Method and modular synthesis device for RNA-based therapeutic agents

Abstract

A method for RNA synthesis using a liquid flow device, the method comprising the steps of introducing a plurality of reactants, 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 inlet ports, reacting at least some of the reactants in reaction channels or wells in the first module of a flow system, retaining or recirculating the DNA in a first reactor module, and flowing the reaction products of the reactants 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 methods of RNA synthesis, particularly but not exclusively to scalable, modular liquid flow systems and methods for use in continuous, automated, or semi-automated RNA synthesis. [Background technology]

[0002] A flow reactor (also called a continuous flow reactor) provides a continuous flow of materials or reactants through a network of conduits connected to form a fluid pathway. Ports and valves enable and disable fluid pathways through various configurations of the conduit network when combined with mixing, reaction, and filtration modules in a flow system to control the synthesis, purification, and formulation of RNA or nucleic acid therapeutics for continuous flow product production. Purposefully written software code, run by a computer system, can direct the control of a full set of process parameters in the flow system, including mixing conditions, temperature, pH, reagent concentration, monitoring, residence time, purity profile, and output. Prior art flow reactors are typically constructed as an assembly of individual modules connected face-to-face to form a unit block that manages the fluid flow. Examples of flow reactors are described in WO 2013 / 050764.

[0003] Flow technology offers more sustainable, flexible, and efficient manufacturing in pharmaceutical manufacturing. Combined with microtechnology and precision-engineered flow systems, they can be constructed and configured as microfactories that integrate multiple unit operations, such as mixing, reaction synthesis, extraction, separation, filtration, and purification. Furthermore, the small volume-to-surface-area ratio inherent in flow mixing and reaction systems allows for precise control of process conditions relative to heat and mass transfer, thereby accelerating processes and increasing productivity within a small equipment footprint. Integrated flow systems that combine these unit operations can be considered microfactories or miniaturized manufacturing systems that reduce factory floor space, lower energy consumption, and improve resource utilization. Therefore, sustainable growth can be achieved with better environmental impact, cost-effectiveness, and flexibility compared to current manufacturing systems commonly used today to produce substances such as prophylactic vaccines.

[0004] Several protocols in molecular biology and biotechnology are increasingly using lab-on-a-chip (LOC) devices. These LOC devices use microfabrication to miniaturize and integrate analytical assays in the laboratory, incorporating microscale synthesis and microscale filtration into a microchip. These microdevices consume less material, resulting in less waste, lower costs, and faster response times. By managing fluid flow through micromixers, microchannels, and filters, various steps of a given protocol can be integrated into the LOC device, enabling cell sorting, mixing, and reactions, such as those for DNA or RNA synthesis. It is important that LOC devices be made of non-reactive materials and preferably transparent to allow visual inspection of real-time reactions on the chip. Transparency also allows for spectral analysis and characterization. A preferred material for rapid prototyping of these 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 vaccine accessibility, particularly in LMICs (low- and medium-income countries), and pandemics such as Ebola and dengue fever have population-impacting (characteristics).

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

[0007] It is therefore an object of the present invention to provide a modular manufacturing platform capable of scalable synthesis and / or filtration of chemical compounds, as well as biomolecules and non-biomolecules, including, inter alia, RNA-based vaccines and other nucleic acid therapeutics. A particular object of the present invention is to provide an integrated, configurable microfactory flow system for use in the preparation of nucleic acid-based vaccine products.

[0008] The present disclosure provides a modular, integrated device inspired by the above methods and laboratory protocols, including a configurable and highly scalable method for rapid, end-to-end production of RNA and nucleic acid materials at the point of use, for example, in a hospital setting.

[0009] In particular, the system includes a combination of flow reactor, "sequential" filtration, and mixing sections. These sections may be integrated in any order appropriate for target RNA, nucleic acid therapeutics, or prophylactic vaccines. The integrated flow system may also include several optional analytical probes, including fiber optic probes, detectors, and light sources for UV-Vis absorbance or fluorescence spectroscopy, used for in situ and in-line control of the synthesis or production of target substances in continuous flow processes.

[0010] A specific objective is to provide a microfluidics-based device and method that integrates modules, including a liquid flow bioreactor module and a liquid flow filtration module, connected via corresponding conduits / flow paths to form a continuous liquid flow pathway, that can be operated continuously and, as will be understood from the present specification, automated using electronic and / or software control utilities involving the use of component actuators such as membranes, pumps, valves, gates, inlet ports, outlet ports, syringe pumps, sensors, etc.

[0011] A specific object is to provide a liquid flow system for use in the reactive synthesis of predetermined chemical components, such as biomolecules or non-biomolecules, by a process that may be continuous or discontinuous, automated, or semi-automated. Another specific object is to provide a modular system in which individual modular units can be connected according to a synthetic pathway. Such units may include ports, valves, and appropriate connections that allow communication and connection between the respective modules. Yet another specific object is to provide a liquid reaction system that can be configured to measure and be responsive to system properties, such as pressure, temperature, pH, amount, or ratio of one or more predetermined chemical components, liquid flow rate, or liquid reaction state.

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

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

[0014] It is an object of the present invention to provide a modular manufacturing platform capable of synthesizing and / or filtering biomolecules and non-biomolecules, including chemical compounds, and in particular RNA and the resulting vaccines. A specific object is to provide devices and systems that can be configured as microfactories for biomolecule synthesis to enable downstream vaccine production.

[0015] A specific object is to provide an apparatus and method for forming an integrated liquid flow system including constituent modular components including liquid flow bioreactor modules and liquid flow filtration modules connected via corresponding conduits / flow paths to form a liquid flow pathway, which can be operated continuously and automated using electronic and / or software control utilities.

[0016] According to a first aspect of the present invention, there is provided a method for RNA synthesis, comprising the steps of: introducing a plurality of reactants, including at least one nucleoside triphosphate (NTP), a reaction buffer, and DNA, a DNA-based compound, or a DNA-based mixture, into a first fluid flow module via a plurality of inlet ports; reacting at least some of the reactants in reaction channels or wells in the first module of the flow system; retaining or recirculating the DNA in a first reactor module and causing the reaction products of the reactants to flow into a first fluid filtration module; and filtering the reaction products in the first filtration module.

[0017] Optionally, the at least one nucleoside triphosphate (NTP) is a solution of at least one nucleoside triphosphate (NTP).

[0018] Optionally, the at least one nucleoside triphosphate (NTP) comprises any one of adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), uridine triphosphate (UTP), or a combination thereof.

[0019] Optionally, the DNA is plasmid DNA. Optionally, the plurality of reactants further comprises any one or combination of an enzyme mixture, a salt solution, and an RNA polymerase.

[0020] Optionally, the method includes recycling 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. Optionally, the method includes providing 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. Optionally, the method includes providing the effluent from the second reactor module to a second liquid-flow filtration module. Optionally, the method includes recycling unreacted NTPs to the inlet region of the first reactor module and unreacted capping enzyme to the inlet region of the second reactor module.

[0021] Optionally, the salt solution or buffer comprises MgCl 2. Optionally, the RNA polymerase comprises T7 polymerase.

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

[0023] According to yet another aspect of the present invention there is provided the use of RNA or RNA-based compounds prepared by the methods 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 comprising a first narrow liquid flow channel having an inlet, a second narrow liquid flow channel having an outlet, and a osmosis membrane positioned along the length of each of the first and second channels to separate the first flow channel from the second flow channel such that permeate liquid can pass from liquid in the first flow channel through the membrane into the second flow channel along the length of the first and second channels.

[0025] Optionally, a majority of the length of the first flow path is disposed adjacent a majority of the length of the second flow path through the membrane.

[0026] In some cases, the pore size of the membrane is in the range 100-1000 kDa, in the range 100-800 kDa, in the range 200-800 kDa or in the range 200-600 kDa, in the range 300 kDa-10 MDa.

[0027] Optionally, the device includes a first plate having a first flow channel formed therein and a second plate having a second flow channel formed therein, the first and second plates sandwiching a membrane between opposing surfaces of each of the plates. Optionally, each of the first and second flow channels includes a series of straight and curved sections. Optionally, the first and second flow channels include respective serpentine profiles along their lengths. Optionally, the first and second flow channels are open along their lengths and are disposed in direct contact with membranes that partially form longitudinal walls or surfaces of the first and second flow channels. Optionally, the pore size of the membrane is less than the average molecular size of an RNA molecule.

[0028] According to yet another aspect of the present invention, there is provided a liquid flow system for use in processing a liquid, comprising a first reactor module having a reaction flow channel or well, 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 disposed in communication with the outlet of the first reactor module, wherein the first filtration module comprises a liquid flow filtration device as claimed in the present application.

[0029] Optionally, the system includes 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] Optionally, the system includes a second filtration module having a liquid filtration region, at least one inlet, and an outlet, the inlet being in communication with the outlet of the second reactor module.

[0031] Optionally, the system includes a liquid injection port 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 reactor module.

[0032] According to yet another aspect of the present invention, there is provided a method of filtering a liquid using a liquid flow filtration apparatus, comprising the steps of: flowing a liquid from an inlet into a first narrow liquid flow channel; forcing a permeating component of the liquid to pass through a membrane extending along the first channel and into a second narrow liquid flow channel; and retaining a retentate component of the liquid within the first channel, wherein the membrane is positioned to separate the first flow channel from the second flow channel along their respective lengths such that permeate liquid can pass from the first flow channel through the membrane into the second flow channel along their respective lengths.

[0033] According to yet another aspect of the present invention, there is provided 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 liquid delivery section for delivering a liquid flow to the channel or well, a first sensor for measuring any one or a combination of pressure, temperature or pH of the liquid in the system, a second sensor for measuring any one or a combination of pressure, temperature or pH of the liquid in the system, a reaction status monitoring device for monitoring a property of the liquid in the system indicative of a state of reaction of at least two chemical components in the liquid in the system, and a control section for receiving data from at least one or a combination of the first sensor, the second sensor and the reaction status monitoring device and controlling at least one property of the liquid in the system.

[0034] Optionally, the system characteristic is any one or combination of: a pressure of the fluid in the system, a temperature of the fluid in the system, a pH of the fluid in the system, a volume or ratio of one or more chemical components of the fluid in the system, or a flow rate of the fluid in the system. Optionally, flowing the fluid comprises pressurizing the fluid in the first flow path. Optionally, the controller comprises a CPU, PCB, PLC, PC, processor chip, or handheld electronic device. Optionally, the additional sensor comprises any one or combination of a temperature sensor, a pH sensor, a pressure sensor, a flow sensor, a flow rate sensor, or a spectroscopic sensor.

[0035] According to yet another aspect of the present invention, there is provided a method for processing a liquid using a liquid flow device, the method comprising the steps of: introducing at least one type of liquid into a reactor module through at least one inlet; using at least one flow delivery part to flow the liquid through or within a reaction flow channel or well, and discharging the liquid at an outlet; measuring at least one or a combination 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 or a combination of properties of the liquid in the liquid flow device using a control unit in response to at least one or a combination of pressure measurement, pH measurement, temperature measurement, and / or reaction state measurement of the chemical components of the liquid.

[0036] Optionally, the system includes a plurality of reactor modules and filtration modules connected in communication with each other. Optionally, the flow delivery unit is at least one pump, optionally a syringe pump. Optionally, the liquid analysis sensor includes a UV-Vis spectrometer for absorbance analysis or fluorescence analysis in the range of 190 nm to 1000 nm.

[0037] Optionally, the method for RNA synthesis includes recycling unreacted NTPs to an inlet region of the first reactor module and recycling unreacted capping enzyme to an inlet region of the second reactor module.

[0038] According to a further aspect of the present invention there is provided RNA prepared by the method claimed in the present application. According to a further aspect of the present invention there is provided the use of RNA prepared by the method of any preceding claim in the preparation of a vaccine.

[0039] According to yet another aspect of the present invention, there is provided a liquid flow apparatus for processing a liquid, comprising a first reactor module having a reaction flow channel or well, at least one inlet and at least one outlet, and a first filtration module having a fluid filtration region, at least one inlet and at least one outlet, wherein the inlet is in communication with the outlet of the first reactor module.

[0040] Optionally, the apparatus includes a second filtration module having a fluid filtration region, at least one inlet, and an outlet, the inlet being in communication with the outlet of the second reactor module.

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

[0042] Optionally, the device includes a third recirculation conduit extending between the outlet region of the second filtration module and the at least one inlet region of the first reaction module. Optionally, the device includes a plurality of inlet ports that allow fluid chemical components to enter the reaction channels or wells. Optionally, the device includes at least one pump connected to at least one inlet of the first reactor module to drive a fluid flow through the reaction channels or into the wells. Optionally, the device includes at least one valve, fluid flow gate, fluid flow port, heating element, fluid reservoir, or holdup reservoir in fluid communication with the device. Optionally, the pump includes a syringe pump.

[0043] Optionally, the device has a plate-like structure such that the first reactor module and the first filtration module are at least partially formed as channels or recessed grooves on or within the plate-like structure. Optionally, the device includes a plurality of sensors located in different liquid flow regions of the device.

[0044] In some cases, the sensor comprises at least one temperature sensor, at least one flow sensor, at least one pressure sensor, at least one pH sensor, at least one flow rate sensor, at least one spectroscopic sensor, at least one optical sensor, at least one optical fiber, or a spectroscopic optical fiber, or any combination thereof.

[0045] Optionally, the device includes a controller connected to at least one pump, valve, flow gate, flow port, heating element, liquid reservoir or holding reservoir, and sensor to control a characteristic of the liquid flow in the device in response to the state of a physical, chemical, or mechanical property of the liquid quantified by the sensor. Optionally, the controller includes a CPU, processor, PCB, PLC, handheld electronic device.

[0046] In some cases, the controller includes a control module including any one or combination of software, electronic components, data storage utilities, wired or wireless communication modules and / or ports, visual display outputs, a user interface, at least one pump, valve, fluid flow gate, fluid flow port, heating element, fluid reservoir or sump, and at least one actuator that activates any one or combination of sensors.

[0047] According to yet another aspect of the present invention, there is provided a liquid flow system for processing liquid, comprising a plurality of devices as claimed in the present application, each of said devices having a final liquid flow outlet connected to a collection section that collects liquid flowing out of each of said devices.

[0048] Optionally, the liquid flow system includes a controller connected to at least one pump, valve, liquid flow gate, liquid flow port, heating element, liquid reservoir or holding reservoir, and sensor for each of the devices, or selected ones of the devices, to control the characteristics of the liquid flow in the device in response to the state of a physical, chemical, or mechanical property of the liquid quantified by the sensor.

[0049] According to yet another aspect of the present invention, there is provided a method for processing a liquid using a liquid flow device, comprising the steps of: introducing at least one liquid into a first reactor module through at least one inlet, causing the liquid to flow through or within a reaction flow channel or well, and discharging the liquid at an outlet of the first reactor module; and discharging at least a portion of the liquid from the outlet through at least one inlet to a filtration module having a liquid filtration region, and discharging the liquid from the first filtration module at an outlet, wherein a reaction between at least two chemical components of the liquid occurs in the first reactor module and filtration of the liquid occurs in the first filtration module.

[0050] Optionally, the method includes using at least one fluid pump to drive a liquid flow through the device. Optionally, the method includes recirculating at least a portion of the liquid from an outlet of the reactor module and / or filtration module via at least one recirculation conduit to at least one inlet region of the reactor module. Optionally, the method includes monitoring a state of a chemical reaction between chemical components occurring in the liquid using at least one sensor. Optionally, the method includes controlling the liquid flow through the device in response to the state of the chemical reaction determined by the sensor. Optionally, the method includes monitoring physical, chemical, and / or mechanical properties of the liquid in the device using at least one sensor. Optionally, the method includes controlling the liquid flow through the device in response to the physical, chemical, and / or mechanical properties of the liquid determined by the sensor. Optionally, controlling the liquid flow includes controlling or deactivating at least one actuator, pump, valve, gate, or port provided in communication with the liquid in the device. In some cases, controlling the liquid flow includes activating or deactivating at least one actuator, pump, valve, gate, or port using a CPU, processor, PCB, PLC, or handheld electronic device.

[0051] According to yet another aspect of the present invention, there is provided a liquid flow filtration device comprising a first narrow liquid flow channel having an inlet, a second narrow liquid flow channel having an outlet, and a osmosis membrane positioned along the length of each of the first and second channels to separate the first flow channel from the second flow channel such that permeate liquid can pass from liquid in the first flow channel through the membrane into the second flow channel along the length of the first and second channels.

[0052] Optionally, the first flow path may include at least one outlet, with an inlet located at or towards a first longitudinal end of the first flow path and an outlet located at or towards a second longitudinal end of the first flow path. Optionally, the second flow path may include at least one inlet, with the inlet located at or towards a first longitudinal end of the second flow path and an outlet located at or towards a second longitudinal end of the second flow path.

[0053] According to yet another aspect of the present application, there is provided a liquid flow system for use in processing a liquid, comprising a first reactor module having a reaction flow channel or well, 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 disposed in communication with the outlet of the first reactor module, wherein the first filtration module comprises a liquid flow filtration device as claimed in the present application.

[0054] According to yet another aspect of the present invention, there is provided a method of filtering a liquid using a liquid flow filtration apparatus, comprising the steps of: flowing a liquid from an inlet into a first narrow liquid flow channel; forcing a permeating component of the liquid to pass through a membrane extending along the first channel and into a second narrow liquid flow channel; and retaining a retentate component of the liquid within the first channel, wherein the membrane is positioned to separate the first flow channel from the second flow channel along their respective lengths such that permeate liquid can pass from the first flow channel through the membrane into the second flow channel along their respective lengths.

[0055] According to yet another aspect of the present invention, there is provided 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 liquid delivery section for delivering a liquid flow to the channel or well, a first sensor for measuring any one or a combination of pressure, temperature or pH of the liquid in the system, a second sensor for measuring any one or a combination of pressure, temperature or pH of the liquid in the system, a reaction status monitoring device for monitoring a property of the liquid in the system indicative of a state of reaction of at least two chemical components in the liquid in the system, and a control section for receiving data from at least one or a combination of the first sensor, the second sensor and the reaction status monitoring device and controlling at least one property of the liquid in the system.

[0056] According to yet another aspect of the present invention, there is provided a method for processing a liquid using a liquid flow device, the method comprising the steps of: introducing at least one type of liquid into a reactor module through at least one inlet; using at least one flow delivery part to flow the liquid through or within a reaction flow channel or well, and discharging the liquid at an outlet; measuring at least one or a combination 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 or a combination of properties of the liquid in the liquid flow device using a control unit in response to at least one or a combination of pressure measurement, pH measurement, temperature measurement, and / or reaction state measurement of the chemical components of the liquid. [Brief explanation of the drawings]

[0057] Specific implementations of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Figure 1] FIG. 1 is a schematic diagram of a continuous reaction filtration apparatus suitable for producing RNA. [Figure 2] FIG. 2 is a perspective view of a reactor module and a filtration module suitable for use in the apparatus of FIG. 1. [Figure 3]FIG. 2 is a plan view of a well bioreactor module, i.e., a batch bioreactor module, suitable for use in the apparatus of FIG. 1. [Figure 4] FIG. 2 is a plan view of a bioreactor module with a reaction channel configuration suitable for use in the device of FIG. 1. [Figure 5A] FIG. 2 is a plan view of a filtration module suitable for use in the device of FIG. 1. [Figure 5B] 5B is an image of a membrane in the filtration module of FIG. 5A. [Figure 6A] 2 is a diagram of a portion of a filtration module having an area configured for centrifugal separation of chemical components suitable for use in the apparatus of FIG. 1; [Figure 6B] 1 is an image of a portion of a filtration module having an area 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] FIG. 8 is an enlarged view of a region of the filtration module of FIG. 7. [Figure 9] FIG. 1 is a plan view of a spiral tangential flow filtration module. [Figure 10A] 1 is a component of a filtration module according to a specific implementation. [Figure 10B] FIG. 10B is a schematic diagram of another portion of the filtration module of FIG. 10A. [Figure 10C] FIG. 10C is another view of a portion of the filtration module of FIGS. 10A and 10B. [Figure 11A] FIG. 10 is a plan view of another filtration module formed as a two-layer microchannel TFF. [Figure 11B] 1 is a schematic diagram of a filtration mechanism and a filtration module according to a specific implementation. [Figure 12] FIG. 1 is a schematic diagram of a channel flow bioreactor module. [Figure 13] 1 is a graph used in the calculations for the design of a liquid flow bioreactor and filtration system. [Figure 14]FIG. 1 is a schematic diagram of a reactor module connected to a filtration module to form a liquid flow device. [Figure 15] FIG. 1 is a perspective view of a TFF module. [Figure 16] 1 is an image of a microscopic analysis of the channel dimensions of a TFF module. [Figure 17] FIG. 1 is a schematic diagram of an overview of the prototyping process. [Figure 18A] 1 is a schematic diagram of a liquid flow system including a reactor module, a series of sensors, a reaction status monitoring arrangement, and a control unit according to a specific implementation. [Figure 18B] FIG. 18B is a schematic diagram of components of a reactive status monitoring configuration suitable as part of the liquid flow system of FIG. 18A. [Figure 18C] 18B is a schematic diagram of a series of sensors, reactive status monitoring components, and controls forming part of the fluid flow system of FIG. 18A according to a specific implementation. [Figure 19] FIG. 19 is a schematic diagram of the architecture of a portion of the microfluidic flow system of FIG. 18, including part of the spectrophotometer configuration. [Figure 20] FIG. 1 is a schematic diagram of a serial communication program used in the control system. [Figure 21] Graph of RNA yield versus ratio of various reactants, including NaCl, MgCl2, NTPs, etc. [Figure 22] 1 is a graph of data analysis (RNA yield versus magnesium ions) and specific reaction time selections according to embodiments of the subject invention. [Figure 23A] FIG. 10 is a schematic diagram of a simulation of the analysis results of the velocity profile. [Figure 23B] 1 is a graph of a velocity analysis graph. [Figure 24] FIG. 1 is a schematic diagram of the velocity of the tangential flow filtration channel in the filtration module. [Figure 25A] 1 is a graph of pressure drop in a bioreactor flow path. [Figure 25B] 1 is a graph of pressure drop with multiple drops. [Figure 26A]Photograph of a mask used in the manufacture of a liquid flow reactor and filtration device. [Figure 26B] 1 is a photograph of a mask of a continuous microfluidic flow reactor. [Figure 27] 1 is an image of a continuous flow reactor sealed with Kapton tape. [Figure 28] This is an image of the completed configuration using two acrylic plates. [Figure 29] This is an image of the 3D printed mold after use. [Figure 30A] FIG. 1 is a first portion of a schematic diagram of an embodiment of the present flow system integrated with a downstream formulation system using a modular flow reactor and installed in a conventional fill-finish vaccine manufacturing line. [Figure 30B] FIG. 1 is a second part of a schematic diagram of an embodiment of the present flow system integrated with a downstream formulation system using a modular flow reactor and installed in a conventional fill-finish vaccine manufacturing line. [Figure 30C] FIG. 10 is a third part of a schematic diagram of an embodiment of the present flow system integrated with a downstream formulation system using a modular flow reactor and installed in a conventional fill-finish vaccine manufacturing line. [Figure 30D] 30A-30D are graphs of absorbance versus wavelength for the output solution of the flow reactor of the embodiment of FIGS. 30A-30D evaluated using UV-Vis spectroscopy compared to a conventional batch protocol. [Figure 31] Photograph of an experimental liquid flow compound synthesis platform. [Figure 32] Python live plot of spectrophotometer data (intensity vs. wavelength). [Figure 33] Images of samples with different concentrations (A: 25 mM / L, B: 50 mM / L, C: 75 mM / L, D: 100 mM / L) used with a microfluidic flow reactor module and continuous filtration system. [Figure 34] 1 is a graph of ultraviolet-visible spectrum analysis. [Figure 35]Here is an image of Python live plotting pH data. [Figure 36] Graphs of predicted plots (left: reaction with 1 mM dNTP, right: reaction with 4 mM dNTP). [Figure 37] Predictive profiler graphs for the first and second experiments are shown (top: reaction with 1 mM dNTP, bottom: reaction with 4 mM dNTP). [Figure 38] Summary of various data from the liquid flow bioreactor and filtration (left: response with 1 mM dNTP, right: response with 4 mM dNTP. LogWorth=-log10(p-value)). [Figure 39] FIG. 1 is a schematic diagram of a scaled-up liquid flow reactor and liquid flow device used in the present system. [Figure 40A] FIG. 10 is a diagram of the source code for absorbance data and real-time plotting (absorbance versus wavelength) in Python. [Figure 40B] This is a diagram of the source code for live plotting pH data in Python. DETAILED DESCRIPTION OF THE INVENTION

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

[0059] A porous medium is prepared and impregnated with a liquid, focusing on the fact that no liquid-liquid interface is formed, and one type of liquid permeates the pore space. Let dp be the particle size and U be the order of velocity. It has been found that (1) is applicable for Reynolds numbers less than 1. That is,

number

[0060] The permeability of a porous medium is a property that depends on pore size and pore structure. Dimensional analysis shows that permeability is a function of porosity, e, and particle size, dp, which represent pore shape and pore size, respectively. The Carman-Kozeny relationship empirically links these quantities as follows, providing the correct dimensionality:

number

number

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

number

[0062] With the volumetric flow rate Q and cross-sectional area A known, the Reynolds number was calculated from the following:

number

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

number

[0064] where A and A t are the cross-sectional areas of the flow path and the connecting pipe, respectively. K b is the loss factor of the bend, and Kc and K e represents the contraction loss factor and expansion loss factor due to the area change. Phillips' K is about 1.2 for a 90 degree bend. b The cross-sectional area of ​​the flow path and the cross-sectional area of ​​the connecting pipe are equal, and K c and K. e Assuming that the maximum possible value of is assumed, the relative minor losses to the measured pressure drop are negligible compared to the measured pressure drop.

[0065] Hooman and Merrikh (M. Bahrami, 2010) developed an analytical solution for flow and pressure drop in porous channels as follows:

number

[0066] The cross-sectional aspect ratio ε of the samples considered in this study is 0.5. Therefore, we do not consider a perfectly rectangular cross section, but can model the sample as a porous medium sandwiched between two parallel plates.

[0067] The appropriate cassette is selected depending on the total sample volume, the required process time, and the desired final sample volume. The following formula is used to calculate the membrane area required to process a sample in a given time:

number

[0068] Fluid flow simulation for prototype testing 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. The CFD was used to analyze the velocity profiles in the transition region and the transition region to study the behavior of the liquid flow. The liquid properties affect the liquid flow in the microchannel. According to the inventors, a minimum dynamic viscosity and low surface tension are required for the selected appropriate liquid.

[0069] Microscale devices have potential in chemical and biochemical engineering, and liquid flow plays an important role in combining them with numerous applications. The mechanisms and principles of single-phase liquid flow have been reviewed using experimental data using different types of liquids and different surface roughnesses. Therefore, at the microscopic scale, the influence of liquid properties such as surface tension and viscosity dominates. (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 inertial to viscous forces acting on a fluid flowing through a channel (i.e., the ratio of the fluid's momentum to the frictional forces imparted by the channel walls). Flows with low Reynolds numbers are laminar, or stratified, in which the individual fluid streams flow parallel to one another and mix only by convective and molecular diffusion. Flows with high Reynolds numbers are turbulent, in which "parcels" of various sizes of fluid exhibit simultaneous random motion in space and time, resulting in rapid mixing throughout the channel. The transition between laminar and turbulent flow typically occurs at Re=2000 for internal flows. (Schulte, TH, Bardell, RL, & Weigl, BH (2002). Microfluidic-technologies-in-clinical-diagnostics_2002_Clinica-Chimica-Acta. 321, 1-10)

[0071] Turbulent flow fields are more complex than laminar flow fields, but they have significant secondary effects. One- and two-dimensional models that describe the time evolution of analytical distributions of these states have been developed. Confocal fluorescence microscopy experiments and three-dimensional numerical modeling can help confirm quantitative descriptions 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 systems In this study, it would be better to build a sensor integration 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 flow diagram illustrating the path toward robust sensor integration in liquid flow devices. This diagram could serve as a guide for integration studies. 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 potential of complementary metal-oxide semiconductor (CMOS) technology for integration and detection. The CMOS image sensor served as an effective reaction detection platform, allowing the CMOS image sensor to monitor real-time photon changes according to the amplification process. The photons were observed and converted into digital units by the CMOS image sensor. In addition, UV spectrum studies, light color intensity detection, and pH analysis were conducted to demonstrate the efficiency of CMOS (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 on a CMOS standard process. Silicon area: 1.12 mm. 2 The completed system was installed in and showed a linearity of 59 mV / pH in the concentration range of pH levels 2-12, making it an excellent alternative for biological and medical applications.

[0073] Although CMOS is a reasonable choice, it is difficult to realize due to cost and time considerations. The integration of each sensor was reviewed for a more realistic approach. Regarding the pH sensor, there is a study 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.). The system utilized an extended-gate ion-responsive field-effect transistor (ISFET) with an integrated pseudo-reference electrode to monitor the pH value within the flow reaction chamber. For the temperature sensor, a serpentine-shaped temperature sensor (line width: 50 mm) and a heater (line width: 400 mm) were integrated under the 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. In this technique, a dedicated manifold aligns the optical fiber with the digital microfluidic device, allowing optical measurements to be performed in the device plane, which provides the inspiration for optical detection.

[0074] The preferred wavelength range for the analysis of the present invention is 190 nm to 1000 nm, and the peak obtained is approximately 260 nm.

[0075] method Continuous flow synthesis and scale-up A schematic diagram of a continuous RNA production process is shown in Figure 1. 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 the modules 10-13 is connected via a respective flow path or conduit. The first bioreactor module 10 is provided with a plurality of fluid supply input ports 14 for introducing respective reactants into the first bioreactor module 10. Each port 14 includes a corresponding syringe pump 15. The first TFF includes a corresponding input / inlet port 16, the second bioreactor module includes a corresponding input / inlet port 17, and the second TFF module includes a corresponding input / inlet port 18. Each of ports 16-18 may be provided with a corresponding associated pump or syringe port for introducing fluids and pressurizing the fluid flow through the narrow reaction channel 24 of each reactor and the narrow filtration channel 25 in each module. Multiple recirculation channels 19, 20, and 21 connect various fluid flow regions (at or near the respective inlet / outlet end regions) of modules 10, 11, 12, and 13 to provide communication for the recirculation of reactants / chemicals. The apparatus of FIG. 1 also includes sensors, reaction status monitoring devices, controls, and other associated electronic and actuator components (not shown), as described elsewhere herein, to provide a fully automated continuous 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 configured to filter large molecular weight chemical components, such as DNA, from the fluid based on molecular size and / or molecular weight. The filtered desired liquid is then recycled to the inlet region of reactor 10 via flow path 19. In this manner, certain chemicals and reactants can be reused to achieve a continuous or semi-continuous process.

[0076] 1 and 2, FIG. 2 shows a variation of the fluidic system of FIG. 1, in which the reactor 10 includes a reaction well 50, as opposed to the narrow reaction channel 24 of FIG. 1. As described with reference to FIG. 1, a plurality of injection ports 14 are connected in communication with the well 50 for supplying desired compounds, agents, solvents, etc., used in the reaction. The filtration modules 11, 12 may include a series of extraction ports 51, 22, which allow extraction of predetermined species, agents, solvents, etc. from the filtration modules, and collection of the final product, e.g., fully capped RNA, at port 22.

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

[0078] Referring to FIG. 39, multiple reactor filtration systems described with reference to FIGS. 1 and 2 (and generally shown as plate-like units 10, 11, 12, and 13) may be connected together as scaled-up flow reactors and devices to form a fully integrated, modular flow reaction filtration system for the production of RNA-based therapeutics and prophylactic vaccines at a scale sufficient for evaluation, clinical trials, and ultimately manufacturing. In particular, appropriate connecting conduits provide communication between the individual plate-like units 10, 11, 12, and 13 so that the output sections (located in output section 22) of each unit can be combined to provide a total output. The aggregated system of FIG. 39 may include a single controller, multiple controllers, corresponding sensors, and reaction status monitoring utilities and units, as described herein with reference to FIGS. 18A-20. Multiple fluidic devices of the present invention may operate in parallel to enable the continuous production of RNA-based therapeutics and prophylactic vaccines. Using this scaling method, it is possible to reach output manufacturing at the scale required by the target market, which could be the catchment area of ​​a local hospital, or where units are installed on a conventional fill-finish line at the manufacturer's production site. Figure 20 is a schematic diagram of the serial communication program used for the control system between the Arduino IDE control utility and the 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] Using RNA synthesis as an example, during the process, a segment of DNA is copied into RNA by the enzyme RNA polymerase. The transcription mixture consists of NTPs (nucleoside triphosphates), DNA, MgCl2 (PO7 -4 The first bioreactor channel is injected with a syringe pump containing Mg (which reacts with ATP to form a precipitate and affect RNA productivity) and T7 polymerase (catalysis). 2+Hydroxy naphthol blue (HNB) is used as a colorimetric indicator for titration of ions. 2+ HNB-Mg 2+ Complexation: Mg 2+ As a result of the reduction of ions, the color changes from purple to sky blue. The mixture resides in the first reactor 10 for approximately 6 hours, during which time the RNA transcription reaction progresses and RNA begins to be produced, along with magnesium pyrophosphate precipitate (Mg2P2O7). All of the above components except DNA flow through the first tangential flow filtration (TFF) module 11, which has a high molecular weight (MW) cutoff membrane. The DNA is filtered and returned to the original inlet port 14 (2) via the recirculation channel 19, where it remains in the first reactor; the rest is filtered and proceeds to the next stage.

[0080] After the first filtration module, the T7 polymerase is recycled to inlet port 14 (4) while the above components are passed downstream. Then, m7G methyltransferase (5' cap) is injected at port 17 and enters the second reactor 12 and flow path 24, where the inlet reacts for 2 hours. The flow-through enters the second TFF section 13, which has a medium MW cutoff membrane. During this filtration step, unreacted NTPs and 5' caps are retained and recycled (via 21 and 20) to enter bioreactors 12 and / or 10. Finally, the purified RNA is extruded at outlet 22. Simultaneously, Mg2PP i Other components such as water, salts, etc. are collected in a separate container or port 51 and optionally discarded or further processed. The output (capped RNA) may then be processed by further processing steps (not described in this description but well known to those skilled in the art) to produce the desired vaccine.

[0081] The goal is to achieve continuous synthesis and continuous purification to obtain RNA material and improve the system's throughput. To maintain continuous reaction in the process, raw materials must be added to the reactor via part 14 to compensate for consumed molecules, which can be achieved by adjusting the actuator syringe pump and a material flow feedback loop. Optimizing the reaction conditions (magnesium concentration, pH, temperature, reaction time, etc.) can sometimes result in substantial improvements in RNA yield.

[0082] Set-Based Concurrent Engineering (SBCE) Method Lean product development methodology was chosen as the primary framework for developing the design. The lean product development methodology is set-based concurrent engineering (SBCE). SBCE is a process of dividing the product to be developed into different subsystems, allowing a set of possible solutions for each subsystem to be developed in parallel. As the design evolves, educated decisions are made using tools such as simulation, prototyping, validation, and other gained knowledge to narrow the set of solutions for each subsystem. According to the SBCE methodology, the first essential step is to divide the product into different subsystems that can be developed independently. Considering the conceptual design of the system that needs to be developed, there are four major identifiable processes that can be grouped into just two functions: a bioreactor and a filtration process. At the same time, the system needs to be integrated with a control system that can control the pressure from the pumps and have sensors located in the critical product areas to measure key parameters. Corresponding to the conceptual design of the system, it was possible to define the different subsystems that could be developed in parallel. Table 1 shows the different subsystems, with a brief description given for each subsystem and the corresponding level of change.

[0083] [Table 1]

[0084] Subsystem design specification determination method Bioreactor Two different bioreactor designs were developed. The first design was inspired by traditional batch processes used in chemistry for RNA synthesis. The design included four inputs 14 for each raw material, connected to a 1 mL main chamber where the reaction occurs by allowing sufficient residence time. The vessels could then be selectively emptied by releasing microfluidic valves at the vessel outlets, allowing new reagents to be introduced into the inlets. In comparison to traditional batch systems, this implies that the device operates in a continuous (perfusion) mode. After the reaction is complete, the product is released into the output channel. Sensors assess whether the next step in the process can proceed or not. Figures 2 and 3 show example bioreactor designs.

[0085] This design requires a complex and well-coordinated control system to control the valves integrated into the device. After the residence time has elapsed, the product needs to be delivered to the rest of the system.

[0086] The second bioreactor design is a microflow reactor shaped as a serpentine liquid conduit, which allows for the mixing of reactants and the continuous synthesis of the desired product, as described in International Publication No. WO 2019 / 193346. The details of this publication are incorporated herein by reference. This embodiment allows for continuous synthesis through controlled liquid flow. In this design, there are four chemical inputs, which are continuously fed into the flow reactor via a T-shaped mixer. The reaction conditions and effectiveness are controlled by the net flow rate into the liquid conduit and the liquid velocity of the individual components through the T-shaped mixer. These are controlled by setting the dispensing rate of the dispensing pump using computer control. Product is formed when the mixture reaches the end of the serpentine-shaped flow bioreactor within a predetermined residence time. In the production section, the flow is temporarily slowed by a feature of the device that functions as a microfluidic well, allowing in-line measurements to be taken using the preferred analytical method for the particular chemical solution, such as in-line UV-Vis spectroscopy. By extracting specific features of the signal from the spectrometer, purpose-built software can parse and analyze the signal and compare it to a reference vector to determine whether the desired product has been formed. Figure 4 shows a schematic diagram of the serpentine flow bioreactor.

[0087] Filtration Module There are two types of filtration: direct flow filtration (DFF) and tangential flow filtration (TFF). While TFF has a liquid flow parallel to the filter, DFF is characterized by a main flow perpendicular to the filter. DFF has a significantly higher risk of clogging, and it has been demonstrated that TFF has a higher filtrate flow rate than DFF as the filtered volume increases. Furthermore, typical applications of TFF are the concentration, diafiltration, and fractionation of biomolecules and the clarification and removal of cells, which are similar to the applications presented in this study. In other words, TFF is the most effective option for this application and is therefore the filtration type of choice.

[0088] The TFF method was selected as a model for the filtration module. Several design solutions have been proposed in research literature and commercial practice. Several other considerations, such as ease of manufacturing, were considered for this subsystem, including its ability to integrate the module into the overall system. The design of the first filtration module features a serpentine-shaped pathway with two channels at the same height, separating the functional parts on the device and intentionally forming a pattern of functional parts, which acts 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) (pp. 216-221). China.) previously used a TFF filter design for biological cell separation that was fast, reusable, and low-cost compared to other solutions.

[0089] The present invention utilizes purposefully selected membranes with sizes appropriate for the separation of biomolecules commonly present in solutions following nucleic acid synthesis. Figures 5A and 5B show a filtration module containing filtration functions within a serpentine microchannel. The size and length of the channel are purposefully designed to allow for the selection of the correct biomolecules in the filtrate and retentate streams. Fabrication of such modules is achieved using microengineering techniques, including photolithography and deep reactive etching of glass and silicon substrates. These techniques are well known, commercially available, comply with pharmaceutical regulations, and allow for low-cost mass production of devices.

[0090] The second filtration module design does not require any filter membranes. It operates primarily using centrifugal force, which causes some particles to move faster than others when the fluid trajectory is curved at a given velocity. Therefore, by having a bend microchannel that splits into two different flow paths, particles of different sizes can be directed into different outlet flow paths (Blatter, C., Jurischka, R., Tahhan, I., Schot, 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 fluid velocities greater than 1 m / s. Under normal conditions, this separation technique can achieve efficiencies of up to 90%, and the lack of a membrane significantly simplifies the filtration process. 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 fluid path is spiral, as described in International Publication No. 2019 / 193346, the details of which are incorporated herein by reference. As explained 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) (pp. 122-129). China.), this design adds a centrifugal effect to the conventional serpentine-shaped filtration module described above. This centrifugal effect increases separation efficiency and reduces the risk of clogging. Figures 9-10C show example designs.

[0092] A fourth design for the filtration module uses a membrane sandwiched between two liquid streams. X. Li et al. (2014) (Continuous-Flow Microfluidic Blood Cell Sorting for Unprocessed Whole Blood Using Surface-Micromachined Microfiltration Membranes. Royal Society of Chemistry) used a filtration membrane for blood cell separation. However, in this invention, the filtration module uses two microchannel layers, sandwiching a filtration membrane between them to form a filtrate stream and a retentate stream. The continuous filtrate stream is collected at the output and sent to downstream modules according to process requirements, while the retentate stream is recycled back 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, while molecules too large to pass through the membrane remain in the upper channel. This design solution offers superior efficiency and, most importantly, new insights in terms of ease of manufacturing. 11A and 11B show a design example of this solution.

[0093] While the four filtration module embodiments described above rely on controlling the pressure differential between the liquid streams and the separation method employed, in a fifth embodiment, the device combines digital microfluidics, macrofluidics, and commonly used molecular biology protocols to separate and extract nucleic acids, particularly DNA. Two such protocols exist: one based on charge, and the other involves attaching magnetic beads to DNA or RNA for purification. For example, in the present invention, magnetic beads, such as Invitrogen DNA-binding beads (Thermofisher Scientific) attached to DNA plasmids, can be retained within the reaction vessel when the vessel is drained and replenished with new solution using a digitally generated magnetic field, as in the bioreactor design of the present invention shown in Figure 2, or forced through the filtration membrane of the filtration module design described above and back to the reaction module through the retentate flow path.

[0094] Embodiment While all designs are modular in multiple configurations of flow systems for the production of nucleic acid-based therapeutics, the serpentine filtration module represents a preferred example due to its ease and low cost of preparation. The overall process for fabricating prototypes for device validation is further described below.

[0095] 7 and 8 show a preferred flow-through filtration module, preferably including a first plate layer 30 and a second plate layer 31, with the first plate layer 30 and the second plate layer 31 positioned with their respective major surfaces facing each other and sandwiching a membrane 36 therebetween to achieve a lamella structure. Each of the plates 30, 31 includes a serpentine flow path having straight portions 34 and bent or curved portions 35 to form respective flow paths 37, 38 in direct contact with and at least partially defined by the intermediate membrane 36. A return reservoir, input reservoir, or buffer reservoir 33 is provided at or toward the longitudinal end of either the first or second flow paths 37, 38. Thus, fluid can flow through the upper plate's flow channel 37 while certain chemical components of the fluid with smaller molecular size can defusing through the membrane 36 and enter the adjacent fluid flow channel 38 in the second plate 31. Thus, the TFF modules of Figures 7 and 8 are configured to separate permeate and retentate, as seen above, which are differentiated by particle size and / or molecular weight.

[0096] Liquid flow simulation method for device studies There are two different dimensions of the bioreactor channels required, as shown in Table 2. Computational fluid dynamics (CFD) software was used to analyze the hypothetical data in the microchannels. A steady-state flow rate is required for the microchannels. [Table 2]

[0097] FLUENT: FLUENT is a computational fluid dynamics (CFD) software that helps deal with liquid flow problems. FLUENT uses the finite volume method to solve the governing equations of fluid flow and offers a number of different physical models, such as 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] A solution can be obtained by following these steps: geometry, mesh, setup, solution, results. The flow path is represented by a 2D CAD design. Then the material properties and boundary conditions are set. Finally, the domain must be meshed. FLUENT converges the problem until it reaches the convergence limit or reaches a predefined number of iterations. a) Geometry The geometry consists of a wall and inlet and outlet boundaries and is shown in FIG. b) Mesh Coarse and fine mesh formats can be used. As shown in Table 3, the mesh density varies based on the refinement index. [Table 3]

[0099] Fluid flow modeling using computational fluid dynamics: Laminar flow is selected for the Reynolds number (Re=0.0269). Air and water (liquid) must be selected as materials. Density and viscosity can be specified as material properties.

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

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

[0102] Prototype construction To fabricate the prototype, various steps were followed. First, a mold was made and then the chip was cast using the mold. To verify each design, various subsystems were fabricated separately. To validate the design, experiments were performed on each subsystem. Various variations of the mold were studied and prepared. The design variations are shown in Figure 14.

[0103] After conducting experiments on each type, some refinements were made between the two variants. · Two templates were recombined into one, facilitating the entire preparation chip fabrication process. The cross-sectional area of ​​the channel was increased from 0.4mm x 0.8mm to 0.5mm x 1mm to avoid the collapse of the channel walls during chip validation. Adding extra space between channels (0.5mm-1mm) and between the mold boundary and the feature improved the chip quality and avoided any defects when removing the PDMS mask from the mold. Finally, we added some walls to the mold boundaries to improve the overall quality of the PDMS chips during casting.

[0104] For vertical TFF devices, the top and bottom layers were considered and prepared separately to create two distinct layers sealed with a filtration membrane in between. Figure 15 shows the designs of the two layers. Table 6 describes the individual features of the two designs. [Table 6]

[0105] Soft lithography and SLA 3D printing molds To perform the desired reaction or filtration, microfluidic chips require highly accurate and precise methods and techniques. The most common method used for microfluidic chips is soft lithography. In this case, the essence of soft lithography is to create a mold, which is cast from PDMS using Kamei, K., Ichhiro, 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. While various techniques can be used for 3D printing, stereolithography was chosen for the protruding parts.

[0106] Various molds were designed and parts were sent to be 3D printed using SLA. SLA printing allows complex shapes to be produced with an accuracy of 10 μm in approximately 24 hours. The highest resolution setting on the SLA machine was used to optimize the print resolution and accuracy for the amount of material needed for the mold and the various shapes and features. A first variation of the mask was analyzed under a microscope to compare dimensions between the CAD file and the 3D printed parts. Figure 16 shows two microscopic analyses of the channel dimensions. [Table 7]

[0107] Finally, molds were printed with two different materials. One was a photopolymer resin and the other was a high-temperature resin. The main difference between these two materials is the maximum temperature before deflection under load: one can reach 250°C, while the other can reach 50°C.

[0108] Filtration membrane selection The selection of specific filtration membranes was achieved in two steps. The first step was characterized by exploring the molecular cutoff size of two different membranes used in the two filtration stages. In practice, each filtration stage addresses different molecules with different molecular weights. As demonstrated herein using the conceptual process, due to the high cost of the drug, a portion of the drug molecules must be filtered and recycled back to the corresponding bioreactor. Table 8 details the individual molecules and their sizes, or molecular weights. [Table 8]

[0109] For the two vertical TFF stages, the first vertical TFF deals with T7 polymerase, while the second vertical TFF deals with M7g+ methyltransferase. Considering the various molecular sizes, a given filtration membrane must have a molecular fraction size three times smaller than the molecules that need to be retained; in this hypothetical example, these molecules are RNA molecules (General Electric, 2014). Additionally, the filter must be large enough to allow the recycled molecules to pass through the filter. Based on this knowledge, we assigned molecular fraction sizes for each membrane as follows: Vertical TFF1 → 300kDa molecular weight cutoff membrane Vertical TFF1 → 500kDa molecular weight cutoff membrane

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

[0111] PDMS mask and casting Once the mold is printed and prepared, it can be used to cast polydimethylsiloxane (PDMS) microfluidics. The product used to fabricate the PDMS chip is Sylgard™ 184 silicone elastomer. This product is provided by two components: a silicone elastomer portion and a curing agent, which is responsible for molecular cross-linking of the elastomer, solidifying it while maintaining its casting properties. However, some preparation is required before dispensing PDMS over the mold. Table 9 lists the curing temperatures along with the corresponding curing times. Mix the silicone elastomer and curing agent in a beaker at a mass ratio of 10:1 (silicone / curing agent) for 10 minutes. After mixing, any air bubbles that may have formed during mixing must be removed using a desiccator. Repeat this process until all air bubbles have disappeared. If tiny bubbles remain, place the beaker at a resting place. The bubbles will pop naturally. Once the mixture is prepared, apply it to the 3D printed mold and let it rest for 10-20 minutes.

[0112] Place the mask PDMS side up in an oven or let it rest at ambient temperature. ·table [Table 9]

[0113] Various temperatures were tested to compare the properties and behavior of PDMS curing. However, the first mold used in this process was unable to support high temperatures. Therefore, ambient temperature curing was tested over a weekend. Finally, once the various features were cast onto the PDMS mask, the open side of this needed to be sealed. Two different methods for sealing the mask were used and tested with different results. (Figure 17 outlines the various steps in the prototyping process to create a continuous-flow bioreactor system.) The first method featured sealing the PDMS mask with another flat layer of PDMS, which allowed for an extra thickness of the overall mask, which was useful for incorporating piping and fittings into the mask. The second method featured sealing the open side of the PDMS mask with Kapton tape. This method allowed for a perfect seal between the features of the PDMS mask and the tape. However, the thinner the mask, the more complicated the integration of the piping and fittings.

[0114] Sensing method The main detection system is designed taking into account the functional components and reactions of continuous-flow microfluidic systems. First, the sensing system of a microfluidic chip is limited by the chip size and the characteristics of the factors that need to be detected. Furthermore, selecting a suitable sensor to obtain accurate and real-time detection results from the chip is another key issue. Detection systems in microfabrication include spectrophotometers, pH sensors, and pressure controllers and sensors. The theory behind spectrophotometers has been explained by L. Zhu, (2005), Nuno Miguel Matos Pires, (2014), and Kihwan Choi, (2015). The theory behind pressure controllers and sensors has also been reviewed by Yung-Shin Sun, (2016).

[0115] Material Selection a) Spectrophotometer The spectrophotometer (USB2000+ Miniature Fiber Optic Spectrometer from Ocean Optics, UK) can store 1000 complete spectra per second and has a detection wavelength range of 190–1100 nm. In addition, the official software is primarily programmed using open-source Python packages tailored to the characteristics of the detection system. This spectrophotometer is ideal for monitoring continuous reactions. b) pH meter The pH meter (HI-1093B pH electrode, Hanna Instruments, Bedfordshire, UK) has a measurement range of 0 to 13. To integrate the pH meter with the microfluidic chip, the dimensions of the pH meter are limited by the design specifications. The diameter of the measuring cell on the microfluidic chip is 5 mm, and the body of the pH meter is 3 mm in diameter. c) Pressure Controllers and Sensors CFD analysis of the microfluidic chip indicates a pressure of 0.27 Bar in the bioreactor and TFF sections. The pressure sensor (MPS Microfluidic High Precision Pressure Sensor from ELVEFLOW, Paris, France) can achieve five measurement ranges from 70 mBar to 7 Bar. It can also detect ultra-small internal volumes down to 7.5 μL, making it applicable to microfluidic chips with a volume of 0.5 mL. The sensor's flow regulation is highly sensitive and responsive, making it suitable for real-time monitoring of minute changes in flow rate. The sensor's companion sensor reader (ELVEFLOW Sensor Reader, Paris, France) offers high speed and is easily integrated into the chip, making measurements simple and feasible. d) Pressure supply source A syringe pump (C3657 C-Series Syringe Pumps, Tricontinent, California, USA) was used as the pressure source to deliver the reactant materials to the microfluidic chip. The pump stroke speed ranged from 1.2 seconds to 100 minutes per stroke, and the pump resolution was 3,000 steps in standard mode and 24,000 steps in high-resolution mode. The system response time was 4 to 6 hours. This syringe pump also enabled steady-state flow rates in the microfluidic system. The pressures of the five inlets that inject the reactant components allowed for efficient and efficient 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, and the temperature of the reaction. At the same time, the inlet pressure must be controlled to adjust the flow rate of the mixed solution in the continuous flow channel to achieve the appropriate ratio of RNA production and the appropriate reaction time. Three detection sites are present on the microfluidic chip: two are used to quantify RNA and Mg2+, and the other is used to measure pH. Pressure sensors are also fabricated off-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, while absorbance measurement at 680 nm can be used to obtain Mg2+ quantification. The absorbance measurements of RNA and Mg2+ can be converted to concentrations using the Beer-Lambert law. In this design, PDMS is used to fabricate the bioreactor, which is sufficiently transparent, enabling effective RNA detection.

[0117] During the reaction, the pH value is the main factor affecting the corresponding thermostable DNA polymerase. A pH of 8.3 to 9.0 can provide optimal results in the system. Increasing the pH during the reaction can also help stabilize the DNA template and improve transcription results. The activity of the recombinase enzyme is significantly affected by the pH of the assay. For device integration, an Arduino microcontroller (ARDUINO UNO REV3 from Arduino, UK) was used as the data acquisition core.

[0118] Integrated Experimental Platform 18A-18C show schematic diagrams of the liquid flow reaction system. The system includes a plurality of sensors configured to measure various characteristics of the liquid flowing through the liquid flow system, including pressure, temperature, pH, flow rate, flow volume, and the like. The system incorporates a plurality of valves, injection ports, pumps, particularly syringe pumps, gates, and the like, to control the flow path of the liquid components through the device. For example, liquid components may be recirculated from outlet ports at the longitudinal ends of the bioreactor and / or filtration module to the inlet ports of the upstream bioreactor and / or filtration module, as needed. The system also includes a controller, which typically includes a CPU, microprocessor, or other suitable electronic processor device. In certain embodiments, the processor is integrated into a PCB or PLC. The controller may include a user interface, sensory data storage utilities, 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, in combination with sensors, injection ports, pumps, valves, and the like, can be utilized by the controller to continuously and automatically control the liquid flow through the liquid flow system. Thus, a continuous reactor system is realized, which allows for continuous input of chemicals and allows for continuous production of the desired reactant product. This system is useful for recycling and reusing chemicals, solvents, etc., minimizing waste and maximizing efficiency. After the equipment is prepared, their layout is designed and the experimental environment is set up. Naturally, the PDMS chip should be fixed by a fixture to prevent it from moving. The positions of other equipment are then determined accordingly.

[0119] Initially, four syringe pumps were used to apply pressure to the four input reservoirs. Typical tubing and fittings are used to deliver small sample volumes to the PDMS chip. Another pump was connected to the second reservoir at the beginning of the second bioreactor (the end of the first TFF), which injected the m7G methyltransferase and regulated the pressure within the chip. Pressure sensors using a feedback loop significantly improved the responsiveness of the flow control. The pressure sensors were designed to measure pressure at the inlet and outlet of the flow channel. The goal was to keep the syringe pumps running while maintaining a constant pressure within the device.

[0120] To detect the concentration, ultraviolet / visible light must pass through a detection chamber at the end of the first bioreactor (the serpentine flow path), and this light is transmitted by an optical fiber. Two micro-optical fibers, each matching a specific wavelength requirement, are installed vertically to each other. The two micro-optical fibers are a source fiber connected to a light source and a collection fiber connected to a small spectrophotometer. During continuous bioreactions, a pH sensor is placed in the buffer to measure the pH value, while a temperature sensor is placed on the board and a heater is placed on the opposite side to adjust the temperature. The pH and temperature sensors 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 describes the following linear relationship between analyte concentration and absorbance at a particular wavelength:

number

[0122] Therefore, the desired spectrophotometer data is the absorption spectrum itself. The Ocean Optics spectrophotometer provides an easy way to access data from Python. This is the Python-Seabreeze package, which includes the Seabreeze library for communicating with the spectrophotometer. This provides a fully working and tested reference implementation of the Ocean Optics USB interface, which means that spectrophotometer data can be read and monitored 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 to examine the effects of different drug ratios and the reaction time and ratio of magnesium ion. 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 10 mM NTP, 10 mM NaCl, and 75 mM MgCl2 with acetate ions. The maximum yield was 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 Mg(OAc)2 ratio is approximately 80 mmol. After 4 hours of reaction, the RNA production plateaued at approximately 1800 mM. The optimal combination of reaction conditions depends on four main factors: the proportions of MgCl2, NaCl, and NTPs, and the reaction time. In yet another experiment, the proportions of MgCl2, NaCl, and NTPs must be monitored and maintained at predetermined levels to maximize RNA production. The most notable difference between the Imperial College and Cranfield University experiments is the design of the reaction system. For manufacturing considerations, a continuous flow system (continues follow system) is designed for the bioreactor process, rather than a fed-batch reaction system. To verify the continuous flow system, this experiment is designed based on the above experiment conducted by the Shattock Group at Imperial College.

[0124] Three levels for the four factors were selected (Table 10) to verify 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 values ​​of DNA and T7 polymerase are reasonable reuse values. Using these factors, a new experimental design is shown in Table 11. The purpose of the experiment is to explore the relationship between RNA production and the four factors and to identify the reaction trend.

[0125] [Table 10]

[0126] [Table 11]

[0127] Results and Discussion Fluid dynamics and CFD analysis Applying the appropriate governing equations to the hydrodynamic study of the bioreactor and TFF sections, the filtrate volume was calculated under steady-flow conditions with constant fluid velocity and appropriate pressure gradients. Equation (6) was used to obtain a prediction of the pressure drop in the microchannel. The selection of the characteristic length 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 the inlet and outlet bend losses. Bend pressure drops can be obtained within the various number of bends in Table 14. [Table 14]

[0129] Hydrodynamic study of the TFF section The Cerman-Kozeny relationship is used to obtain the permeability constant and solve for the fluid parameters across the micropore channel under appropriate boundary conditions, where various particle sizes and pore sizes are applied as the input particle size is varied in Table 15. [Table 15]

[0130] The pressure drop for the geometry can be predicted from Equation 10. A limitation of the equation is that mathematical errors occur when the unit of measurement is microns. Pressure drop divided by the length of the micropore flow path (shown in Table 16).

number

[0131] The results obtained in this study are only estimates based on geometry, fluid conditions, and boundary conditions. According to membrane selection parameters for Pall products, the filtrate volumes shown in Table 17 can be estimated. [Table 17]

[0132] computational fluid dynamics analysis Velocity profile of bioreactor flow channel As shown by Figures 23A and 23B, there is velocity in the X direction throughout the bioreactor flow path. There are two different colors representing 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 increases due to the high pressure applied by the first pump, and then decreases to a constant velocity of approximately 0.00033 ms^-1. This velocity is approximately twice as fast as predicted.

[0133] Tangential flow filtration channel Figure 24 shows the velocities in the tangential flow filtration channel. The colors indicate that the individual velocities within the microchannels are close to the inlet velocity, ranging from 0.0001327 ms^-1 to 0.0001858 ms^-1. Furthermore, the data in Table 18 show that the outlet 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 flow path. Colors indicate individual pressure values. Figure 25B shows that the pressure drop is a result of the presence of multiple bends in the bioreactor flow path. These varying results were considered in the design modifications.

[0135] Experimental results PDMS mask verification Bioreactor Various variations of the mold have been recognized through the study of this application, and the resulting developments have been observed, which will now be described.

[0136] a) Type 1 generation First, we tested both bioreactors with the first variant. We selectively experimented with different sealants, Kapton tape and a second PDMS layer, and obtained results. Both reactors were allowed to cure at 35°C for two days. The two reactors were first sealed with another flat layer of PDMS. Green water was introduced into one of the inlets of each mask using a syringe. Figures 26A and 26B show initial results on the behavior of the liquid in each mask. The two figures show that, for the batch bioreactor, the green 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 hardens, as seen in the blue area in Figure 26A. Furthermore, Figure 26B shows that a sample of green water passed through the T-junction of the mask and followed the path of the main channel. However, the sample avoided some areas. This can be explained by the size of the channel walls, which could not support the pressure during the hardening process and collapsed.

[0137] Next, we tested a continuous flow reactor sealed with Kapton tape. Figure 27 shows the behavior of the fluid in this configuration. The fluid (dark green) followed the path without any problems. The seal performed better than the previous seal. However, due to the size of the channels, curing still did not occur properly, and some parts of the mask did not peel properly from the mold. This resulted in some defective areas in the mask.

[0138] From all the considerations made during various verifications, the design was modified. In addition, it was decided to create a platform. In the platform, the PDMS mask can be placed between two acrylic plates, which increases the pressure between the two PDMS layers and therefore prevents the channels from collapsing when verifying the mask. These two acrylic plates also allow the attachments and piping to be connected to the chip.

[0139] Figure 28 shows the Kapton sealed mask placed between the two plates and connected to the piping, fittings and some sensing equipment to allow for initial pH and absorbance testing and measurements.

[0140] b) Type 2 generation To print the second generation mold, a high-temperature resistant material was used, and the mask was cast at a curing temperature of 70 °C for 4 hours. As shown in Figure 29, a used mold was used, and the side that directly contacted the mold bonded to the mold. Small sections of the PDMS mask could not be peeled off, making it impossible to remove from the mask. A thin layer of PDMS remained attached to the mask, especially between the channels. Curing was successful, but the mask failed to peel from the mold. After attempting to clean the mold as much as possible with isopropanol, another prototype was obtained, cured at 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 to the PDMS layer that was in direct contact with the high-temperature resistant material. The walls could also be affected by the instability of curing uniformity across the mask.

[0141] Vertical Flow Filtration Module A single vertical TFF mold variation was printed using a high-temperature resistant material. For the bioreactor mask, the same process was followed: a PDMS sample was poured into a foil paper receiver and placed in an oven with two masks to compare various aspects and results of the process. Figures 30A and 30B show the mask and the sample cured in the oven. For the bioreactor mask, the PDMS that came into contact with the mold bonded to it. 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 any default settings and showed no change when peeled from the foil. Additionally, the sample perfectly molded to the shape of the foil, as shown in Figure 30B.

[0142] From the various considerations above, some analysis can be performed and results obtained. The chemistry and protocol are not at fault as the foil samples have been used successfully. The same default occurred several times with different masks. Therefore, high-temperature materials have a negative effect on PDMS mask casting and should not be used in such experiments. The original material used in the first run should be continued and cured for 48 hours with the curing parameters set to ambient temperature to ensure the PDMS mask is cast properly and to avoid any deflection under load away from the mold. Despite the above results, a new platform was created, as shown in FIG.

[0143] Based on the integrated experimental plan, all equipment was assembled in the laboratory and an experimental platform was constructed to verify the effectiveness of the system. The experimental platform constructed in the laboratory is shown in Figure 31. The PDMS chip was fixed in place by fixtures, and the pump and chip were connected by piping and fittings. Two optical fibers were installed perpendicular to each other: a source fiber connected to a light source and a collection fiber connected to a compact spectrophotometer (Ocean Optics USB2000+).

[0144] Absorbance measurement and digital output High accuracy absorbance measurement To accurately measure absorbance, a baseline must be established. First, a spectrum of the background emission source is recorded without any sample. Then, the experiment is repeated with the sample added to the absorption path. The first spectrum is then subtracted from the second spectrum to obtain the pure absorption spectrum of the sample. To counteract the effects of noise, the background spectrum should be subtracted from both the sample spectrum and the reference spectrum. Therefore, equation (9) should become:

number

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

[0146] UV-Vis data analysis Using the Python-Seabreeze API, it is easy to obtain real-time data from a UV-Vis spectrophotometer and plot a graph of intensity versus wavelength. As shown in Figure 32, the light intensity reaches its peak value at a wavelength of approximately 580 nm. To confirm the relationship between absorbance and wavelength at different concentrations, vitamin B12 solutions with different concentrations, A (25 mM / L), B (50 mM / L), C (75 mM / L), and D (100 mM / L), were used (see Figure 33). The UV-Vis spectrum results are shown in Figure 34. The 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 needs to be calibrated when used for the first time. Two standard buffer solutions are used to calibrate the pH sensor, these are pH 4.0 and 7.0 buffer solutions. After the calibration step provided by DFROBOT, the calibration is completed.

[0148] Real-time pH plot The next step feature was to validate the pH sensor using acidic and alkaline solutions. The results in Figure 35 show that a real-time graph changes dynamically with pH change, demonstrating its validity.

[0149] Consider Evaluation of important results The investigation and results are an effective attempt to implement lab-on-a-chip technology to realize continuous flow reactions while maximizing RNA yield. Applying the SBCE method allowed us to establish an entire design framework, which was followed throughout this project. The modular design of the device with separate functional subsystems allows for the inclusion of individual innovative solutions for the operation of the mixing, reaction, and filtration / purification sections. Figures 30A-30C show a schematic diagram of the integrated system, including a downstream module for formulation. In particular, Figures 30A-30C illustrate an embodiment of this flow system integrated with a modular flow reactor for the downstream formulation system (Module 2) (e.g., WO 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, and installed in a conventional fill-finish vaccine production line.

[0150] The synthesis performance of the device is shown in Figure 30D, which is a graph of absorbance versus wavelength for the output solution of the flow reactor of the embodiment of Figures 30A-30D. Figure 40A is a source code illustration of the absorbance data and real-time plotting (absorbance versus wavelength) in Python, and Figure 40B is a source code illustration of the live plotting pH data in Python.

[0151] The product was evaluated using UV-Vis spectroscopy for a conventional batch protocol graph of the flow reactor output solution evaluated using UV-Vis spectroscopy for a conventional batch protocol. The presence of a high peak in the graph indicating high RNA concentration levels indicates the significantly higher productivity of the method 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 higher peak in the flow protocol compared to the batch protocol corresponds to the higher concentration of nucleic acid present in solution. The dotted lines correspond to samples of known concentration of RNA and are included to aid in the comparison of what is obtained with the batch and flow protocols.

[0152] Two different design solutions have been developed for the reactor that allows for the conversion of RNA production from a largely manual batch process (traditional RNA synthesis) to a continuous flow format. Indeed, the continuous flow reactor of the present invention is an innovative and proven reactor that functions flawlessly. The present invention represents a process transformation of the prior art that implements well-developed equipment and processes and demonstrates operation and performance in addressing rapid scale-up and high-productivity production of RNA-based materials in an automated, computer-controlled continuous flow format.

[0153] The filtration module was extensively analyzed to meet the requirements of continuous flow processing and the ability to integrate with flow reactors, while being easy to manufacture and scale using common manufacturing methods. While tangential flow filtration is a common technique that has proven effective for RNA purification processes (A. Eon-Duval et al., 2002), the present invention includes a novel filtration device purposefully designed and constructed for use in continuous flow systems, which is easy to manufacture and can be integrated into continuous flow systems, enabling the production of a scalable, cost-effective device. The system can be integrated as a microfactory, which, upon use, can enable automated and flexible production of therapeutic nucleic acid-based materials. Additionally, the modular design allows the filtration system to recycle certain components, such as enzymes and plasmid DNA, thereby significantly reducing the overall cost of the manufactured material.

[0154] A novel integrated platform was developed to test and experiment on the entire system. The system was prototyped using an acrylic pressure plate. It also included all of the pump ports, sensors, and inlet and outlet mountings. This allowed for testing continuous flow reactions and filtration with rapid feedback from various process elements and steps. Furthermore, a modular system was prototyped, allowing for an easy-to-use framework for conducting experiments. 3D printing was the tool of choice for fabricating the molds due to its flexibility, low cost, and short lead time. The modular configuration can be easily translated into commercial production using techniques such as micromachining of glass, metals such as stainless steel, and polymethyl methacrylate (PMMA) (acrylic) substrates, as well as photolithography and deep reactive ion etching for substrates including photosensitive glass and silicon. Furthermore, injection molding can be used for the PMMA and polycarbonate materials of the structure for large-scale module fabrication.

[0155] UV-visible spectroscopy demonstrates the ability of the present invention to provide reliable online and in situ analysis of the contents of reaction solutions in real time, which also provides a closed feedback loop to manage fluid flow and adjust temperature, pH, and reactant dispensing in the flow system to maintain overall process conditions.

Claims

1. A step of introducing a plurality of reactants into a first liquid flow module through at least one inlet, the plurality of reactants including at least one type of nucleoside triphosphate (NTP), a reaction buffer, and DNA; flowing a liquid through a narrow reaction channel of the first liquid flow module; reacting at least some of the reactants in the narrow reaction channel to produce a reaction product; filtering the reaction product in a first filtration module; recirculating at least a portion of the liquid from the outlet of the narrow reaction channel and / or the filtration module via at least one recirculation conduit to the inlet region of at least one of the first liquid flow modules; A method for synthesizing RNA by a continuous flow process, comprising:

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

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

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

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

6. 6. The method of claim 1, further comprising the step of recycling at least some of the reactants from the outlet of the first filtration module to the inlet region of the narrow reaction channel.

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

8. 8. The method of claim 7, further comprising the step of supplying the liquid exiting the second reaction flow path to a second liquid flow filtration module.

9. 9. The method of claim 8, further comprising the step of recycling unreacted NTP to the inlet region of the narrow reaction channel and recycling unreacted capping enzyme to the inlet region of the second reaction channel.

10. The salt solution is MgCl ２ The method of any one of claims 6 to 9 when dependent on claim 5, comprising:

11. The method of any one of claims 6 to 10 when dependent on claim 5, wherein the RNA polymerase comprises T7 polymerase.

12. 12. The method of any one of claims 1 to 11, comprising providing a modular system in which the first fluid flow module comprises suitable connections that allow communication and connection with other component modules that form part of the modular system, such modules being connectable according to a desired synthetic pathway.

13. Use of an RNA or RNA-based compound prepared by the method of any one of claims 1 to 12 in the preparation of a vaccine or therapeutic.

14. a first reactor module having a first narrow reaction channel, at least one liquid inlet, and at least one liquid outlet, the at least one liquid inlet configured to receive a plurality of reactants including at least one nucleoside triphosphate (NTP), a reaction buffer, and DNA; at least one flow liquid delivery unit configured to deliver liquid to the narrow reaction channel of the first reactor module, wherein at least some of the reactants react in the narrow reaction channel when the reactants flow through the narrow reaction channel; a first filtration module having a fluid filtration region, at least one inlet in communication with the outlet of the first reactor module, and at least one outlet; at least one recirculation conduit for recirculating at least a portion of the liquid from the outlet of the first reactor module and / or the first filtration module to the region of the at least one inlet of the reactor module; A modular apparatus for RNA continuous flow synthesis comprising:

15. 15. The apparatus of claim 14, wherein the recirculation conduit extends between a region of the outlet of the first reactor module and the at least one inlet of the first reactor module.

16. a second reactor module having a narrow elongated reaction flow channel and at least one inlet and outlet, the inlet communicating with the first filtration module; a second filtration module having a fluid filtration region and at least one inlet and outlet, the inlet communicating with the outlet of the second reactor module; 16. The apparatus of claim 14 or 15, comprising:

17. a second recirculation conduit extending between a region of the outlet of the first filtration module and the outlet of the first reactor module; 17. The apparatus of claim 16, wherein optionally a third recirculation conduit extends between the area of ​​the outlet of the second filtration module and the inlet of the first reactor module.

18. the first filtration module comprising: a first narrow elongated flow path having an inlet portion; a second narrow elongated flow path having an outlet portion; an osmosis membrane disposed along the length of each of the first and second narrow passages to separate the first narrow passage from the second narrow passage so that permeate can pass from the liquid in the first narrow passage through the osmosis membrane into the second narrow passage along the length of the first and second narrow passages; Including, 15. The device of claim 14, wherein a majority of the length of the first narrow elongated channel is disposed adjacent to a majority of the length of the second narrow elongated channel via the permeable membrane.

19. 19. The device of claim 18, wherein the pore size of the permeable membrane is in the range of 100 to 1000 kDa, in the range of 100 to 800 kDa, in the range of 200 to 800 kDa, or in the range of 200 to 600 kDa, in the range of 300 kDa to 10 MDa.

20. 20. The device according to claim 18, comprising a first plate in which the first narrow flow path is formed and a second plate in which the second narrow flow path is formed, wherein the permeation membrane is sandwiched between the opposing surfaces of the first and second plates.

21. 21. The device of any one of claims 18 to 20, wherein each of the first and second narrow elongated flow paths comprises a series of straight and curved sections.

22. 22. The apparatus of claim 21, wherein the first and second narrow elongated channels include respective serpentine profiles along their lengths.

23. 23. A device according to any one of claims 18 to 22, wherein the first and second narrow elongate channels are open along their lengths and are arranged in direct contact with the permeable membrane which forms in part the longitudinal walls or faces of the first and second narrow elongate channels.

24. The device according to any one of claims 18 to 23, wherein the pore size of the permeable membrane is less than the average molecular size of an RNA molecule.

25. 25. The apparatus of any one of claims 14 to 24, including a first sensor that measures any one or combination of pressure, temperature or pH of the liquid in the apparatus.

26. a reaction status monitoring device for monitoring a property of the liquid in the apparatus indicative of a state of reaction of at least two chemical components of the liquid in the apparatus; a control unit that receives data from the at least one or a combination of the first sensor and the reaction status monitoring device and controls at least one characteristic of the liquid in the apparatus; 26. The apparatus of claim 25, comprising:

27. 27. The apparatus of claim 26, wherein the controller comprises any one or combination of a CPU, PCB, PLC, PC, processor chip, and handheld electronic device.

28. The characteristics of the device are: the pressure of the liquid in the device; the temperature of the liquid in the device; the pH of the liquid in the device; the volume or ratio of one or more chemical components of said liquid in said device; the flow rate of the liquid in the device 28. The device of claim 27, wherein the device is any one of the following:

29. 29. The device of any one of claims 25 to 28, further comprising a second sensor that measures any one or combination of pressure, temperature, pH of the liquid in the device.

30. 30. The apparatus of claim 29, wherein the first sensor and / or the second sensor comprises a UV-Vis spectrometer for absorbance analysis or fluorescence analysis in the range 190 nm to 1000 nm.

31. The device according to any one of claims 14 to 30, wherein the flow delivery unit is at least one pump, optionally a syringe pump.

Images