Method for synthesizing therapeutic drugs using RNA and a modular synthesis apparatus.

Abstract

This invention provides a method for RNA synthesis using a liquid flow apparatus. [Solution] The method includes the steps of introducing a plurality of reactants, including at least one type of nucleoside triphosphate (NTP), a reaction buffer, and DNA, a DNA-based compound, or a DNA-based mixture, into a first liquid flow module via a plurality of inflow ports; reacting at least some of the reactants in a reaction channel or well in the first module of the flow system; retaining or recirculating the DNA in the first reactor module to allow the reaction products of the reactants to flow into a first fluid filtration module; and filtering the reaction products in the first filtration module.

Description

[Technical Field]

[0001] The present invention relates to a method for RNA synthesis, and more particularly to a modularized liquid flow system and liquid flow method that can be used for continuous RNA synthesis, automated RNA synthesis, or semi-automated RNA synthesis, and whose scale can be changed. [Background technology]

[0002] A flow reactor (also called a continuous flow reactor) provides a continuous flow of material or reactant through a conduit network of conduits connected to form liquid passages. By enabling or deactivating liquid passages through ports and valves in various configurations of the conduit network, when a mixing reaction filtration module is assembled in the flow system, communication is enabled to control the synthesis, purification, and formulation of RNA or nucleic acid therapeutics for the production of products in a continuous flow format. By operating purpose-created software code through a computer system, it is possible to instruct the control of an entire set of process parameters within the flow system. Process parameters include mixing state, temperature, pH, reagent concentration, monitoring, residence time, purity profile, and yield. Examples of prior art flow reactors are typically formed as assemblies of individual modules connected face-to-face to form unit blocks that manage the flow of liquid. An example of a flow reactor is described in International Publication No. 2013 / 050764.

[0003] Flow technology provides more sustainable, flexible, and efficient manufacturing in pharmaceutical production. Combined with microtechnology and precision engineering flow systems, it can be constructed and configured as a microfactory integrating multiple unit operations such as mixing, reaction synthesis, extraction, separation, filtration, and purification. Furthermore, the small volume-to-surface area ratio unique to flow mixing reaction systems allows for precise control of process states with respect to heat and mass transfer, resulting in faster processes and increased productivity in a smaller equipment footprint. These integrated flow systems, combining unit operations, can be considered microfactories or miniaturized manufacturing systems that reduce factory floor space, lower energy consumption, and improve resource utilization. Therefore, compared to current manufacturing systems commonly used today for producing substances such as preventive vaccines, it is possible to achieve sustainable growth with superior environmental impact, cost-effectiveness, and flexibility.

[0004] Several protocols exist in molecular biology and biotechnology that increasingly utilize lab-on-a-chip (LOC) devices. These LOC devices employ microfabrication to miniaturize and integrate laboratory analytical assays, integrating micro-synthesis and micro-filtration into microchips. These microdevices consume less material, minimize waste, reduce costs, and offer faster response times. By controlling the liquid flow through micromixers, microchannels, and filters, various steps of a given protocol can be integrated into the LOC device, enabling cell sorting, mixing, and, for example, allowing reactions for DNA or RNA synthesis. It is important that LOC devices are formed from non-reactive materials and are preferably transparent to allow real-time visual inspection of reactions on the chip. Transparency also enables spectral analysis and investigation. A preferred material used for rapidly fabricating prototypes for such applications is PDMS (polydimethylsiloxane).

[0005] In 2019, the World Health Organization identified 10 global threats to global health. Eight of these 10 threats address pandemics and, in particular, the accessibility of vaccination in LMICs (low- and medium-income countries), and pandemics such as Ebola and dengue fever have a population impact.

[0006] Therefore, there is an urgent need for equipment and methods suitable for use in the manufacture of vaccines and vaccine precursors that can meet the demands for large-scale, low-cost, and rapid bulk synthesis. In particular, there is a need for a compound manufacturing platform that can be used to meet urgent (i.e., within a few days) vaccine demand, is easy to store and deploy worldwide, is highly productive, and is cost-effective to implement. [Overview of the project]

[0007] Therefore, an object of the present invention is to provide a modular manufacturing platform capable of performing scale-modular synthesis and / or filtration of compounds, including RNA vaccines and other nucleic acid therapeutics, as well as biomolecules and non-biomolecules. A particular object of the present invention is to provide a configuration-modular integrated microfactory flow system used for the preparation of nucleic acid vaccine products.

[0008] This disclosure provides a modular, integrated device influenced by laboratory protocols and a configuration that is highly scalable and adaptable, including a method for rapidly producing RNA and nucleic acid materials to the final stage when in use, for example, in hospital installations.

[0009] In particular, this system includes a combination of a flow reactor, a continuous filtration unit, and a mixing unit. These units may be integrated in any order suitable for target RNA, nucleic acid therapeutics, or preventive vaccines. This integrated flow system may also include several appropriately selectable analytical probes, including optical fiber probes, detectors, and light sources for UV-Vis absorbance or fluorescence spectroscopy, used for insightful control and in-line control of the synthesis or production of target substances in a continuous flow process.

[0010] The specific objective is to provide a microfluidics-based apparatus and method for integrating modules including a fluid bioreactor module and a fluid filtration module, which can be operated continuously and automated using electronically controlled utilities and / or software-controlled utilities involved in the use of components such as membranes, pumps, actuators, valves, gates, supply ports, outlet ports, syringe pumps, sensors, etc., connected via corresponding conduits / flow channels to form a continuous fluid flow path.

[0011] A specific objective is to provide a liquid flow system used for the reaction synthesis of predetermined chemical components, such as biomolecules and non-biomolecules, through processes that may be continuous or discontinuous, automated or semi-automated. Another specific objective is to provide a modular system in which individual modular units can be connected according to the synthesis pathway. Such units may include ports, valves, and appropriate connectors that enable communication and connection between each module. Yet another specific objective is to provide a liquid reaction system that can be configured to be responsive to system characteristics such as liquid pressure, temperature, pH, volume, the ratio of one or more predetermined chemical components, liquid flow rate, and the reaction state of the liquid.

[0012] Another specific objective is to provide a fluid filtration apparatus and method capable of separating the chemical components of a liquid in an automated fluid flow system or a semi-automated fluid flow system. The objective is to provide a filtration system that can be operated continuously by appropriate electronic control, liquid supply, recirculation, and / or hydraulic liquid transport. Yet another specific objective is to provide a fluid flow system having one or more reactor modules and filtration modules connected to form a network.

[0013] Another objective is to provide a method and apparatus for synthesizing RNA. Yet another objective is to provide an apparatus and method for synthesizing RNA from DNA. Yet another objective is to provide a form of synthetic RNA that can be used in the preparation of vaccines.

[0014] One object of the present invention is to provide a modular manufacturing platform capable of synthesizing and / or filtering biomolecules and non-biomolecules, particularly RNA and the vaccines subsequently obtained. A specific object is to provide apparatus and systems that can be configured as a microfactory used for biomolecular synthesis, enabling downstream vaccine production.

[0015] The specific objective is to provide an apparatus and method for forming a fluid integration system that includes element module components, including a fluid bioreactor module and a fluid filtration module, which are connected via corresponding conduits / flow channels to form a fluid path, and which can be operated continuously and automated using electronic and / or software control utilities.

[0016] A first aspect of the present invention provides an RNA synthesis method comprising the steps of: introducing a plurality of reagents, including at least one nucleoside triphosphate (NTP), a reaction buffer, and DNA, a DNA-based compound, or a DNA-based mixture, into a first liquid flow module via a plurality of inflow ports; reacting at least some of the reagents in a reaction channel or well in the first module of the flow system; retaining or recirculating the DNA in the first reactor module to allow the reaction products of the reagents to flow into a first fluid filtration module; and filtering the reaction products in the first filtration module.

[0017] In some cases, at least one type of nucleoside triphosphate (NTP) is a solution of at least one type of nucleoside triphosphate (NTP).

[0018] In some cases, at least one nucleoside triphosphate (NTP) may contain one or a combination of adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), or uridine triphosphate (UTP).

[0019] In some cases, the DNA is plasmid DNA. In some cases, the reagents further include one or a combination thereof of an enzyme mixture, a salt solution, or RNA polymerase.

[0020] In some cases, the method includes the step of recirculating at least some of the plurality of reactants from the outlet of the first filtration module to the inlet region of the first reactor module. In some cases, the method includes the step of feeding at least a portion of the filtered reaction product from the first filtration module to a second liquid flow reactor module, in combination with introducing a capping enzyme into the second reactor module. In some cases, the method includes the step of feeding the liquid flowing out from the second reactor module to a second liquid flow filtration module. In some cases, the method includes the step of recirculating unreacted NTP to the inlet region of the first reactor module and recirculating unreacted capping enzyme to the inlet region of the second reactor module.

[0021] In some cases, the salt solution or buffer contains MgCl2. In some cases, the RNA polymerase contains T7 polymerase.

[0022] According to another aspect of the present invention, there is provided an RNA or RNA-based compound prepared by the method claimed in the present application.

[0023] According to yet another aspect of the present invention, there is provided the use of an RNA or RNA-based compound prepared by the method claimed in the present application in the preparation of a vaccine.

[0024] According to yet another aspect of the present invention, there is provided a liquid flow filtration device including a first elongated liquid flow channel having an inlet, a second elongated liquid flow channel having an outlet, and a permeable membrane disposed to partition the first channel from the second channel along the lengths of the first and second channels such that permeate can pass from the liquid in the first channel through the membrane to the second channel along the lengths of the first and second channels.

[0025] In some cases, most of the length of the first channel is disposed adjacent to most of the length of the second channel through the membrane.

[0026] Depending on the case, the pore size of the membrane may be in the range of 100-1000kDa, 100-800kDa, 200-800kDa, 200-600kDa, or 300kDa-10MDa.

[0027] Depending on the configuration, the apparatus may include a first plate on which a first channel is formed, and a second plate on which a second channel is formed, if any, with a membrane sandwiched between the opposing surfaces of the first and second plates. Depending on the configuration, each of the first and second channels may include a series of straight and curved sections. Depending on the configuration, the first and second channels may include their respective meandering profiles in their longitudinal directions. Depending on the configuration, the first and second channels may be open along their lengths and positioned in direct contact with a membrane that partially forms the longitudinal walls or surfaces of the first and second channels. Depending on the configuration, the pore size of the membrane may be less than the average molecular size of RNA molecules.

[0028] A further aspect of the present invention provides a fluid flow system for processing a liquid, comprising a reaction flow channel or well, a first reactor module having at least one inlet and at least one outlet, a fluid filtration region, and a first filtration module having at least one inlet and at least one outlet, provided in communication with the outlet of the first reactor module, wherein the first filtration module includes a fluid filtration device as claimed in this application.

[0029] Depending on the circumstances, the system may include a second reactor module having a reaction flow channel or well, at least one inlet, and an outlet, the inlet being in communication with the first filtration module.

[0030] Depending on the circumstances, the system may include a liquid filtration area and a second filtration module having at least one inlet and an outlet, wherein the inlet is provided in communication with the outlet of the second reactor module.

[0031] Optionally, the system includes a liquid injection port provided in communication with the inlet region of the first filtration module. Optionally, the system includes a first recirculation conduit extending between the outlet region of the first reactor module and at least one inlet of the first reactor module. Optionally, the system includes a second recirculation conduit extending between the outlet region of the first filtration module and the outlet of the first reactor module. Optionally, the system includes a third recirculation conduit extending between the outlet region of the second filtration module and the inlet of the first reaction module.

[0032] A further aspect of the present invention provides a method for filtering a liquid using a liquid flow filter, comprising the steps of: flowing a liquid from an inlet into a first narrow liquid flow channel; forcing a permeate component of the liquid to flow through a membrane extending along the first channel into a second narrow liquid flow channel; and retaining a stagnant component of the liquid in the first channel, wherein the membrane is positioned to separate the first channel from the second channel along their respective lengths so that the permeate can pass from the first channel through the membrane into the second channel along their respective lengths.

[0033] A further aspect of the present invention provides a flow system comprising: at least one reactor module having a reaction liquid flow channel or well, at least one liquid inlet, and at least one liquid outlet; at least one flow delivery unit for flowing liquid into the channel or well; a first sensor for measuring one or a combination thereof of pressure, temperature, or pH of the liquid in the system; a second sensor for measuring one or a combination thereof of pressure, temperature, or pH of the liquid in the system; a reaction state monitoring device for monitoring properties of the liquid in the system indicating the reaction state of at least two chemical components of the liquid in the system; and a control unit for receiving data from at least one or a combination thereof of the first sensor, the second sensor, and the reaction state monitoring device for controlling at least one property of the liquid in the system.

[0034] Depending on the circumstances, the system characteristics may be one or a combination of the following: the pressure of the liquid in the system, the temperature of the liquid in the system, the pH of the liquid in the system, the volume or ratio of one or more chemical components in the liquid in the system, or the flow rate of the liquid in the system. Depending on the circumstances, the step of flowing the liquid may include pressurizing the liquid in a first flow path. Depending on the circumstances, the control unit may include a CPU, PCB, PLC, PC, processor chip, or handheld electronic device. Depending on the circumstances, further sensors may include one or a combination of a temperature sensor, pH sensor, pressure sensor, flow rate sensor, fluid flow rate sensor, or spectroscopic sensor.

[0035] A further aspect of the present invention provides a method for processing a liquid using a liquid flow device, comprising the steps of: introducing at least one type of liquid into a reactor module through at least one inlet; flowing the liquid through a reaction flow channel or well, or flowing within a reaction flow channel or well, using at least one flow supply unit to discharge the liquid at an outlet; measuring at least one or a combination thereof of pressure, temperature, or pH of the liquid in the liquid flow device; monitoring the reaction state of chemical components in the liquid in the liquid flow device; and controlling at least one characteristic of the liquid in the liquid flow device using a control unit in accordance with at least one or a combination thereof of measuring the pressure, pH, temperature, and / or reaction state of the chemical components of the liquid.

[0036] Depending on the configuration, the system may include multiple reactor modules and filtration modules connected in a communication state. Depending on the configuration, the flow delivery unit may be at least one pump, which may be a syringe pump. Depending on the configuration, the liquid analysis sensor may include a UV-Vis spectrometer for absorbance analysis or fluorescence analysis in the range of 190 nm to 1000 nm.

[0037] In some cases, the RNA synthesis method includes the steps of recirculating unreacted NTPs to the inflow region of the first reactor module and recirculating unreacted capping enzymes to the inflow region of the second reactor module.

[0038] In yet another aspect of the present invention, RNA prepared by the method claimed in this application is provided. In yet another aspect of the present invention, the use of RNA prepared by the method of any prior claim in the preparation of a vaccine is provided.

[0039] A liquid flow device is provided, comprising a reaction flow channel or well, a first reactor module having at least one inlet and at least one outlet, and a fluid filtration region, a first filtration module having at least one inlet and at least one outlet, wherein the inlet is provided in communication with the outlet of the first reactor module.

[0040] Depending on the circumstances, the apparatus may include a second filtration module having a fluid filtration region, at least one inlet, and an outlet, wherein the inlet is provided in communication with the outlet of the second reactor module.

[0041] Optionally, the device includes a liquid injection port provided in communication with the inlet region of the second reactor module. Optionally, the device includes a first recirculation conduit extending between the outlet region of the first reactor module and the inlet region of at least one of the first reactor modules.

[0042] Optionally, the apparatus includes a third recirculation conduit extending between the outflow area of ​​the second filtration module and the inflow area of ​​at least one of the first reaction modules. Optionally, the apparatus includes a plurality of inflow ports that allow fluid chemical components to flow into the reaction channel or well. Optionally, the apparatus includes at least one pump connected to at least one inflow of the first reactor module to deliver a liquid flow into the reaction channel or into the well. Optionally, the apparatus includes at least one valve, liquid flow gate, liquid flow port, heating element, liquid storage unit, or stagnant liquid reservoir provided in communication with the liquid in the apparatus. Optionally, the pump includes a syringe pump.

[0043] In some cases, the device has a plate-like structure in which a first reactor module and a first filtration module are at least partially formed as flow channels or recessed grooves provided on or within the plate-like structure. In some cases, the device includes a plurality of sensors arranged in different liquid flow regions of the device.

[0044] Depending on the circumstances, the sensor may include at least one temperature sensor, at least one flow sensor, at least one pressure sensor, at least one pH sensor, at least one fluid volume sensor, at least one spectroscopic sensor, at least one optical sensor, at least one optical fiber or spectroscopic optical fiber, or a combination thereof.

[0045] Depending on the circumstances, the apparatus may include a control unit connected to at least one pump, valve, liquid flow gate, liquid flow port, heating element, liquid storage unit or retention reservoir, and sensor, which controls the characteristics of the liquid flow in the apparatus according to the physical, chemical, or mechanical properties of the liquid quantified by the sensor. Depending on the circumstances, the control unit may include a CPU, processor, PCB, PLC, or handheld electronic device.

[0046] Depending on the circumstances, the control unit may include a control module that includes software, electronic components, data storage utilities, wired or wireless communication modules and / or ports, a visual display output unit, a user interface, at least one pump, valve, liquid flow gate, liquid flow port, heating element, liquid storage unit or retention reservoir, and at least one actuator or combination thereof that operates one or a combination thereof of a sensor.

[0047] A liquid processing system is provided, comprising a plurality of devices as defined in the claims of this application, and a final liquid flow outlet for each of the devices, each of which is connected to a collection unit that collects the liquid flowing out of each of the devices.

[0048] Depending on the circumstances, the liquid flow system may include a control unit connected to at least one pump, valve, liquid flow gate, liquid flow port, heating element, liquid storage unit or retention reservoir and a sensor, which controls the characteristics of the liquid flow in the apparatus according to the state of the physical, chemical, or mechanical properties of the liquid quantified by the sensor.

[0049] A method for treating a liquid using a liquid flow device is provided, comprising the steps of: introducing at least one type of liquid into a first reactor module through at least one inlet, flowing the liquid through a reaction flow channel or well, or flowing within a reaction flow channel or well, to discharge the liquid at an outlet of the first reactor module; and flowing at least a portion of the liquid from the outlet through at least one inlet to a filtration module having a liquid filtration region, to discharge the liquid from the first filtration module at an outlet, wherein a reaction occurs between at least two chemical components of the liquid in the first reactor module, and the liquid is filtered in the first filtration module.

[0050] Depending on the circumstances, the method may include the step of flowing a liquid through the apparatus using at least one fluid pump. Depending on the circumstances, the method may include the step of recirculating at least a portion of the liquid from the outlet of the reactor module and / or filtration module to the area of ​​at least one inlet of the reactor module via at least one recirculation conduit. Depending on the circumstances, the method may include the step of monitoring the state of chemical reactions between chemical components occurring in the liquid using at least one sensor. Depending on the circumstances, the method may include the step of controlling the liquid flow through the apparatus in accordance with the state of chemical reactions identified by the sensor. Depending on the circumstances, the method may include the step of monitoring the physical, chemical and / or mechanical properties of the liquid in the device using at least one sensor. Depending on the circumstances, the method may include the step of controlling the liquid flow in the apparatus in accordance with the physical, chemical and / or mechanical properties of the liquid identified by the sensor. Depending on the circumstances, the step of controlling the liquid flow may include controlling or stopping the operation of at least one actuator, pump, valve, gate or port provided in communication with the liquid in the apparatus. Depending on the circumstances, the step of controlling the liquid flow may include the step of operating or deactivating at least one actuator, pump, valve, gate, or port using a CPU, processor, PCB, PLC, or handheld electronic device.

[0051] A further aspect of the present invention provides a liquid flow filtration apparatus comprising a first narrow liquid flow channel having an inlet, a second narrow liquid flow channel having an outlet, and a permeable membrane positioned along the respective lengths of the first and second channels to separate the second channel from the first channel, so that a permeate can pass from the liquid in the first channel through the membrane to the second channel along the length of the first and second channels.

[0052] Depending on the circumstances, the first flow path may include at least one outlet, wherein the inlet is located at or near the first longitudinal end of the first flow path, and the outlet is located at or near the second longitudinal end of the first flow path. Depending on the circumstances, the second flow path may include at least one inlet, wherein the inlet is located at or near the first longitudinal end of the second flow path, and the outlet is located at or near the second longitudinal end of the second flow path.

[0053] A further aspect of this application is provided, a fluid flow system for use in processing a liquid, comprising a reaction flow channel or well, a first reactor module having at least one inlet and at least one outlet, a fluid filtration region, and a first filtration module having at least one inlet and at least one outlet, provided in communication with the outlet of the first reactor module, wherein the first filtration module includes a fluid filtration device as claimed in this application.

[0054] A further aspect of the present invention provides a method for filtering a liquid using a liquid flow filter, comprising the steps of: flowing a liquid from an inlet into a first narrow liquid flow channel; forcing a permeate component of the liquid to flow through a membrane extending along the first channel into a second narrow liquid flow channel; and retaining a stagnant component of the liquid in the first channel, wherein the membrane is positioned to separate the first channel from the second channel along their respective lengths so that the permeate can pass from the first channel through the membrane into the second channel along their respective lengths.

[0055] A further aspect of the present invention provides a liquid flow system comprising: at least one reactor module having a reaction liquid flow channel or well, at least one liquid inlet, and at least one liquid outlet; at least one flow delivery unit for flowing liquid into the channel or well; a first sensor for measuring one or a combination thereof of pressure, temperature, or pH of the liquid in the system; a second sensor for measuring one or a combination thereof of pressure, temperature, or pH of the liquid in the system; a reaction state monitoring device for monitoring properties of the liquid in the system indicating the reaction state of at least two chemical components of the liquid in the system; and a control unit for receiving data from at least one or a combination thereof of the first sensor, the second sensor, and the reaction state monitoring device for controlling at least one property of the liquid in the system.

[0056] A further aspect of the present invention provides a method for processing a liquid using a liquid flow device, comprising the steps of: introducing at least one type of liquid into a reactor module through at least one inlet; flowing the liquid through a reaction flow channel or well, or flowing within a reaction flow channel or well, using at least one flow supply unit to discharge the liquid at an outlet; measuring at least one or a combination thereof of pressure, temperature, or pH of the liquid in the liquid flow device; monitoring the reaction state of chemical components in the liquid in the liquid flow device; and controlling at least one characteristic of the liquid in the liquid flow device using a control unit in accordance with at least one or a combination thereof of measuring the pressure, pH, temperature, and / or reaction state of the chemical components of the liquid. [Brief explanation of the drawing]

[0057] Hereinafter, specific examples of the present invention will be described with reference to the attached drawings. [Figure 1] This is a schematic diagram of a continuous reaction filtration apparatus suitable for RNA production. [Figure 2] This is a perspective view of a reactor module and a filtration module suitable for use in the apparatus shown in Figure 1. [Figure 3]This is a plan view of a well bioreactor module, or batch bioreactor module, suitable for use in the apparatus shown in Figure 1. [Figure 4] This is a plan view of a bioreactor module with a reaction channel shape suitable for use in the apparatus shown in Figure 1. [Figure 5A] This is a plan view of a filtration module suitable for use in the apparatus shown in Figure 1. [Figure 5B] Figure 5A shows an image of the membrane inside the filtration module. [Figure 6A] This is a partial diagram of a filtration module having a region configured for centrifugal separation of chemical components, suitable for use in the apparatus shown in Figure 1. [Figure 6B] This is an image of a portion of a filtration module that has a region for centrifugal separation of chemical components. [Figure 7] This is a vertical tangential flow filtration (VTFF) module used in this liquid flow device. [Figure 8] Figure 7 is an enlarged view of the filtration module area. [Figure 9] This is a plan view of a spiral tangential flow filtration module. [Figure 10A] These are components of a filtration module related to a specific implementation example. [Figure 10B] Figure 10A is a schematic diagram of another part of the filtration module. [Figure 10C] This is another diagram of a part of the filtration module shown in Figures 10A and 10B. [Figure 11A] This is a plan view of another filtration module formed as a two-layer microchannel TFF. [Figure 11B] This is a schematic diagram of a filtration mechanism and filtration module relating to a specific implementation example. [Figure 12] This is a schematic diagram of a flow-type bioreactor module. [Figure 13] This graph was used in the design calculations for a fluid bioreactor and filtration system. [Figure 14]This is a schematic diagram of a reactor module connected to a filtration module to form a liquid flow device. [Figure 15] This is a perspective view of the TFF module. [Figure 16] This is a microscopic analysis image of the flow path dimensions of the TFF module. [Figure 17] This is a schematic diagram outlining the prototype manufacturing process. [Figure 18A] This is a schematic diagram of a liquid flow system including a reactor module, a series of sensors, a reaction state monitoring configuration, and a control unit, relating to a specific implementation example. [Figure 18B] Figure 18A is a schematic diagram of components of a suitable reaction state monitoring configuration as part of the liquid flow system. [Figure 18C] This is a schematic diagram of a series of sensors, a reaction state monitoring configuration, and a control unit that form part of the liquid flow system shown in Figure 18A, which relates to a specific implementation example. [Figure 19] Figure 18 is a schematic diagram of a portion of the architecture of the microfluidic flow system, including a part of the spectrophotometer configuration. [Figure 20] This is a schematic diagram of a serial communication program used in a control system. [Figure 21] This graph shows the relationship between RNA production and the proportions of various reagents, including NaCl, MgCl2, and NTPs. [Figure 22] This graph shows data analysis related to the embodiment of the invention (RNA production in relation to magnesium ions) and the selection of a specific reaction time. [Figure 23A] This is a schematic diagram of the simulation results of the velocity profile analysis. [Figure 23B] This is a graph of the velocity analysis. [Figure 24] This is a schematic diagram of the velocity of the tangential flow filtration channel in the filtration module. [Figure 25A] This is a graph of the pressure drop in the bioreactor flow path. [Figure 25B] This is a graph of pressure drop undergoing multi-stage descent. [Figure 26A]This is a photograph of a mask used in the manufacture of liquid flow reactors and filtration systems. [Figure 26B] This is a photograph of the mask for a continuous microfluidic flow reactor. [Figure 27] This is an image of a continuous flow reactor sealed with Kapton tape. [Figure 28] This is an image of a composition completed using two acrylic plates. [Figure 29] This is an image of the 3D printed mold after use. [Figure 30A] This is the first part of a schematic diagram of an embodiment of this flow system, which is integrated with a downstream formulation system using a modular flow reactor and attached to a conventional filled-finish vaccine manufacturing line. [Figure 30B] This is the second part of a schematic diagram of an embodiment of this flow system, which is integrated with a downstream formulation system using a modular flow reactor and attached to a conventional filled-and-finished vaccine manufacturing line. [Figure 30C] This is the third part of a schematic diagram of an embodiment of the flow system, which is integrated with a downstream formulation system using a modular flow reactor and attached to a conventional filled-and-finished vaccine manufacturing line. [Figure 30D] These are graphs of the absorbance of the production solution of the flow reactor in embodiments Figures 30A to 30D against wavelength, evaluated using ultraviolet-visible spectroscopy compared to a conventional batch protocol. [Figure 31] This is a photograph of an experimental liquid-flow compound synthesis platform. [Figure 32] This is a live plot of spectrophotometer data (intensity against wavelength) generated using Python. [Figure 33] These are images of samples with different concentrations (A: 25 mM / L, B: 50 mM / L, C: 75 mM / L, D: 100 mM / L) used in conjunction with a microfluidic flow reactor module and a continuous filtration system. [Figure 34] This is a graph of ultraviolet-visible spectral analysis. [Figure 35]This is an image of the pH data plot generated by Python. [Figure 36] This is a graph of the prediction plot (the left shows the response when dNTP is 1mM, and the right shows the response when dNTP is 4mM). [Figure 37] The graphs of the predictive profilers for the first and second experiments are shown (the top graph shows the response with dNTP 1mM, and the bottom graph shows the response with dNTP 4mM). [Figure 38] This is an overview of various data from the liquid flow bioreactor and filtration (left: reaction with dNTP 1mM, right: reaction with dNTP 4mM, LogWorth = -log10(p-value)). [Figure 39] This is a schematic diagram of the scaled-up liquid flow reactor and liquid flow device used in this system. [Figure 40A] This figure shows the source code for real-time plotting (absorbance against wavelength) using Python, along with absorbance data. [Figure 40B] This is a diagram of the source code for the Python live plot of pH data. [Modes for carrying out the invention]

[0058] Detailed Description of Preferred Embodiments of the Invention Principles of fluid dynamics in microporous channels Advances in microfabrication have made it possible to construct microchannels with dimensions in the micrometer range. Since microchannels are typically incorporated into these microsystems, it is crucial to determine the characteristics of the liquid flow in these microchannels, which are used in the design of various microflow devices. Understanding the liquid behavior in porous media, particularly microporous media that can be used in lab-on-a-chip designs for synthetic RNA vaccine production, is severely limited due to technical constraints.

[0059] A porous medium is prepared and impregnated with a liquid that does not form an interface between liquids, allowing one type of liquid to spread throughout the pore space. Let dp be the particle size and U be the order of velocity. It has been found that (1) is applicable to Reynolds numbers of 1 or less. That is,

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[0060] The permeability of a porous medium is a property that depends on the pore size and pore structure. Dimensional analysis has shown that permeability is a function of porosity (e) and particle size (dp), where these represent pore shape and pore size, respectively. The Carman-Kozeny relation empirically links these quantities as follows, while maintaining dimensionality.

number

number

[0061] Bahrami et al. (M. Bahrami, 2006) developed a general model for predicting pressure drop in microchannels of arbitrary cross-sections. The selection of characteristic length is arbitrary and does not affect the final solution. According to Bahrami et al.'s model, the pressure drop of a flow that has finished changing from laminar flow in a microchannel of arbitrary cross-section can be obtained as follows.

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[0062] Given that the volumetric flow rate Q and cross-sectional area A are known, the Reynolds number was calculated from the following.

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[0063] Minor losses ΔPmin (other pressure drops related to the measured pressure drop) are inlet losses, outlet losses, and bend losses. These losses are usually derived from conventional relationships used on a macroscopic scale. Phillips (reference) showed that minor pressure losses can be derived from the following:

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[0064] Here, A and A t These are the cross-sectional areas of the flow path and connecting pipe, respectively. b is the loss coefficient for the curved section, and Kc and K e This represents the reduction and expansion loss factors due to the change in area. Phillips is approximately 1.2 for a 90-degree bend. b This was recommended. The cross-sectional area of ​​the flow path and the cross-sectional area of ​​the connecting pipe are equal, K c and K e Assuming that the possible value of is maximized, the relative minor loss with respect to the measured pressure drop can be ignored compared to the measured pressure drop.

[0065] Hooman and Merrikh (M. Bahrami, 2010) developed the following analytical methods for flow and pressure drop in porous channels.

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[0066] The cross-sectional aspect ratio ε of the sample tested in this study is 0.5. Therefore, although a perfectly rectangular cross-section is not considered, the sample can be modeled as a porous medium sandwiched between two parallel plates.

[0067] Select the appropriate cassette according to the total sample volume, required process time, and desired final sample volume. Use the following formula to calculate the membrane area required to process the sample within the given time.

number

[0068] Liquid flow simulation for prototype evaluation The height of the channel and the type of liquid affect the performance of the microchannel. The computational fluid dynamics (CFD) software FLUENT was used to simulate the microchannel. From the CFD, the velocity profiles between the transitional and completed transitional regions were analyzed to examine the behavior of the liquid flow. The properties of the liquid affect the liquid flow within the microchannel. According to the inventors, a minimum kinematic viscosity and low surface tension are necessary, depending on the selected, appropriate liquid.

[0069] Because micro-devices have potential in chemical and biochemical engineering, liquid flow plays a crucial role in integrating micro-devices with a wide range of applications. The mechanisms and principles of single-phase liquid flow were reviewed using experimental data with different types of liquids and surface roughnesses. Therefore, at the microscopic level, the influence of liquid properties such as surface tension and viscosity prevails. (Nawi, MNM, Manaf, AA, Arshad, MR, & Sidek, O. (2013). Numerical simulation of the microchannel for the microfluidic based flow sensor. Proceedings-2012 IEEE International Conference on Control System, Computing and Engineering, ICCSCE 2012, 345-348.)

[0070] The Reynolds number is the ratio of the inertial force to the viscous force acting on a fluid flowing through a channel (i.e., the ratio of the fluid's momentum to the frictional force exerted by the walls of the channel). Flows with a small Reynolds number are laminar flows, or layered flows, where each fluid flows parallel to the others and mixes only by convective diffusion and molecular diffusion. Flows with a large Reynolds number are turbulent flows, where fluid "parcels" of various sizes move in a spatially and temporally random manner, resulting in rapid mixing across the channel. The transition between laminar and turbulent flow typically occurs at Re=2000 in internal flows. (Schulte, TH, Bardell, RL, & Weigl, BH (2002). Microfluidic-technologies-in-clinical-diagnostics_2002_Clinica-Chimica-Acta.321,1-10)

[0071] Turbulent fields are more complex than laminar fields, but they exhibit significant secondary effects. One-dimensional and two-dimensional models illustrating the temporal evolution of the analytical distribution of such conditions have been improved. Confocal fluorescence microscopy observations and three-dimensional numerical analysis modeling can aid in confirming the quantitative explanation of reaction-diffusion processes near walls. (Schulte, TH, Bardell, RL, & Weigl, BH (2002). Microfluidic-technologies-in-clinical-diagnostics_2002_Clinica-Chimica-Acta.321,1-10)

[0072] Sensor integration with flow system In this study, it would be preferable to construct a sensor-integrated platform to acquire and monitor data throughout the RNA transcription process. Gruber, P. (Gruber, P., Marques, M., Szita, N. and Mayr, T. (2017). Integration and application of optical chemical sensors in microbioreactors. Lab on a Chip, 17(16), pp.2693-2712.) proposed a flowchart illustrating the path toward robust sensor integration in liquid flow devices. This diagram could serve as a guide for considering integration. Various integrated platform systems have been designed and developed for real-time reaction monitoring (Wang, T., Kim, S. and An, J. (2017). A novel CMOS image sensor system for quantitative loop-mediated isothermal amplification assays to detect food-borne pathogens. Journal of Microbiological Methods, 133, pp.1-7.). Wang's method demonstrated the high potential of complementary metal-oxide-semiconductor (CMOS) technology for integration and detection. The CMOS image sensor acts as an effective reaction detection platform, allowing for real-time monitoring of photon changes according to the amplification process. Photons were observed by the CMOS image sensor and converted into digital units. In addition, UV spectroscopy, detection of light color intensity, and pH analysis were performed to demonstrate the correctness of the CMOS efficiency (Wang, T., Devadhasan, J., Lee, D. and Kim, S. (2016). Real-time DNA Amplification and Detection System Based on a CMOS Image Sensor. Analytical Sciences, 32(6), pp.653-658.).Lopez-Huerta (Lopez-Huerta, F., Woo-Garcia, R., Lara-Castro, M., Estrada-Lopez, J. and Herrera-May, A. (2013). An Integrated ISFET pH Microsensor on a CMOS Standard Process. Journal of Sensor Technology, 03(03), pp.57-62.) proposed an integrated ISFET pH microsensor for a standard CMOS process. Silicon region: 1.12 mm. 2 The completed system was incorporated and exhibited linearity of 59 mV / pH in the concentration range of pH levels 2 to 12, making it an excellent alternative for biological and medical applications.

[0073] While CMOS is a reasonable choice, its implementation is difficult considering the cost and time involved. A more realistic approach was taken, and the idea of ​​integrating each sensor was reconsidered. Regarding the pH sensor, it is known that there is research on real-time feedback control of pH within microfluidics using integrated sensing and actuation (Welch, D. and Christen, J. (2014). Real-time feedback control of pH within microfluidics using integrated sensing and actuation. Lab on a Chip, 14(6), p.1191.). In this system, an extended-gate ion-response field-effect transistor (ISFET) with an integrated pseudo-reference electrode was used to monitor the pH value within the liquid-flow reaction chamber. Regarding the temperature sensor, a serpentine-shaped temperature sensor (wire width: 50 mm) and a heater (wire width: 400 mm) were incorporated into the lower center of the reaction chamber (Sun, H., Olsen, T., Zhu, J., Tao, J., Ponnaiya, B., Amundson, S., Brenner, D. and Lin, Q. (2015). A bead-based microfluidic approach to integrated single-cell gene expression analysis by quantitative RT-PCR. RSC Advances, 5(7), pp.4886-4893.). Choi, K., Mudrik, J., and Wheeler, A. (2015). (A guiding light: spectroscopy on digital microfluidic devices using in-plane optical fiber waveguides. Analytical and Bioanalytical Chemistry, 407(24), pp.7467-7475.) presented a novel method for in-plane digital microfluidic spectroscopy. This technique enables optical measurements within the device's plane by aligning the optical fiber with the digital microfluidic device using a dedicated manifold, thus providing an idea for optical detection.

[0074] The preferred wavelength range for analysis in this invention is 190 nm to 1000 nm. The resulting peak is approximately 260 nm.

[0075] method Continuous flow synthesis and scaling up Figure 1 shows a schematic diagram of a continuous RNA production process. As shown in Figure 1, the fluid flow device includes a first bioreactor module 10, a first tangential flow filtration (TFF) module 11, a second bioreactor module 12, and a second tangential flow filtration (TFF) module 13. Each of modules 10-13 is connected via its respective flow path or conduit. The first reactor module 10 is provided with multiple fluid supply ports 14 for introducing each reagent into the first bioreactor 10. Each port 14 includes a corresponding syringe pump 15. The first TFF includes a corresponding inlet / inlet port 16, the second bioreactor module includes a corresponding inlet / inlet port 17, and the second TFF module includes a corresponding inlet / inlet port 18. Each of ports 16-18 may be provided with a corresponding auxiliary pump or syringe port for introducing liquid and pressurizing it to flow through the narrow reaction channel 24 of each reactor and the narrow filtration channel 25 in each module. Multiple recirculation channels 19, 20, 21 connect and communicate various liquid flow regions of modules 10, 11, 12, 13 (regions in their respective inlet / outlet end regions or regions in their respective inlet / outlet end regions) to reflux the reagents / pharmaceuticals. The apparatus in Figure 1 also includes sensors, reaction state monitoring devices, control units, and other auxiliary electronic and actuator components (not shown) as described elsewhere in this specification, to provide a fully automated continuous liquid-flow synthesis platform for the production of compounds including biological and non-biological species. The outlet end of the narrow reaction channel 24 of the first bioreactor 10 includes a gate 23, which is configured to filter large molecular weight chemical components such as DNA from the liquid based on molecular size and / or molecular weight. Subsequently, the filtered desired liquid is recirculated through the flow path 19 to the inlet region of the reactor 10. In this way, a continuous or semi-continuous process can be achieved by reusing the predetermined chemicals and reactants.

[0076] Referring to Figures 1 and 2, Figure 2 shows a modification of the fluid system of Figure 1, in which the reactor 10 includes a reaction well 50 in contrast to the narrow reaction channel 24 of Figure 1. As described with reference to Figure 1, several injection ports 14 are connected to and communicate with the well 50 for supplying desired compounds, drugs, solvents, etc., used in the reaction. Filtration modules 11, 12 may also include a series of extraction ports 51, 22, which allow for the extraction of predetermined species, drugs, solvents, etc., from the filtration module, and at port 22, the final product, such as fully capped RNA, can also be collected.

[0077] As can be seen from the above, the specific details of the flow path profile, ports, gates, inlets, outlets, and the use of pumps and fluid flow control actuators can be implemented using the configurations described herein.

[0078] Referring to FIG. 39, a plurality of reactant filtration systems described with reference to FIGS. 1 and 2 (and shown in their entirety as plate-like units 10, 11, 12, 13) may be connected together as a scaled-up liquid flow reactor and liquid flow device to form a fully integrated modular liquid flow reaction filtration system for the production of therapeutic agents and prophylactic vaccines using RNA, at a scale sufficient for evaluation, clinical trials, and ultimately manufacturing. In particular, the output sections (located at output section 22) of each unit are connected to communicate with each other through appropriate connecting conduits so that the total output can be obtained collectively. The integrated system of FIG. 39 may include a single control unit, multiple control units, corresponding sensors, a reaction state monitoring utility, and units as described herein with reference to FIGS. 18A - 20. By operating a plurality of fluid devices of the present invention in parallel, continuous production of therapeutic agents and prophylactic vaccines using RNA can be achieved. By using this scaling method, it is possible to reach the production of products at the scale required by the target market that can be served by regional hospitals, or the target market where the units are attached to conventional filling and finishing lines at the manufacturer's production site. FIG. 20 is a schematic diagram of a serial communication program used in a control system between an Arduino IDE control utility and a Python IDE control utility. In a possible embodiment, an Arduino board can be used. In a preferred embodiment, an industrial edge computing SBC (Single Board Computer) node is used.

[0079] As an example, RNA synthesis is used, but during the process, segments of DNA are copied into RNA by the enzyme RNA polymerase. The transcription mixture is composed of NTP (nucleoside triphosphate), DNA, MgCl2 (which reacts to form a precipitate and affects RNA productivity), and T7 polymerase (for catalysis), and these are each injected into the first bioreactor flow path by a syringe pump. Mg -4 reacts to form a precipitate and affects RNA productivity), T7 polymerase (for catalysis), and these are each injected into the first bioreactor flow path by a syringe pump. Mg 2+Hydroxynaphthol blue (HNB) is used as a colorimetric quantitative indicator for ion titration. First, HNB and Mg 2+ By combining with ions, HNB-Mg 2+ Form a complex. Mg 2+ As a result of the decrease in ions, the color changes from purple to sky blue. The mixture remains in the first reactor 10 for approximately 6 hours, during which time the RNA transcription reaction proceeds continuously, and RNA begins to be produced, accompanied by a magnesium pyrophosphate precipitate (Mg2P2O7). All of the above components except DNA flow through the first tangential flow filtration (TFF) module 11. Module 11 has a membrane with a large molecular weight (MW). The DNA is filtered and refluxed to the original inflow port 14(2) via the recirculation channel 19 and remains in the first reactor, while the rest is filtered and proceeds to the next stage.

[0080] After the first filtration module, the above components are passed downstream while T7 polymerase is recirculated to the inflow port 14(4). Then, m7G methyltransferase (5' cap) is injected into port 17 and flows into the second reactor 12 and channel 24, where the inflow remains and reacts for 2 hours. The pass-through material flows into the second TFF section 13, which has a membrane with a moderate fractionation MW. In this filtration stage, unreacted NTPs and 5' caps are retained and recirculated (via 21 and 20) and flow into bioreactors 12 and / or 10. Finally, the purified RNA is pushed out at the efflux section 22. Simultaneously, Mg2PP i Other components, such as water and salts, are collected in a separate container, i.e., port 51, and may be discarded or post-processed as needed. The product (capped RNA) may then be processed by further processing procedures (not described herein, but well known to those skilled in the art) to produce the desired vaccine.

[0081] The objective is to obtain RNA material through continuous synthesis and purification, thereby improving the system's throughput. To maintain the continuous reaction of the process, it is necessary to add raw materials to the reactor via part 14 to replenish the molecules consumed. This can be achieved through adjustment of the actuator syringe pump and a feedback loop for material flow. Substantial improvement in RNA yield may be possible by optimizing the reaction conditions (magnesium concentration, pH, temperature, reaction time, etc.).

[0082] Set-Based Concurrent Engineering (SBCE) method The Lean product development methodology has been chosen as the primary framework for developing the design. Lean product development is set-based concurrent engineering (SBCE). SBCE is a process of dividing the product to be developed into different subsystems, thereby allowing the development of a set of possible solutions for each subsystem in parallel. As the design progresses, the set of solutions for each subsystem is narrowed down by knowledge-based judgment using tools such as simulation, prototyping, verification, and other acquired knowledge. According to the SBCE method, the first essential step is to divide the product into different subsystems that can be developed individually. Considering the conceptual design of the system to be developed, there are four main distinguishable processes that can be grouped into just two functions: the bioreactor and the filtration process. Simultaneously, the system needs to be integrated with a control system that can have sensors in key product parts to control pressure from the pump and measure important parameters. Corresponding the conceptual design of the system made it possible to define different subsystems that could be developed in parallel. Table 1 shows the different subsystems, along with a brief description of each subsystem and the corresponding level of transformation.

[0083] [Table 1]

[0084] Subsystem design specification determination method Bioreactor Two different bioreactor designs were being developed. The first design is influenced by conventional batch processing used in the chemical field for RNA synthesis. The design includes four input sections 14 for each raw material, each connected to a 1 mL main chamber where the reaction takes place, allowing for a sufficient residence time. Subsequently, a microfluidic valve is opened at the outlet of the container to selectively drain the product from the container and introduce new reagents into the inflow section. If equivalent to a conventional batch system, this suggests that the device operates in continuous (perfusion) mode. After the reaction is complete, the product is released and flows into the output channel. A sensor evaluates whether the process can proceed to the next step or not. Figures 2 and 3 show examples of bioreactor designs.

[0085] This design example requires a complex and well-tuned control system to manage the valve integrated with the device. After the residence time has elapsed, the product needs to be supplied to the rest of the system.

[0086] A second bioreactor design is a microflow reactor shaped as a serpentine liquid conduit, enabling the mixing of reagents and the continuous synthesis of the desired product, as described in International Publication No. 2019 / 193346. Details of this International Publication are incorporated herein by reference. In this embodiment, continuous synthesis is possible by controlling the liquid flow. In this design, there are four drug input ports, which are continuously supplied to the flow reactor via a T-mixer. The reaction state and effectiveness are controlled by the net inflow into the liquid conduit and the liquid velocity of the individual components passing through the T-mixer. These are controlled by setting the dispensing rate of the dispensing pump using computer control. The product is formed when the mixture reaches the end of the serpentine flow bioreactor within a predetermined residence time. In the production section, the liquid flow is temporarily slowed by a functional section of a device that functions as a microliquid-holding well, and measurements are obtained in the line using preferred analytical methods for specific chemical solutions, such as in-line UV-Vis spectroscopy. By extracting specific features of the signal from the spectrometer, the signal is parsed and analyzed with purposefully designed software and compared with a reference vector to determine whether the desired product has indeed been formed. Figure 4 is a schematic diagram of the meandering flow bioreactor.

[0087] Filtration module There are two types of filtration: direct flow filtration (DFF) and tangential flow filtration (TFF). In TFF, the liquid flow is parallel to the filter, whereas in DFF, there is a main flow perpendicular to the filter. DFF is highly susceptible to clogging, and it has been demonstrated that the filtrate flow rate of TFF is greater than that of DFF when the amount of filtered material increases. Furthermore, typical applications of TFF include biomolecular concentration, diafiltration and fractionation, and cell clarification and removal, which are similar to the applications in this project. In other words, TFF is the most effective choice for the applications in this project, and therefore the preferred filtration method.

[0088] For the filtration module, the TFF method was selected as one model. Several design solutions have been proposed in research literature and commercial practice. Several other factors, such as ease of manufacture, were considered for this subsystem, and its ability to integrate as a module into the overall system was taken into consideration. A feature of the first filtration module design is a serpentine path with two channels at the same height, where the functional part is separated on the device, and the pattern of the functional part is intentionally formed to function as a filtration membrane. X. Chen et al. (2007) (Microfluidic Chip for Blood Cell Separation and Collection Based on Crossflow Filtration. Sensors and Actuator B 130 (2008) (p.216-p.221). China.) conventionally used a TFF filter design for the separation of living cells that is fast, reusable, and low cost compared to other solutions.

[0089] The present invention utilizes a deliberately selected membrane with a size suitable for separating biomolecules commonly present in solutions after nucleic acid synthesis. Figures 5A and 5B show a filtration module including a filtration function within a serpentine microchannel. The size and length of the channel are deliberately designed to allow selection of the correct biomolecules in the filtrate flow and the retained liquid flow. Such modules are manufactured using microengineering techniques, including photolithography and deep reactive etching of glass or silicon substrates. These techniques are well-known, industrially available, compliant with pharmaceutical regulations, and have low costs for mass production of devices.

[0090] The second filtration module design does not require any filter membrane. The second filtration module design operates primarily using centrifugal force, which causes some particles to move faster than others when the liquid trajectory is a curved path at a given velocity. Therefore, by having a single bent microchannel that splits into two different channels, particles of different sizes can be directed to different outflow channels (Blatter, C., Jurischka, R., Tahhan, I., Schoth, A., Kerth, P., Menz, W. (2005). Microfluidic Blood / Plasma Separation Unit Based on Microchannel Bend Structures. Proceedings of the 3rd Annual International IEEE EMBS. Hawaii.). It is important to note that this filtration method only works under appropriate conditions, such as when the liquid velocity exceeds 1 m / s. Under normal conditions, this separation method can achieve up to 90% efficiency and significantly simplifies the filtration process due to the absence of a membrane. Figures 6A and 6B show the CAD design of this design solution.

[0091] The third design is very similar to the first design module described above. The main difference is that the liquid pathway is spiral, as described in International Publication No. 2019 / 193346. Details of this International Publication are incorporated herein by reference. As described by Z. Geng et al. (2012) (Continuous Blood Separation Utilizing Spiral Filtration Microchannel with Gradually Varied Width and Micro-Pillar Array. Sensors and Actuators B 180 (2013) (p.122-p.129). China.), this design adds a centrifugal effect to the conventional serpentine filtration module described above. This centrifugal effect increases separation efficiency and reduces the risk of blockage. Figures 9 to 10C show design examples.

[0092] The fourth design of the filtration module uses a membrane sandwiched between two liquid flows. X. Li et al. (2014) (Continuous-Flow Microfluidic Blood Cell Sorting for Unprocessed Whole Blood Using Surface-Micromachined Microfiltration Membranes. Royal Society of Chemistry.) uses a filtration membrane for blood cell separation. However, in this invention, the filtration module uses two microfluidic channel layers, with the filtration membrane sandwiched between the two microfluidic channel layers to form a filtrate flow and a stagnant liquid flow. The continuous filtrate flow is collected in the output section and sent to the downstream module according to process requirements, while the stagnant liquid flow is recirculated and returned to the reactor module. Liquid flows from the reactor through the upper channel to the TFF process. Due to the pressure difference, some molecules in the liquid pass through the membrane and reach the lower channel, but molecules that are too large to pass through the membrane remain in the upper channel. This design solution provides excellent efficiency, and most importantly, offers a new perspective on ease of manufacturing. Figures 11A and 11B show design examples of this solution.

[0093] The four embodiments of the filtration module described above depend on the control of the pressure difference between the liquid flows and the separation method used. However, in the fifth embodiment, the device combines digital microfluidic technology, macrofluidic technology, and commonly used molecular biology protocols to separate and extract nucleic acids, particularly DNA. There are two such protocols: one is charge-based, and the other involves attaching magnetic beads to DNA or RNA for purification. For example, in the present invention, when draining from the container and replenishing with new solution, magnetic beads such as Invitrogen DNA-binding beads (Thermofisher Scientific), which are attached to DNA plasmids, can be retained in the reaction vessel using a digitally generated magnetic field, or forced through the filtration membrane of the filtration module design described above and refluxed to the reaction module through the retained liquid channel, as in the bioreactor design of the present invention shown in Figure 2.

[0094] Embodiment While all designs are modular flow systems for the manufacture of nucleic acid-based therapeutics, the serpentine filtration module is a preferred example due to its ease of preparation and low cost. The entire process for manufacturing prototypes to validate the device is described further below.

[0095] Figures 7 and 8 show a preferred fluidized bed filtration module, which preferably includes a first plate-like layer 30 and a second plate-like layer 31, where the main surfaces of the first plate-like layer 30 and the second plate-like layer 31 are positioned opposite each other, with a membrane 36 sandwiched between them to realize a lamella structure. Each of the plates 30 and 31 includes a meandering flow path having a straight portion 34 and a curved portion, or curved portion 35, to directly contact the interlayer membrane 36 and to form respective flow paths 37 and 38, which are at least partially formed by the interlayer membrane 36. A recovery reservoir, an input reservoir, and a buffer reservoir 33 are provided at either the longitudinal end of the first or second flow paths 37 and 38, or towards the longitudinal end. Therefore, the liquid can flow through the channel 37 of the upper plate while the predetermined chemical components of the liquid with small molecular sizes can defuse through the membrane 36 into the adjacent liquid flow channel 38 of the second plate 31. Thus, the TFF modules of Figures 7 and 8 are configured to separate permeate and retained liquid, distinguished by particle size (particle size) and / or molecular weight, as can be seen from the above.

[0096] Liquid flow simulation method for device evaluation As shown in Table 2, there are two different dimensions required for the bioreactor channel. Computational fluid dynamics (CFD) software was used to analyze the virtual data in the microchannel. A steady-state flow rate is required for the microchannel. [Table 2]

[0097] FLUENT: FLUENT is computational fluid dynamics (CFD) software that helps in dealing with fluid flow problems. FLUENT uses the finite volume method to solve the governing equations of fluids and provides a number of different physical models, including laminar or turbulent flow, viscous or inviscid, compressible or incompressible. For geometry and grid generation, it runs GAMBIT, a preprocessor that forms a bundle with FLUENT.

[0098] The solution can be obtained by following the steps of geometry, meshing, setup, solution, and result. The flow path is represented in a 2D CAD design drawing. Then, material properties and boundary conditions are set. Finally, the region must be meshed. FLUENT converges the object until it reaches a convergence range or a predetermined number of iterations. a) Geometry The geometry consists of walls and inlet and outlet boundaries, as shown in Figure 12. b) Mesh Coarse and dense mesh formats can be used. As shown in Table 3, the mesh density varies based on the refining index. [Table 3]

[0099] Liquid flow modeling using computational fluid dynamics: For the Reynolds number (Re=0.0269), laminar flow was selected. Air and water (liquid) must be selected as the materials. Density and viscosity can be defined as material properties.

[0100] The following boundary conditions shown in Table 4 were assigned using FLUENT. [Table 4]

[0101] c) Solving The mesh is exported to FLUENT along with the specified physical properties and initial conditions. When the solution converges or the specified number of iterations is reached, FLUENT exports the data. As shown in Table 5, the total flow time is 14,400 seconds (4 hours), which means that the CFD software needs to record data every 60 seconds and repeat 240 times. Subsequently, the CFD software analyzes the velocity, energy, and continuity in the X and Y directions. As a result, convergence was observed after 38 iterations, as shown in Figure 13. [Table 5]

[0102] Prototype construction Various steps were followed to produce the prototype. First, a mold was created, and the chip was cast using the mold. Various subsystems were individually manufactured to verify each design. Experiments were conducted on each subsystem to confirm the design. Various modifications of the mold were considered and prepared. The design modifications are shown in Figure 14.

[0103] After conducting experiments on each type, several improvements were made between the two variations. By combining two template plates into one, the entire chip manufacturing process was simplified. • By increasing the cross-sectional area of ​​the channel from 0.4mm x 0.8mm to 0.5mm x 1mm, the collapse of the channel walls during chip verification was avoided. By adding extra space between the flow channels (0.5mm~1mm) and between the mold boundary and the functional part, the quality of the chip was improved and any problems that might occur when removing the PDMS mask from the mold were avoided. Finally, we added some walls to the mold boundaries to improve the overall quality of the PDMS chip during casting.

[0104] For the vertical TFF device, the upper and lower layers were considered and prepared separately, and two different layers were fabricated that are sealed together with an intermediate filtration membrane. Figure 15 shows the designs of the two layers. Table 6 describes the individual functional parts of the two designs. [Table 6]

[0105] Molds made using soft lithography and SLA 3D printing. To perform the desired reaction or filtration operation, microfluidic technology chips require highly precise and accurate methods and techniques. The most common method used for microfluidic technology chips is soft lithography. In this case, the essence of soft lithography lies in mold fabrication, for which PDMS is cast using Kamei, K., ichiro, Mashimo, Y., Koyama, Y., Fockenberg, C., Nakashima, M., Nakajima, M.,...Chen, Y. (2015). 3D printing of soft lithography mold for rapid production of polydimethylsiloxane-based microfluidic devices for cell stimulation with concentration gradients. Biomedical Microdevices. The mold is 3D printed from a CAD design of the desired part. Various techniques can be used for 3D printing, but for protrusions, stereolithography was selected.

[0106] Various molds were designed, and the parts were sent and 3D printed using SLA. SLA printing allows for the production of complex shapes with an accuracy of 10 μm in approximately 24 hours. The printing resolution and accuracy were optimized by setting the highest resolution on the SLA machine based on the amount of material required for the mold and the various shapes and features. The first modified mask was analyzed using a microscope, and the dimensions of the CAD file and the 3D printed part were compared. Figure 16 shows two microscopic analyses of the channel dimensions. [Table 7]

[0107] Finally, the mold was printed using two different materials. Both materials are photopolymer resins, one of which is a high-temperature resin. The main difference between these two materials is the maximum temperature before load deflection. While one can reach 250°C, the other can only reach 50°C.

[0108] Selection of filtration membranes The selection of specific filtration membranes was achieved in two steps. The first step involved exploring the molecular fractionation sizes of two different membranes to be used in the two filtration stages. In practice, each filtration stage deals with different molecules with different molecular weights. As shown herein using a conceptual process, the cost of the drug is high, so it is necessary to filter out a portion of the drug molecules and reflux them into the corresponding bioreactor for reuse. Table 8 details the individual molecules and their molecular weights. [Table 8]

[0109] Regarding the two vertical TFF steps, the first vertical TFF is the step that deals with T7 polymerase, while the second vertical TFF is the step that deals with M7g+ methyltransferase. Considering the various molecular sizes, the default filtration membrane needs to have a molecular fraction size three times smaller than the molecules that need to remain, and in this hypothetical example, such molecules are RNA molecules (General Electric, 2014). In addition, the filter needs to be large enough to allow the reused molecules to pass through. Based on these findings, the molecular fraction sizes of each membrane were assigned as described below. • Vertical TFF1 → Film with molecular weight cutoff of 300 kDa • Vertical TFF1 → Film with molecular weight cutoff of 500 kDa

[0110] The second step in the process was to select a suitable supplier for the membrane. Therefore, due to time constraints, we selected 1m x 1m flat sheet membranes from Synder filtration. We obtained three types of membranes with different pore sizes: 300kDa, 400kDa, and 500kDa. This allowed us to conduct various verifications and experiments and analyze the filtration effect of each membrane.

[0111] PDMS masks and casting Once the mold is printed and prepared, polydimethylsiloxane (PDMS) microfluidics can be cast using this mold. The product used to prepare the PDMS chips is Sylgard™ 184 silicone elastomer. This product consists of two components: one silicone elastomer portion and a curing agent that plays a role in forming molecular crosslinks of the elastomer. The curing agent solidifies the elastomer while maintaining its casting properties. However, some preparation is required before applying the PDMS across the mold. Table 9 shows the curing temperature along with the corresponding curing time. Mix the silicone elastomer and hardener in a beaker in a mass ratio of 10:1 (silicone / hardener) for 10 minutes. After mixing, it is necessary to remove any air bubbles using a desiccator. Repeat this process until all air bubbles have disappeared. If tiny bubbles remain, place the beaker down. The bubbles will burst naturally. Once the mixture is prepared, apply it to the 3D printed mold and let it set for 10-20 minutes.

[0112] Place the mask in the oven with the PDMS facing upwards, or allow it to settle at ambient temperature. ·table [Table 9]

[0113] Various temperatures were tested to compare the characteristics and curing process of PDMS. However, the first mold used in this process could not withstand high temperatures. Therefore, curing at ambient temperature was continued even over the weekend. Finally, with various functional parts cast onto the PDMS mask, the open end needed to be sealed. Two different methods were used to seal the mask, and different results were obtained from the tests (Figure 17 is an overview of the various steps in the prototype fabrication process for creating a continuous flow bioreactor system). The first method was characterized by sealing the PDMS mask with another flat layer of PDMS. This method allowed for an increase in the overall excess thickness of the mask, which was useful for incorporating piping and mounting parts into the mask. The second method was characterized by sealing the open side of the PDMS mask with Kapton tape. This method allows for complete sealing between the functional part of the PDMS mask and the tape. However, if the mask is thin, the assembly of the piping and mounting parts becomes more complicated.

[0114] Sensing method The main detection system is designed by considering the functional components and reactions of a continuous flow microfluidic technology system. First, the sensing system for a microfluidic technology chip is limited by the chip size and the characteristics of the factors that need to be detected. Furthermore, selecting a sensor suitable for obtaining accurate, real-time detection results for the chip is another important challenge. The detection system for a microfluidic plant includes a spectrophotometer, pH sensor, and pressure controller and sensor. The theoretical background of the spectrophotometer is explained by L. Zhu, (2005), Nuno Miguel Matos Pires, (2014), and Kihwan Choi, (2015). The theoretical background of the pressure controller and sensor was also examined by Yung-Shin Sun, (2016).

[0115] Material Selection a) Spectrophotometer The spectrophotometer (Ocean Optics' USB2000+Miniature Fiber Optic Spectrometer) can store 1000 complete spectra per second, and its detection wavelength range is 190-1100 nm. In addition, since it uses official software, programming primarily relies on open-source Python packages tailored to the characteristics of the detection system. This spectrophotometer is well-suited for monitoring continuous reactions. b) pH meter The pH meter (HI-1093B pH electrode from Hanna Instruments, Bedfordshire, UK) has a measurement range of 0 to 13. To integrate the pH meter with the microfluidic technology chip, design specifications impose limitations on the dimensions of the pH meter. The diameter of the measurement cell on the microfluidic technology chip is 5 mm², and the body of the pH meter has a diameter of 3 mm. c) Pressure controllers and sensors CFD analysis of the microfluidic technology chip shows that the pressure in the bioreactor and TFF section is 0.27 Bar. The pressure sensor (ELVEFLOW (Paris, France) MPS Microfluidic High Precision Pressure Sensor) can achieve five measurement ranges from 70 mBar to 7 Bar. It can also detect ultra-small internal volumes down to 7.5 μL and can be applied to microfluidic chips with a volume of 0.5 mL. The flow rate adjustment of this sensor is highly sensitive and responsive, making it suitable for real-time monitoring of minute changes in flow rate. High speed can be achieved by using the sensor reader (ELVEFLOW (Paris, France) Sensor Reader) that the sensor is paired with, and integration into the chip is easy, making measurements simple and feasible. d) Pressure supply source A syringe pump (C3657 C-Series Syringe Pumps from Tricontinent, California, USA) is used as the pressure source to supply the reactant material to the microfluidic chip. The pump stroke speed ranges from 1.2 seconds to 100 minutes per stroke, and the pump resolution is 3,000 steps in standard mode and 24,000 steps in high-resolution mode. The system reaction time is 4 to 6 hours. Such a syringe pump can also achieve a steady flow rate in the microfluidic system. The pressure at the five inlets into which the reactant components are injected can be used to control the ratio of the reactant, resulting in efficient and waste-free production of the RNA vaccine.

[0116] method The key reaction parameters that need to be monitored are the concentrations of RNA and Mg2+, the pH value of the reaction, and the temperature. Simultaneously, the inlet pressure needs to be controlled to adjust the flow rate of the mixed solution in the continuous flow channel to achieve an appropriate ratio of RNA production and to optimize the reaction time. Three detection points are located on the microfluidic chip; two are used for RNA and Mg2+ quantification, and the other for pH measurement. A pressure sensor is fabricated outside the chip. Changes in Mg2+ and RNA concentration levels can be used to detect the reaction rate. Converting absorbance to concentration is a common method for quantifying RNA and Mg2+. UV absorbance measurement at 260 nm is the gold standard for RNA quantification, and Mg2+ quantification can be obtained using absorbance measurement at 680 nm. The absorbance measurements of RNA and Mg2+ can be converted to concentration using the Beer-Lambert law. In this design, PDMS is used to fabricate the bioreactor, and PDMS is sufficiently transparent. This enables effective RNA detection.

[0117] During the reaction, the corresponding thermostable DNA polymerase is the main cause of the pH value. A pH of 8.3 to 9.0 can yield optimal results in the system. Furthermore, increasing the pH value during the reaction can help stabilize the DNA template and improve transcription results. The activity of recombinant enzymes is greatly affected by the assay pH. For device integration, an Arduino microcontroller (Arduino Uno Rev3 from Arduino, UK) was used as the core for data acquisition.

[0118] Integrated experimental platform Figures 18A to 18C show schematic diagrams of the liquid-flow reaction system. The system includes multiple sensors configured to measure various properties of the liquid flowing within the liquid-flow system, including pressure, temperature, pH, flow rate, and flow volume. The system incorporates multiple valves, injection ports, pumps, particularly syringe pumps and gates, to control the flow paths of the liquid components in the apparatus. For example, if necessary, the liquid components may be recirculated from an outlet port at the longitudinal end of the bioreactor and / or filtration module and returned to the inlet port of the upstream bioreactor and / or filtration module. The system further includes a control unit, which typically includes a CPU, microprocessor, or other suitable electronic processing device. In certain embodiments, the processor is integrated into a PCB or PLC. The control unit is provided with a user interface, a sensing data storage utility, software, a visual display device, communication ports and / or modules, at least one analog-to-digital converter and / or a data source library. These components, combined with sensors, injection ports, pumps, valves, etc., can be used by the control unit to continuously and automatically control the liquid flow in the liquid-flow system. Therefore, a continuous reactor system used for the continuous injection of chemicals is realized, enabling the continuous production of the desired reagent product. This system is effective in minimizing waste and maximizing efficiency by recirculating and reusing chemicals, solvents, etc. After the equipment is prepared, its layout is designed and the experimental environment is set up. Naturally, the PDMS chip should be secured in place by a fixture to prevent movement. Then, the positions of other instruments are determined accordingly.

[0119] Initially, four syringe pumps were used to apply pressure to the four input containers. Piping and mounting are typically used to deliver minute amounts of sample to the PDMS chip. Another pump is connected to a second container at the front of the second bioreactor (the end of the first TFF), which allows for the injection of m7G methyltransferase and adjustment of the pressure in the chip. The use of a pressure sensor with a feedback loop significantly improves the responsiveness of the fluid flow control. Pressure sensors are envisioned to measure pressure at both the inlet and outlet of the flow path. The goal is to keep the syringe pumps running while maintaining a constant pressure within the device.

[0120] To perform concentration detection, ultraviolet / visible light must pass through a detection chamber at the end of the first bioreactor (serpentine channel), and this light is transmitted by optical fiber. Two micro-optical fibers, each suited to specific wavelength requirements, are positioned vertically to each other: a light source fiber connected to a light source and a collecting fiber connected to a miniature spectrophotometer. During continuous bioreactions, a pH sensor is intended to measure the pH value in a buffer, while a temperature sensor can be placed on the board with a heater on the opposite side to regulate the temperature. Both the pH sensor and temperature sensor are equipped with BNC connectors, which can be connected to an Arduino. Therefore, data can be obtained from these sensors.

[0121] Data acquisition and monitoring Spectrophotometer data The Lambert-Beer law explains the following linear relationship between the concentration of a substance and the absorbance at a specific wavelength.

number

[0122] Therefore, the desired spectrophotometer data is the absorption spectrum itself. Ocean Optics has implemented an easy method to access data from Python using their spectrophotometer. This method is provided by the Python-Seabreeze package, which includes the Seabreeze library for communicating with the spectrophotometer. This ensures that Ocean Optics' USB interface is fully functional and a tested reference implementation is available, making it possible to read and monitor spectrophotometer data using Python. The connection between Python and the spectrophotometer is shown in Figure 19.

[0123] After connecting the pH sensor to the Arduino via a BNC connector, sensor data can be obtained, and real-time data can be checked on the serial monitor. The code used for this is shown in Figure 19. The same mechanism is used to obtain temperature sensor data. Five full-factorial experiments were conducted, focusing on the effects of different drug ratios and the reaction time and ratio of magnesium icons. The experiments were conducted based on a fed-batch system with a volume of 500 μL. Background experimental data is shown below. Several conclusions can be drawn from the data. Figure 21 shows the maximum yield obtained from a mixture of NTP 10 mM, NaCl 10 mM, MgCl2 75 mM, and acetate ions. The maximum yield is approximately 900 mM RNA, which can be considered the optimal combination. Figure 22 shows the optimal reaction time for RNA production. RNA production can reach its maximum level when the proportion of Mg(OAc)2 is approximately 80 mmol. After 4 hours of reaction, the RNA production reaches a stable state at approximately 1800 mM. The optimal combination of reaction conditions depends on four main factors: the ratios of MgCl2, NaCl, and NTP, as well as the reaction time. Furthermore, in other experiments, it is necessary to monitor and maintain the ratios of MgCl2, NaCl, and NTP at predetermined levels to obtain the maximum RNA yield. The most significant difference between the experiments at Imperial College and Cranfield University lies in the design of the reaction system. For production considerations, a continuous flow system (continues-follow system) for the bioreactor process is designed, rather than a fed-batch reaction system. To validate the continuous flow system, this experiment is planned based on the above-mentioned experiment conducted by the Shattock Group at Imperial College.

[0124] Three levels were selected for the four factors (Table 10) to determine the optimal ratio for RNA production in a continuous flow system. The selected factors refer to the results of experiments in a fed-batch system. In addition, the DNA and T7 polymerase values ​​were considered to be reusable values. Using these factors, a new experimental plan is shown in Table 11. The objective of the experiment is to explore the relationship between RNA production and the four factors and to identify the response trends.

[0125] [Table 10]

[0126] [Table 11]

[0127] Results and Review Fluid dynamics and CFD analysis Appropriate governing equations were applied to the hydrodynamic analysis of the bioreactor and TFF sections to calculate the filtrate volume under steady flow conditions where the liquid velocity is constant and an appropriate pressure gradient is present. Equation (6) was used to predict the pressure drop in the microchannels. The selection of characteristic lengths was arbitrary and does not affect the final solution, as shown in Tables 12 and 13. [Table 12] [Table 13]

[0128] Other pressure drops related to the measured pressure drop are inlet and outlet bend losses. Bend pressure losses can be obtained within the range of various bend numbers shown in Table 14. [Table 14]

[0129] Fluid dynamics analysis of the TFF portion The permeability constant is obtained, and the Cerman-Kozeny relation is used to solve for fluid parameters across the micropore channel under appropriate boundary conditions. Here, since the input particle size changes in Table 15, various particle sizes and pore sizes are applicable. [Table 15]

[0130] The pressure drop for the aforementioned shape can be predicted from Equation 10. However, the formula has a limitation: mathematical errors occur when the unit of measurement is microns. The pressure drop divided by the length of the micropore channel is shown in Table 16.

number

[0131] The results obtained in this study are merely predictions based on shape, liquid state, and boundary conditions. The filtrate volume shown in Table 17 can be estimated according to the membrane selection parameters of Pall's products. [Table 17]

[0132] computational fluid dynamics analysis Bioreactor channel velocity profile As shown in Figures 23A and 23B, a velocity exists in the X direction throughout the bioreactor flow path. There are two different colors that indicate the same velocity ranging from 0.0001 ms⁻¹ to 0.0002 ms⁻¹. This is because yellow indicates the positive direction and green indicates the negative direction. Figure 23B shows that the velocity initially increased due to the high pressure applied by the first pump, and then decreased to a constant velocity of approximately 0.00033 ms⁻¹. This velocity is about twice as high as the predicted result.

[0133] Tangential flow filtration channel Figure 24 shows the velocity of the tangential flow filtration channel. The colors indicate that the individual velocities within the microchannels are close to the inflow velocity, ranging from 0.0001327 ms⁻¹ to 0.0001858 ms⁻¹. Furthermore, Table 18 shows that the outflow velocity decreases slightly from 0.000145 ms⁻¹ to 0.000138 ms⁻¹, as desired. [Table 18]

[0134] Isobaric representation of bioreactor pressure Figure 25 shows the pressure drop in the bioreactor channel. The colors indicate individual pressure values. Figure 25B shows that the pressure drops as a result of the presence of numerous bends in the bioreactor channel. These various results were considered in design modifications.

[0135] Experimental results PDMS Mask Verification Bioreactor Various modifications of the type have been understood through the examination of this application, and progress has also been made in the results, which will now be explained.

[0136] a) First Generation First, both of the first modifications of the two bioreactors were examined. Experiments were selectively conducted with different encapsulants: Kapton tape and a second PDMS layer, and results were obtained. Both reactors were allowed to cure at 35°C for two days. The two reactors were initially sealed with another flat PDMS layer. Green water was introduced into one of the inlets of each mask using a syringe. Figures 26A and 26B show the initial results regarding the behavior of the liquid in each mask. The two figures show that, on the batch bioreactor, the water reaches the same height in the middle of the reactor as it is introduced into the mask. This is explained by the collapse of the middle of the reactor as it cured, as can be seen in the blue area of ​​Figure 26A. Furthermore, Figure 26B shows that the green water sample followed the path of the main channel through the T-junction of the mask. However, the sample avoided several areas. This can be explained by the size of the channel walls, i.e., their inability to withstand the pressure during the curing process and subsequent collapse.

[0137] Next, we examined a continuous flow reactor sealed with Kapton tape. Figure 27 shows the behavior of the liquid in this configuration. The liquid (dark green) followed its path without any problems. The sealing functioned better than the sealing described above. However, due to the size of the flow path, curing did not occur properly again, and some parts of the mask were not properly detached from the mold. This resulted in the formation of several defect areas in the mask.

[0138] Based on all the considerations made during various verifications, the design was modified. In addition, it was decided to fabricate a support platform. On the platform, the PDMS mask can be placed between two acrylic plates, which increases the pressure between the two PDMS layers and thus prevents the flow path from collapsing during mask verification. The mounting part and piping can also be connected to the chip using these two acrylic plates.

[0139] Figure 28 shows a Kapton-sealed mask positioned between two plates, connected to piping, mounting points, and several sensing devices, enabling the initial pH and absorbance tests and measurements.

[0140] b) Type 2 generation High-temperature resistant material was used to print the second generation, and the mask was cast at a curing temperature of 70°C for 4 hours. As shown in Figure 29, using the mold after use, the side that was in direct contact with the mold had bonded to the mold. It was not possible to peel off minute parts of the PDMS mask and remove it from the mask. In particular, a thin layer of PDMS remained stuck to the mask between the channels. Although curing was performed properly, the mask could not be peeled off the mold. After attempting to clean the mold as much as possible with isopropanol, another prototype was obtained by curing at a curing temperature of 100°C for 35 minutes. However, the same problem occurred. This problem can be explained by the fact that the high-temperature resistant material from the 3D printer bonded with the PDMS layer that was in direct contact with this high-temperature resistant material. The walls can also be said to be affected by the instability of the overall curing uniformity of the mask.

[0141] Vertical flow filtration module A single modified vertical TFF type was printed using a high-temperature resistant material. For the bioreactor mask, the same process was followed, with the PDMS sample poured into the foil paper receiving section and placed in an oven along with two masks to compare various aspects of the process and the results. Figures 30A and 30B show the masks and the samples cured in the oven. In the case of the bioreactor mask, the PDMS that came into contact with the mold bonded to the mold. When peeled from the mold, the mask cracked and broke, as shown in Figure 30A. On the other hand, the sample cured in the foil paper cured without using any default settings, and nothing happened when peeled from the foil. In addition, as shown in Figure 30B, the sample perfectly cast the shape of the foil.

[0142] Based on the various considerations described above, it is possible to perform some analysis and obtain results. Since the sample in the foil was used without any problems, it cannot be said that there were any issues with the chemistry or protocol. The same default occurred several times with different masks. Therefore, high-temperature materials negatively affect PDMS mask casting and should not be used in such experiments. The initial material used in the first attempt should be continued, and the curing parameter should be set to ambient temperature and cured for 48 hours to ensure that the PDMS mask is properly cast and to avoid any load deflection that would cause it to separate from the mold. Despite the results described above, a new platform was created, as shown in Figure 31.

[0143] Based on the integrated experimental plan, all equipment was gathered in the laboratory, and an experimental platform was constructed to verify the system's effectiveness. The experimental platform fabricated in the laboratory is shown in Figure 31, where the PDMS chip was secured by fixtures to prevent movement, and the pump and chip were connected by piping and mounting parts. Two optical fibers were installed perpendicular to each other: a light source fiber connected to a light source and a collecting fiber connected to a small spectrophotometer (Ocean Optics USB2000+).

[0144] Absorbance measurement and digital output High accuracy absorbance measurement To accurately measure absorbance, a baseline must be established. First, the spectrum of the background light source is recorded without the sample. Next, the experiment is repeated by adding the sample to the absorption path. Then, the first spectrum is subtracted from the second spectrum to obtain the pure absorption spectrum of the sample. To cancel out the effects of noise, the background spectrum should be subtracted from both the sample spectrum and the reference spectrum. Therefore, equation (9) should be as follows:

number

[0145] Here, I sample is the light intensity after passing through the sample, and I B This is the background light intensity recorded by a spectrophotometer without a sample, with the light source turned off or blocked. REF This is the reference light intensity.

[0146] UV-Vis data analysis Using the Python-Seabreeze API, it is easy to acquire real-time data from a UV-Vis spectrophotometer and plot the intensity against wavelength. As shown in Figure 32, the light intensity reaches its peak value at a wavelength of approximately 580 nm. To examine the relationship between absorbance and wavelength at different concentrations, vitamin B12 solutions of different concentrations—A (25 mM / L), B (50 mM / L), C (75 mM / L), and D (100 mM / L)—were used, respectively (see Figure 33). The UV-Vis spectral results are shown in Figure 34, where absorbance increases with increasing sample concentration, reaching a maximum at a wavelength of 550 nm.

[0147] pH sensor calibration and digital output pH sensor calibration To ensure accuracy, the pH probe must be calibrated before first use. Two standard buffer solutions were used to calibrate the pH sensor: pH 4.0 and pH 7.0. Calibration was completed after the calibration steps provided by DFROBOT.

[0148] Plotting pH values ​​in the results The next step involved verifying the effectiveness of the pH sensor using acidic and alkaline solutions. The results in Figure 35 show that the graph changes dynamically in real time according to the pH change, thereby demonstrating its validity.

[0149] Consider Evaluation of important results The investigation and the results obtained demonstrate an effective attempt to achieve continuous flow reactions while maximizing RNA output by implementing lab-on-a-chip technology. By applying the SBCE method, the entire design framework could be constructed, and this framework was followed from the beginning to the end of this project. The modular design of the device with divided functional subsystems allows for the inclusion of individual innovative solutions for the operation of the mixing, reaction, and filtration / purification sections. Figures 30A to 30C show schematic diagrams of the integrated system including downstream modules for formulations. In particular, Figures 30A to 30C illustrate an embodiment of this flow system that integrates a downstream formulation system (Module 2) (e.g., International Publication No. 2013 / 050764 and M. Jreissat, 2016, "A novel flow system for the concurrent product and process design of emulsion-based formulations", Brunel University London) and an integrated UV-Vis probe using a modular flow reactor, and is attached to a conventional filled-finish vaccine manufacturing line.

[0150] The synthesis results of the devices are shown in Figure 30D, which is a graph of the absorbance of the production solution of the flow reactor as a function of wavelength in the embodiments shown in Figures 30A to 30D. Figure 40A is a diagram of the source code for the absorbance data and the real-time plot (absorbance as a function of wavelength) in Python, and Figure 40B is a diagram of the source code for the real-time plot pH data in Python.

[0151] The product is evaluated using UV-Vis spectroscopy on the graph of the conventional batch protocol for the is-producing solution of the flow reactor, which was evaluated using UV-Vis spectroscopy on the graph of the conventional batch protocol. The presence of high peaks in the graph indicating high concentrations of RNA suggests that the productivity of this method is significantly higher compared to the batch protocol. The UV-Vis spectra shown in Figure 30D compare the RNA material obtained using the conventional batch protocol and the continuous flow protocol of the present invention. The high peaks in the flow protocol compared to the batch protocol correspond to the higher concentration of nucleic acids present in the solution. The dotted lines correspond to samples with known concentrations of RNA and are included to aid in the comparison of what is obtained by the batch and flow protocols.

[0152] Two different design solutions were developed for reactors that enable the conversion of RNA production from a largely manual batch process (conventional RNA synthesis) to a continuous flow format. In fact, the continuous flow reactor of the present invention is an innovative and proven reactor that functions without issue. The present invention represents a prior art process change that demonstrates the operation and performance when implementing well-developed equipment and processes to address rapid scaling and the highly productive production of RNA-based materials in an automated, computer-controlled continuous flow format.

[0153] The filtration module was thoroughly investigated and analyzed to ensure it is easy to manufacture and scale using common manufacturing methods while meeting the requirements for continuous flow processing and integration with a flow reactor. Tangential flow filtration is a common technique that has been demonstrated to be effective in RNA purification processes (A. Eon-Duval et al., 2002). However, this invention includes a novel filtration device that is intentionally designed and constructed to be used as a continuous flow system, is easy to manufacture, can be integrated into a continuous flow system, is scalable, and enables the manufacture of a cost-effective device. This system can be integrated as a microfactory that enables automated and flexible manufacturing of therapeutic nucleic acid-based materials when in use. In addition, the modular design allows for the recycling of enzymes and specific components such as plasmid DNA using this filtration system, thereby significantly reducing the total cost of the manufactured material.

[0154] A novel integrated platform was developed for testing and experimentation of the entire system. The system was prototyped using an acrylic pressure plate. The system includes all the ports for the pump, sensors, and mounting parts for the inlet and outlet. This allows for verification of continuous flow reactions and filtration while obtaining rapid feedback from various elements and stages of each process. Furthermore, a modular system was prototyped to enable an easy-to-use framework for conducting experiments. 3D printing was chosen as the tool for manufacturing the molds due to its high flexibility, low cost, and short lead time. The modular configuration can be easily transitioned to commercial production using techniques such as microfabrication of glass, metals such as stainless steel, and polymethyl methacrylate PMMA (acrylic) substrates, as well as photolithography and deep reactive ion etching on substrates containing photosensitive glass and silicon. In addition, injection molding can be used for the PMMA and polycarbonate materials of the structure for large-scale manufacturing of modules.

[0155] The ability of this invention to provide reliable online and insightful analysis of the contents of reaction solutions in real time using ultraviolet-visible spectroscopy has been demonstrated. This also provides a closed feedback loop to manage liquid flow and adjust temperature, pH, and the dispensing of reactants in the flow system to maintain the overall process state.

Claims

1. The steps include introducing at least one type of nucleoside triphosphate (NTP), a reaction buffer, and multiple reagents comprising DNA, a DNA-based compound, or a DNA-based mixture into a first liquid flow module via multiple inflow ports, The steps include reacting at least some of the reagents in a reaction channel or well in the first module of the flow system, The steps include: retaining or recirculating the DNA in the first reactor module and allowing the reaction product of the reagent to flow into the first fluid filtration module; The steps include filtering the reaction product in the first filtration module and RNA synthesis method including

2. The method according to claim 1, wherein the at least one nucleoside triphosphate (NTP) is a solution of at least one nucleoside triphosphate (NTP).

3. The method according to claim 1 or 2, wherein the at least one nucleoside triphosphate (NTP) comprises one or a combination thereof of adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP).

4. The method according to any one of claims 1 to 3, wherein the DNA is plasmid DNA.

5. The method according to any one of claims 1 to 4, wherein the plurality of reagents further comprises one of an enzyme mixture, a salt solution, RNA polymerase, or a combination thereof.

6. The method according to any one of claims 1 to 5, comprising the step of recirculating at least some of the plurality of reactants from the outflow area of ​​the first filtration module to the inflow area of ​​the first reactor module.

7. The method according to any one of claims 1 to 6, comprising the step of supplying at least a portion of the filtered reaction product from the first filtration module to the second liquid flow reactor module, in combination with introducing a capping enzyme into the second reactor module.

8. The method according to claim 7, further comprising the step of supplying the liquid discharged from the second reactor module to a second liquid flow filtration module.

9. The method according to claim 8, further comprising the steps of recirculating unreacted NTPs to the inlet region of the first reactor module and recirculating unreacted capping enzymes to the inlet region of the second reactor module.

10. The aforementioned salt solution is MgCl ２ The method according to any one of claims 1 to 9, which is dependent on claim 5, including the method described in claim 5.

11. The method according to any one of claims 1 to 10, in the case of claim 5, wherein the RNA polymerase includes T7 polymerase.

12. RNA or RNA-based compound prepared by the method described in any one of claims 1 to 11.

13. Use of RNA or RNA-based compounds prepared by the method described in any one of claims 1 to 11 during the preparation of the vaccine.

14. A first narrow liquid flow channel having an inlet, A second narrow liquid flow channel having an outlet section, A permeable membrane is arranged along the respective lengths of the first and second channels so as to separate the second channel from the first channel, allowing the permeate to pass from the liquid in the first channel through the membrane into the second channel along the length of the first and second channels. A liquid flow filtration system including a liquid flow filter.

15. The apparatus according to claim 14, wherein the majority of the length of the first channel is arranged adjacent to the majority of the length of the second channel via the membrane.

16. The apparatus according to claims 14 and 15, wherein the pore size of the membrane is in the range of 100 to 1000 kDa, 100 to 800 kDa, 200 to 800 kDa, or 200 to 600 kDa, or 300 kDa to 10 MDa.

17. The apparatus according to any one of claims 14 to 16, comprising a first plate on which the first flow channel is formed and a second plate on which the second flow channel is formed, wherein the film is sandwiched between the opposing surfaces of the first and second plates.

18. The apparatus according to any one of claims 14 to 17, wherein each of the first and second flow paths includes a series of straight and curved portions.

19. The apparatus according to claim 18, wherein the first and second flow channels include their respective longitudinal meandering profiles.

20. The apparatus according to any one of claims 14 to 19, wherein the first and second channels are open along their respective lengths and are arranged in direct contact with the membrane that partially forms the longitudinal walls or surfaces of the first and second channels.

21. The apparatus according to any one of claims 14 to 20, wherein the pore size of the membrane is less than the average molecular size of RNA molecules.

22. A first reactor module having a reaction flow channel or well, at least one inlet, and at least one outlet, A first filtration module having a fluid filtration region, at least one inlet provided in a manner that communicates with the outlet of the first reactor module, and at least one outlet. A liquid flow system used for the aforementioned processing of a liquid, including, The first filtration module includes a liquid flow filtration apparatus according to any one of claims 13 to 21. Liquid flow system.

23. The system according to claim 22, comprising a second reactor module having a reaction flow channel or well, at least one inlet, and an outlet, wherein the inlet is provided in communication with the first filtration module.

24. The system according to claim 23, further comprising a second filtration module having a liquid filtration region, at least one inlet, and an outlet, wherein the inlet is provided in communication with the outlet of the second reactor module.

25. The system according to any one of claims 22 to 24, further comprising a liquid injection port provided in a manner that communicates with the inlet region of the first filtration module.

26. The system according to any one of claims 22 to 25, comprising a first recirculation conduit extending between the outflow region of the first reactor module and the at least one inflow region of the first reactor module.

27. The system according to any one of claims 22 to 26, further comprising a second recirculation conduit extending between the outflow region of the first filtration module and the outflow region of the first reactor module.

28. The system according to claim 27, as dependent on claim 24, further comprising a third recirculation conduit extending between the outflow region of the second filtration module and the inflow region of the first reaction module.

29. The steps include: flowing liquid from the inlet into the first narrow liquid flow channel, The steps include forcing the permeable component of the liquid to pass through a membrane extending along the first flow path and flow into a second narrow liquid flow path, A method for filtering a liquid using a liquid flow filter, comprising the step of retaining the stagnant components of the liquid in the first flow path, The membrane is positioned to separate the first channel from the second channel along their respective lengths, so that the permeate can pass from the first channel through the membrane into the second channel along the respective lengths of the first and second channels. method.

30. The method according to claim 29, wherein the step of flowing the liquid includes the step of pressurizing the liquid in the first flow path.

31. It is a flow system, A reactor module having a reaction liquid flow channel or well, at least one liquid inlet, and at least one liquid outlet, A flow delivery unit that delivers liquid into the aforementioned flow path or well, A first sensor for measuring one or a combination thereof of the pressure, temperature, or pH of the liquid in the system, A second sensor for measuring one or a combination thereof of the pressure, temperature, and pH of the liquid in the system, A reaction state monitoring device that monitors the properties of the liquid in the system, indicating the reaction state of at least two chemical components of the liquid in the system, A control unit that receives data from the first sensor, the second sensor, and at least one or a combination thereof of the reaction state monitoring device, and controls at least one characteristic of the liquid in the system. A flow system that includes this.

32. The above characteristics of the system are, The pressure of the liquid in the system, The temperature of the liquid in the system, pH of the liquid in the system, The volume or ratio of one or more chemical components of the liquid in the system, Flow rate of the liquid in the system The system according to claim 31, which is one of the above or a combination thereof.

33. The system according to claim 32, wherein the control unit includes a CPU, PCB, PLC, PC, processor chip, and handheld electronic device.

34. The system according to claim 33, wherein the further sensors include one or a combination thereof of a temperature sensor, a pH sensor, a pressure sensor, a flow sensor, a fluid volume sensor, or a spectroscopic sensor.

35. A method for processing liquid using a liquid flow device, The steps include introducing at least one type of liquid into the reactor module through at least one inlet, The steps include: flowing the liquid through a reaction flow channel or well, or using at least one flow delivery unit to flow the liquid through the reaction flow channel or well, and discharging the liquid at the outlet; A step of measuring at least one or a combination thereof of the pressure, temperature, or pH of the liquid in the liquid flow device, The steps include monitoring the reaction state of the chemical components in the liquid in the liquid flow device, A step of controlling at least one characteristic of the liquid in the liquid flow device using a control unit in accordance with at least one or a combination thereof of the measurement of the pressure, pH, temperature and / or the reaction state of the liquid's chemical components. A method that includes this.

36. The system according to claim 35, comprising a plurality of reactor modules and filtration modules connected and joined together in a communication state.

37. The system according to claim 35, wherein the flow delivery unit is at least one pump, which may be a syringe pump.

38. The system according to claim 35, wherein the liquid analysis sensor includes an ultraviolet-visible spectrometer for absorbance analysis or fluorescence analysis in the range of 190 nm to 1000 nm.

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