deprotecting agents, capping reagents, washing reagents, etc. The reagents loaded in each reagent bottle 4 are pumped into the synthesis column 11 by a pump in the automated synthesizer 1 to carry out each step of the reaction, thereby achieving continuous flow preparation of morpholine oligonucleotides. As an example, the automated synthesizer 1 is connected to the reagent bottles 4 via polytetrafluoroethylene tubing. The reagent bottles 4 are transparent glass bottles or brown glass bottles.

[0048] In the preparation of morpholino oligonucleotides, morpholinophosphoramide protected by a protecting group is first loaded onto a solid support and packed into a synthesis column 11. The temperature of the heating device 2 is controlled at 30-60℃ to maintain the temperature inside the synthesis column 11 at 30-60℃. The temperature of the isothermal device 3 is also controlled at 30-60℃ to maintain the temperature of the reagent circulating in the outlet tube 13 at 30-60℃. Then, the deprotecting agent loaded in the reagent bottle 4 is pumped through the pump in the automated synthesizer 1 at a first flow rate of 150-240. The first flow rate is pumped at 120-150 cm / h to 3-7 cv into synthesis column 11, followed by a second flow rate of 120-150 cm / h to 7-10 cv into synthesis column 11, with the difference between the first and second flow rates not less than 30 cm / h, to remove the protecting group on the amino group of the morpholine ring. Then, the cleaning reagent loaded in reagent bottle 4 is pumped into synthesis column 11 at a flow rate of 100-400 cm / h for 2-5 cv for cleaning. After cleaning, the cleaning reagent is pumped into synthesis column 1 again... The morpholinophosphoramide monomer reagent and activating reagent loaded in reagent bottle 4 are simultaneously pumped into synthesis column 11 at a flow rate of 150-300 cm / h for mixing. The synthesis column and the post-column heating are maintained at 30-60℃. The reagents are circulated between outlet tube 13, inlet tube 12 and synthesis column 11 for 60-120 min to carry out the coupling reaction. Then, the cleaning reagent loaded in reagent bottle 4 is pumped into the synthesis column at a flow rate of 100-400 cm / h at a flow rate of 2-5 cv. The column is cleaned in column 11. After cleaning, the capping reagent loaded in reagent bottle 4 is pumped into the synthesis column 11 at a flow rate of 60-240 cm / h at a flow rate of 0.5-3 cv to carry out the capping reaction, thus forming a base. Then, the cleaning reagent loaded in reagent bottle 4 is pumped into the synthesis column 11 at a flow rate of 100-400 cm / h at a flow rate of 2-5 cv to carry out the cleaning. After cleaning, each base is cycled through the above steps to finally produce morpholino oligonucleotide.

[0049] The embodiments described above are some, but not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

Claims

1. A continuous flow apparatus for the preparation of morpholino oligonucleotides, characterized in that, The application relates to an automatic synthesizer, which comprises: an automatic synthesizer with a synthesis column, a liquid inlet pipe connected between the inlet of the synthesis column and the automatic synthesizer, and a liquid outlet pipe connected between the outlet of the synthesis column and the automatic synthesizer; a heating device wrapped around the surface of the synthesis column; and a constant temperature device wrapped around the outer wall of the liquid outlet pipe. The liquid inlet pipe and the liquid outlet pipe are metal pipes. The length of the constant temperature device wrapped around the liquid outlet pipe is not less than 1 / 2 of the length of the liquid outlet pipe. The diameter of the liquid outlet pipe is 0.006-0.05 of the length of the liquid outlet pipe.

2. The continuous flow production device of claim 1, wherein, The volume of the liquid outlet pipe is not more than 80% of the volume of the synthesis column.

3. The continuous flow production device of claim 2, wherein, The height of the heating device wrapped around the surface of the synthesis column is not less than the height of the solid phase carrier loaded in the synthesis column.

4. The continuous flow production device of claim 3, wherein, The heating device and the constant temperature device both comprise a temperature probe.

5. The continuous flow production apparatus according to any one of claims 1 to 4, wherein, The heating device is selected from a heating bag or a heating sheet.

6. The continuous flow production device of claim 1, wherein, The constant temperature device is selected from an oil bath device.

7. The continuous flow production device of claim 1, wherein, The automatic synthesizer is further connected with a plurality of reagent bottles.

8. The continuous flow production device of claim 7, wherein, ​ 9. The continuous flow production device of claim 7, wherein, ​ 10. The continuous flow production device of claim 1, wherein, ​

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