Method and modular device for synthesizing RNA-based therapeutic agents

Abstract

A method of RNA synthesis via a fluid flow device. The method may involve: introducing a plurality of reactants into a first fluid flow module via a plurality of inlet ports, the plurality of reactants including: at least one nucleoside triphosphate (NTP), a reaction buffer, and DNA, a DNA-based compound, or a DNA-based mixture; allowing at least some of the reactants to react within a reaction channel or well within a first module of the flow system; retaining the DNA at the first reactor module or recirculating the NDA at the first reactor module and allowing a reaction product flow of the reactants to the first fluid filtration module; and filtering the reaction product in the first filtering module.

Description

[0001] This application is a divisional application of a Chinese patent application with an international application number of PCT / GB2021 / 051132, a title of invention of "Methods and Modular Devices for the Synthesis of RNA-Based Therapeutic Agents", and an application number of 202180046367.5 after entering the Chinese national phase, which was filed on May 12, 2021. Technical Field

[0002] The present invention relates to methods of RNA synthesis and, in particular, although not exclusively, to scalable and modular fluid flow systems and methods for continuous, automated or semi-automated RNA synthesis. Background Art

[0003] A flow reactor (alternatively referred to as a continuous flow reactor) provides a continuous flow of materials or reactants through a network of conduits connected to form fluid channels. When assembling the mixing, reaction, and filtration modules of a flow system, fluid communication is achieved through various configurations of the conduit network by enabling or disabling fluid channels with ports and valves to control the conditions for the synthesis, purification, and formulation of RNA or nucleic acid therapeutic agents, thereby manufacturing products in a continuous flow format. A computer system operating purposefully constructed software code enables directed control of the entire set of process parameters within the flow system. These parameters include mixing conditions, temperature, pH, reagent concentration, monitoring, residence time, purity profile, yield, etc. Example prior art flow reactors are typically formed as assemblies of individual modules that are connected face-to-face to form a monolithic block through which fluid is directed. An example flow reactor is described in WO 2013 / 050764.

[0004] Flow technology provides more sustainable, flexible, and efficient pharmaceutical manufacturing production. Combining microtechnology and precision engineering, flow systems can be constructed and configured as microfactories integrating multiple unit operations (e.g., mixing, reaction synthesis, extraction, separation, filtration, and purification). Additionally, the typically small volume-to-surface area ratio in flow mixing and reaction systems allows for precise control of process conditions related to heat and mass transfer, thereby accelerating processes and increasing productivity with a small device footprint. An integrated flow system combining these unit operations can be regarded as a microfactory or scaled-down production system that reduces the use of factory floor space, reduces energy consumption, and improves resource utilization. Thus, compared to current manufacturing systems commonly used for producing substances for prophylactic vaccines, sustainable growth can be achieved with better environmental impact, cost-effectiveness, and flexibility.

[0005] In molecular biology and biotechnology, there are several protocols that increasingly use lab-on-a-chip (LOC) devices. Such LOC devices use microfabrication techniques to miniaturize and integrate laboratory assays and perform small-scale synthesis or filtration in a small chip. These small devices consume less material, generate less waste, reduce costs, and also allow for faster reaction times. By guiding fluids through micromixers, microchannels, filters, the various steps of a given protocol can be integrated into the LOC device, and the various steps of a given protocol allow for cell sorting, mixing, and enabling reactions for, e.g., the synthesis of DNA or RNA. Importantly, the LOC device is formed of reaction-inert material and preferably transparent to enable real-time visual inspection of the reactions in the chip. Additionally, the transparent device also enables spectroscopic analysis and studies. A preferred material for rapid prototyping for such applications is PDMS (polydimethylsiloxane).

[0006] In 2019, the World Health Organization identified ten global threats to global health. Particularly in LMICs (low- and middle-income countries) where epidemics such as Ebola or Dengue have / are affecting the population there, eight of these ten threats are related to epidemics and the availability of vaccines.

[0007] Therefore, there is an urgent need for devices and methods suitable for the manufacture of vaccines and vaccine precursors that can meet the requirements of large-scale, low-cost, and rapid batch synthesis. In particular, there is a need for a chemical compound manufacturing platform that can be used to meet emergency vaccine needs (i.e., within a few days, easy to store and deploy worldwide, and with high productivity and operational cost-effectiveness). Summary of the Invention

[0008] Accordingly, an object of the present invention is to provide a modular manufacturing platform that is capable of scalable synthesis and / or filtration of chemical compounds as well as biomolecules and non-biological molecules, particularly including RNA-based vaccines or other nucleic acid therapies. A particular object of the present invention is to provide a configurable and integrated microfactory flow system for the preparation of nucleic acid-based vaccine manufacturing.

[0009] The present disclosure provides a modular and integrated device inspired by these methods and laboratory protocols, which include configurable and highly scalable methods for the rapid and ultimately point-of-use (e.g., within a hospital environment) manufacture of RNA and nucleic acid substances.

[0010] In particular, the system includes a combination of a flow reactor unit, a "continuous" filtration unit, and a mixing unit. These units can be integrated in any order suitable for the target RNA, nucleic acid therapeutic or prophylactic vaccine. This integrated flow system can include several optional analytical detectors (including fiber optic detectors), detectors, and light sources for UV-Vis absorbance or fluorescence spectroscopy to enable in-situ and on-line control of the synthesis or manufacture of the target substance during continuous flow.

[0011] A specific objective is to provide microfluidic-based devices and methods that integrate modules including a fluid flow bioreactor module and a fluid flow filtration module interconnected via respective conduits / channels to create a continuous fluid flow path, which can be continuously operated and automated using electronic and / or software control tools (involving the use of membranes, component actuators such as pumps, valves, gates, delivery ports, outlet ports, syringe pumps, sensors, etc.), as will be understood.

[0012] A specific objective is to provide a fluid flow system for the reaction synthesis of selected chemical components such as biomolecules and non-biological molecules via a process that can be continuous or discontinuous and can be automatic or semi-automatic. Another specific objective is to provide a modular system in which the individual module units can be interconnected according to the synthesis pathway. Such units can include ports, valves, and appropriate connections to enable fluid communication and interconnection between the individual modules. Another specific objective is to provide a reaction system for fluids that can be configured to measure and respond to the characteristics of the system (e.g., the pressure, temperature, pH of the fluid, the volume or proportion of one or more selected chemical components of the fluid, the flow rate of the fluid, and the reaction state of the fluid).

[0013] Another specific objective is to provide a fluid flow filtration device and method in which the chemical components of a fluid can be separated within an automatic or semi-automatic fluid flow system. The objective is to provide a filtration system that can be continuously operated via appropriate electronic control and fluid delivery, recirculation, and / or drive via fluid pressure. Another specific objective is to provide a fluid flow system having one or more reactor modules and filtration modules interconnected to form a network.

[0014] An additional objective is to provide methods and devices for synthesizing RNA. Another specific objective is to provide devices and methods for synthesizing RNA from DNA. Yet another objective is to provide a form of synthetic RNA that can be used for preparing vaccines.

[0015] An object of the present invention is to provide a modular manufacturing platform capable of synthesizing and / or filtering chemical compounds, as well as biomolecules and non-biological molecules (in particular, including RNA and subsequent vaccines). A specific object of the present invention is to provide devices and systems configurable as microfactories for biomolecule synthesis to achieve downstream vaccine manufacturing.

[0016] A specific object of the present invention is to provide devices and methods for forming a fluid flow integration system, the fluid flow integration system including component modular parts, the component modular parts including a fluid flow bioreactor module and a fluid flow filtration module, the fluid flow bioreactor module and the fluid flow filtration module being interconnected via respective conduits / channels to create a fluid flow path that can be operated continuously and can be automated using electronic and / or software control tools.

[0017] According to a first aspect of the present invention, there is provided a method for RNA synthesis, the method comprising: introducing a plurality of reactants via a plurality of inlet ports into a first fluid flow module, the plurality of reactants including at least one nucleoside triphosphate (NTP), a reaction buffer, and DNA, a DNA-based compound, or a DNA-based mixture; allowing at least some of the reactants to react in reaction channels or wells within the first module of the flow system; retaining the DNA at or recycling the NDA at the first reactor module, and allowing the reaction product stream of the reactants to flow into a first fluid filtration module; and filtering the reaction product within the first filtration module.

[0018] Optionally, at least one nucleoside triphosphate (NTP) is a solution of at least one nucleoside triphosphate (NTP).

[0019] Optionally, at least one nucleoside triphosphate (NTP) includes any one of adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), uridine triphosphate (UTP) or a combination of adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), uridine triphosphate (UTP).

[0020] Optionally, the DNA is plasmid DNA. Optionally, the plurality of reactants further includes any one of an enzyme mixture, a salt solution, RNA polymerase or a combination of an enzyme mixture, a salt solution, RNA polymerase.

[0021] 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 conveying at least some of the filtered reaction product from the first filtration module into a second fluid flow reactor module and inputting a capping enzyme into the second reactor module. Optionally, the method includes conveying the fluid output from the second reactor module to a second fluid flow filtration module. Optionally, the method includes recycling any unreacted NTP to the inlet region of the first reactor module and recycling any unreacted capping enzyme to the inlet region of the second reactor module.

[0022] Optionally, the salt solution or buffer comprises MgCl 2 . Optionally, the RNA polymerase comprises T7 polymerase.

[0023] According to another aspect of the invention, there is provided an RNA or RNA-based compound prepared by the method as described herein.

[0024] According to another aspect of the invention, there is provided the use of an RNA or RNA-based compound prepared by the method as described herein in the preparation of a vaccine.

[0025] According to another aspect of the invention, there is provided a fluid flow filtration device comprising: a first elongate fluid flow channel having an inlet; a second elongate fluid flow channel having an outlet; a permeable membrane positioned to separate the first channel from the second channel along respective lengths of the first and second channels such that permeate can pass from the fluid within the first channel through the membrane into the second channel along the lengths of the first and second channels.

[0026] Optionally, most of the length of the first channel is positioned adjacent to most of the length of the second channel via the membrane.

[0027] Optionally, the pore size of the membrane is in the range of 100 kDa to 1000 kDa, 100 kDa to 800 kDa, 200 kDa to 800 kDa or 200 kDa to 600 kDa, 300 kDa to 10 MDa.

[0028] Optionally, the device includes a first plate in which a first channel is formed and a second plate in which a second channel is formed, with a membrane sandwiched between opposite faces of the first and second plates respectively. Optionally, each of the first and second channels includes a series of straight portions and curved portions. Optionally, the first and second channels include respective serpentine profiles in their longitudinal directions. Optionally, the first and second channels are open along their lengths and are positioned in direct contact with the membrane, which partially defines the longitudinal walls or faces of the first and second channels. Optionally, the pore size of the membrane is smaller than the average molecular size of RNA molecules.

[0029] According to another aspect of the present invention, there is provided a fluid flow system for processing a fluid, the fluid flow system comprising: a first reactor module having a reaction flow channel or well, at least one inlet and at least one outlet; a first filtration module having a fluid filtration region, at least one inlet and at least one outlet arranged to be in fluid communication with the outlet of the first reactor module; wherein the first filtration module includes a fluid flow filtration device as described herein.

[0030] 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 arranged to be in fluid communication with the first filtration module.

[0031] Optionally, the system includes a second filtration module having a fluid filtration region, at least one inlet and an outlet, the inlet being arranged to be in fluid communication with the outlet of the second reactor module.

[0032] Optionally, the system includes a fluid injection port arranged to be in fluid communication with the inlet region of the first filtration module. Optionally, the system includes a first recirculation conduit extending between a region of the outlet 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 a region of the outlet of the first filtration module and the outlet of the first reactor module. Optionally, the system includes a third recirculation conduit extending between a region of the outlet of the second filtration module and the inlet of the first reaction module.

[0033] According to another aspect of the present invention, there is provided a method of filtering a fluid using a fluid flow filtration device, the method comprising: driving a fluid through a first elongate fluid flow channel from an inlet; forcing a permeate component of the fluid through a membrane extending along the first channel and into a second elongate fluid flow channel; and retaining a retentate component of the fluid within the first channel; wherein the membrane is positioned to separate the first channel from the second channel along respective lengths of the first and second channels such that the permeate can pass from the first channel through the membrane into the second channel along respective lengths of the first and second channels.

[0034] According to another aspect of the present invention, there is provided a flow system, the flow system comprising: at least one reactor module having a reaction fluid flow channel or well, at least one fluid inlet and at least one fluid outlet; at least one flow driver for driving fluid through the channel or well; a first sensor for measuring any one of pressure, temperature or pH of the fluid within the system or a combination of pressure, temperature or pH of the fluid within the system; a second sensor for measuring any one of pressure, temperature, pH of the fluid within the system or a combination of pressure, temperature, pH of the fluid within the system; reaction state monitoring means for monitoring a property of the fluid within the system which indicates a reaction state of at least two chemical components of the fluid within the system; and a control unit for receiving data from at least one of the first sensor, the second sensor and the reaction state monitoring means or a combination of the first sensor, the second sensor and the reaction state monitoring means, and controlling at least one property of the fluid within the system.

[0035] Optionally, the property of the system is any one of the following or a combination of the following: the pressure of the fluid within the system; the temperature of the fluid within the system; the pH of the fluid within the system; the volume or proportion of one or more chemical components of the fluid within the system; the flow rate of the fluid within the system. Optionally, the step of driving the fluid comprises: pressurizing the fluid within the first channel. Optionally, the control unit comprises a CPU, a PCB, a PLC, a PC, a processor chip, a handheld electronic device. Optionally, the additional sensors comprise any one of a temperature sensor, a pH sensor, a pressure sensor, a flow rate sensor, a flow sensor or a spectral sensor or a combination of a temperature sensor, a pH sensor, a pressure sensor, a flow rate sensor, a flow sensor or a spectral sensor.

[0036] According to another aspect of the present invention, there is provided a method of treating a fluid using a fluid flow device, the method comprising: introducing at least one fluid into a reactor module via at least one inlet; using at least one flow driver to drive the fluid through a reaction flow channel or well or using at least one flow driver to drive the fluid within the reaction flow channel or well, and outputting the fluid at an outlet; measuring at least one of the pressure, temperature or pH of the fluid within the fluid flow device or a combination of the pressure, temperature or pH of the fluid within the fluid flow device; monitoring the reaction state of the chemical components within the fluid within the fluid flow device; and in response to at least one of the measurement of the pressure of the fluid, the measurement of the pH of the fluid, the measurement of the temperature of the fluid and / or the measurement of the reaction state of the chemical components of the fluid or a combination of the measurement of the pressure of the fluid, the measurement of the pH of the fluid, the measurement of the temperature of the fluid and / or the measurement of the reaction state of the chemical components of the fluid, using a control unit to control at least one characteristic of the fluid within the fluid flow device.

[0037] Optionally, the system comprises a plurality of reactor modules and filtration modules fluidly coupled together. Optionally, the flow driver is at least one pump, and optionally, the flow driver is an injection pump. Optionally, the fluid analysis sensor comprises a UV-Vis spectrometer for absorbance or fluorescence analysis in the range of 190 nm to 1000 nm.

[0038] Optionally, the method of RNA synthesis comprises: recycling any unreacted NTP to the inlet region of the first reactor module and recycling any unreacted capping enzyme to the inlet region of the second reactor module.

[0039] According to another aspect of the present invention, there is provided RNA prepared by the method described herein. According to another aspect of the present invention, there is provided the use of RNA prepared by the method according to any one of the preceding claims in the preparation of a vaccine.

[0040] According to another aspect of the present invention, there is provided a fluid flow device for treating a fluid, the fluid flow device comprising: a first reactor module having a reaction flow channel or well, at least one inlet and at least one outlet; a first filtration module having a fluid filtration region, at least one inlet and at least one outlet, the inlet being arranged to be in fluid communication with the outlet of the first reactor module.

[0041] Optionally, the device comprises a second filtration module having a fluid filtration region, at least one inlet and an outlet, the inlet being arranged to be in fluid communication with the outlet of the second reactor module.

[0042] Optionally, the device includes a fluid injection port configured to be in fluid communication with the inlet region of the second reactor module. Optionally, the device includes a first recirculation conduit that extends between the region of the outlet of the first reactor module and the region of at least one inlet of the first reactor module.

[0043] Optionally, the device includes a third recirculation conduit that extends between the region of the outlet of the second filtration module and the region of at least one inlet of the first reaction module. Optionally, the device includes a plurality of inlet ports to allow input of fluid chemical components into the reaction channels or wells. Optionally, the device includes at least one pump, the at least one pump being coupled to at least one inlet of the first reactor module to drive fluid through the reaction channels or drive fluid within the wells. Optionally, the device includes at least one valve, fluid flow gate, fluid flow port, heating element, fluid storage device or storage container configured to be in fluid communication with the fluid within the device. Optionally, the pump includes an injection pump.

[0044] 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 grooves disposed on or within the plate-like structure. Optionally, the device includes a plurality of sensors positioned at different fluid flow regions of the device.

[0045] Optionally, the sensors include at least one temperature sensor, at least one flow rate sensor, at least one pressure sensor, at least one pH sensor, at least one flow sensor, at least one spectroscopic sensor, at least one optical sensor, at least one optical fiber or spectroscopic fiber, or any combination of at least one temperature sensor, at least one flow rate sensor, at least one pressure sensor, at least one pH sensor, at least one flow sensor, at least one spectroscopic sensor, at least one optical sensor, at least one optical fiber or spectroscopic fiber.

[0046] Optionally, the device includes a control unit coupled to at least one pump, valve, fluid flow gate, fluid flow port, heating element, fluid storage device or storage container, and sensors to control the characteristics of fluid flow within the device in response to the state of the physical, chemical or mechanical properties of the fluid determined by the sensors. Optionally, the control unit includes a CPU, processor, PCB, PLC, handheld electronic device.

[0047] Optionally, the control unit includes a control module, and the control module includes any one of the following or a combination of the following: software; electronic components; data storage means; wired or wireless communication modules and / or ports; visual display outputs; user interfaces; at least one actuator for actuating any one of at least one pump, valve, fluid flow gate, fluid flow port, heating element, fluid storage device or storage container and sensor, or a combination of at least one pump, valve, fluid flow gate, fluid flow port, heating element, fluid storage device or storage container and sensor.

[0048] According to another aspect of the present invention, there is provided a fluid flow system for processing fluids, the fluid flow system comprising: a plurality of devices as claimed in the claims herein, the final fluid flow outlet of each of the devices being coupled to a collection unit to combine the fluid outputs from each of the devices.

[0049] Optionally, the fluid flow system includes a control unit, the control unit being coupled to at least one pump, valve, fluid flow gate, fluid flow port, heating element, fluid storage device or storage container, and sensors of each device or selected devices, to control the characteristics of fluid flow within the devices in response to the state of the physical, chemical or mechanical characteristics of the fluid determined by the sensors.

[0050] According to another aspect of the present invention, there is provided a method of processing fluids using a fluid flow device, the method comprising: introducing at least one fluid into a first reactor module via at least one inlet, and allowing the fluid to flow through or within a reaction flow channel or well, and outputting the fluid at an outlet of the first reactor module; driving at least a portion of the fluid from the outlet through a filtration module having a fluid filtration region via at least one inlet, and outputting the fluid from the first filtration module at an outlet; wherein, within the first reactor module, a reaction occurs between at least two chemical components of the fluid, and filtration of the fluid occurs within the first filtration module.

[0051] Optionally, the method includes using at least one fluid pump to drive fluid through the device. Optionally, the method includes: recirculating at least a portion of the fluid from an outlet of the reactor module and / or the filtration module to a region of at least one inlet of the reactor module via at least one recirculation conduit. Optionally, the method includes: monitoring the state of a chemical reaction occurring between chemical components within the fluid using at least one sensor. Optionally, the method includes: controlling the flow of fluid through the device in response to the state of the chemical reaction identified by the sensor. Optionally, the method includes: monitoring the physical, chemical, and / or mechanical properties of the fluid within the device using at least one sensor. Optionally, the method includes: controlling the flow of fluid within the device in response to the physical, chemical, and / or mechanical properties of the fluid identified by the sensor. Optionally, the step of controlling the fluid flow includes: controlling or deactivating at least one actuator, pump, valve, gate, or port that is arranged to be in fluid communication with the fluid within the device. Optionally, controlling the fluid flow includes: using a CPU, processor, PCB, PLC, or handheld electronic device to actuate or deactivate at least one actuator, pump, valve, gate, or port.

[0052] According to another aspect of the present invention, there is provided a fluid flow filtration device, the fluid flow filtration device comprising: a first elongate fluid flow channel having an inlet; a second elongate fluid flow channel having an outlet; a permeable membrane positioned to separate the first channel from the second channel along respective lengths of the first and second channels such that permeate can pass from the fluid within the first channel into the second channel via the membrane along the lengths of the first and second channels.

[0053] Optionally, the first channel includes at least one outlet; and wherein the inlet is arranged at or towards a first longitudinal end of the first channel, and the outlet is arranged at or towards a second longitudinal end of the first channel. Optionally, the second channel may include at least one inlet; and wherein the inlet is arranged at or towards a first longitudinal end of the second channel, and the outlet is arranged at or towards a second longitudinal end of the second channel.

[0054] According to another aspect of the present application, there is provided a fluid flow system for treating a fluid, the fluid flow system comprising: a first reactor module having a reaction flow channel or well, at least one inlet, and at least one outlet; a first filtration module having a fluid filtration region, at least one inlet arranged to be in fluid communication with an outlet of the first reactor module, and at least one outlet; wherein the first filtration module includes the fluid flow filtration device as described herein.

[0055] According to another aspect of the present invention, there is provided a method of filtering a fluid using a fluid flow filtration device, the method comprising: driving a fluid through a first elongate fluid flow channel from an inlet; forcing a permeate component of the fluid through a membrane extending along the first channel and into a second elongate fluid flow channel; and retaining a retentate component of the fluid within the first channel; wherein the membrane is positioned to separate the first channel from the second channel along respective lengths of the first and second channels such that the permeate can pass from the first channel through the membrane into the second channel along the respective lengths of the first and second channels.

[0056] According to another aspect of the present invention, there is provided a fluid flow system comprising: at least one reactor module having a reaction fluid flow channel or well, at least one fluid inlet, and at least one fluid outlet; at least one flow driver for driving fluid through the channel or well; a first sensor for measuring any one of pressure, temperature, or pH of the fluid within the system or a combination of pressure, temperature, or pH of the fluid within the system; a second sensor for measuring any one of pressure, temperature, or pH of the fluid within the system or a combination of pressure, temperature, or pH of the fluid within the system; reaction state monitoring means for monitoring a characteristic of the fluid within the system, the characteristic indicating a reaction state of at least two chemical components of the fluid within the system; and a control unit for receiving data from at least one of the first sensor, the second sensor, and the reaction state monitoring means or a combination of the first sensor, the second sensor, and the reaction state monitoring means, and controlling at least one characteristic of the fluid within the system.

[0057] According to another aspect of the present invention, there is provided a method of processing a fluid using a fluid flow device, the method comprising: introducing at least one fluid into a reactor module via at least one inlet; using at least one flow driver to drive the fluid through a reaction flow channel or well or using at least one flow driver to drive the fluid within a reaction flow channel or well and outputting the fluid at an outlet; measuring at least one of pressure, temperature, or pH of the fluid within the fluid flow device or a combination of pressure, temperature, or pH of the fluid within the fluid flow device; monitoring a reaction state of chemical components within the fluid within the fluid flow device; and using a control unit to control at least one characteristic of the fluid within the fluid flow device in response to at least one of a measurement of the pressure of the fluid, a measurement of the pH of the fluid, a measurement of the temperature of the fluid, and / or a measurement of the reaction state of the chemical components of the fluid or a combination of a measurement of the pressure of the fluid, a measurement of the pH of the fluid, a measurement of the temperature of the fluid, and / or a measurement of the reaction state of the chemical components of the fluid. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Specific embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0059] Figure 1 is a schematic diagram of a continuous reaction and filtration device applicable to RNA production;

[0060] Figure 2 is applicable to Figure 1 a perspective view of the reactor and filtration module of the device;

[0061] Figure 3 is applicable to Figure 1 a plan view of the well or batch bioreactor module of the device;

[0062] Figure 4 is applicable to Figure 1 a plan view of the reaction channel-shaped bioreactor module of the device;

[0063] Figure 5A is applicable to Figure 1 a plan view of the filtration module of the device;

[0064] Figure 5B is Figure 5A an image of the membrane inside the filtration module;

[0065] Figure 6A is a diagram of a part of the filtration module that has a region configured for centrifugal separation of chemical components applicable to Figure 1 the device;

[0066] Figure 6B is an image of a part of the filtration module that has a region for centrifugal separation of chemical components;

[0067] Figure 7 is a vertical tangential flow filtration (VTFF) module applicable to this fluid flow device;

[0068] Figure 8 is Figure 7 an enlarged view of the region of the filtration module;

[0069] Figure 9 is a plan view of the helical tangential flow filtration module;

[0070] Figure 10A is a component of the filtration module according to a specific implementation;

[0071] Figure 10B is Figure 10A a schematic diagram of another component of the filtration module;

[0072] Figure 10C is Figure 10A and Figure 10B another schematic diagram of the components of the filtration module;

[0073] Figure 11A is a plan view of an additional filtration module formed as a double-layer microchannel TFF;

[0074] Figure 11B is a schematic diagram of a filtration mechanism and a filtration module according to a specific implementation;

[0075] Figure 12 is a schematic diagram of a channel flow bioreactor module;

[0076] Figure 13 is a graph for calculating the design of a fluid flow bioreactor and a filtration system;

[0077] Figure 14 is a schematic diagram of a reactor module coupled to a filtration module to form a fluid flow device;

[0078] Figure 15 is a perspective view of a TFF module;

[0079] Figure 16 is an image of a microscopic analysis of the channel dimensions of a TFF module;

[0080] Figure 17 is a schematic diagram of an overview of a prototype construction process;

[0081] Figure 18A is a schematic diagram of a fluid flow system according to a specific implementation, the fluid flow system including a reactor module, a series of sensors, a reaction state monitoring arrangement, and a control unit;

[0082] Figure 18B is suitable as Figure 18A a schematic diagram of components of a reaction state monitoring arrangement that is part of a fluid flow system;

[0083] Figure 18C is a schematic diagram of a series of sensors, a reaction state monitoring arrangement, and a control unit that are part of a fluid flow system formed according to a specific implementation Figure 18A ;

[0084] Figure 19 is a schematic diagram of the architecture of a part of a microfluidic flow system including a spectrometer arrangement that is part of FIG. 18;

[0085] Figure 20 is a schematic diagram of a serial communication program of a control system;

[0086] Figure 21 is a graph of the relationship between RNA yield and the ratio of various reactants including NaCl, MgCl 2 , NTP, etc.;

[0087] Figure 22 A graph selected based on data analysis (RNA production vs. magnesium ions) and specific reaction times according to aspects of the present invention;

[0088] Figure 23A A schematic diagram simulating the result of velocity distribution analysis;

[0089] Figure 23B A graph of the velocity analysis curve;

[0090] Figure 24 A schematic diagram of the velocity in the tangential flow filtration channel within the filtration module;

[0091] Figure 25A A graph of the pressure drop in the bioreactor channel;

[0092] Figure 25B A graph of the pressure drop as a multi-step decline;

[0093] Figure 26A A photograph of a mask for manufacturing a fluid flow reactor and a filtration device;

[0094] Figure 26B A photograph of a mask for a continuous microfluidic flow reactor;

[0095] Figure 27 An image of a continuous flow reactor sealed with Kapton tape;

[0096] Figure 28 An image of a complete setup using two acrylic plates;

[0097] Figure 29 An image of a 3D printed mold after use;

[0098] Figure 30A The first part of a schematic diagram of an exemplary embodiment of the present flow system integrated with a downstream formulation system that uses a modular flow reactor attached to a conventional fill and finish vaccine production line;

[0099] Figure 30B The second part of a schematic diagram of an exemplary embodiment of the present flow system integrated with a downstream formulation system that uses a modular flow reactor attached to a conventional fill and finish vaccine production line;

[0100] Figure 30C The third part of a schematic diagram of an exemplary embodiment of the present flow system integrated with a downstream formulation system that uses a modular flow reactor attached to a conventional fill and finish vaccine production line;

[0101] Figure 30Dwas evaluated using UV-Vis spectroscopy Figures 30A to 30D Graph of the relationship between the absorbance and wavelength of the output solution of the flow reactor of the Figures 30A to 30D embodiment, compared with the graph of the relationship between the absorbance and wavelength of the output solution of the flow reactor of the

[0102] Figure 31 is a photograph of an experimental fluid flow chemistry compound synthesis platform;

[0103] Figure 32 is a real-time graph of spectrometer data (intensity vs. wavelength) in Python;

[0104] Figure 33 is an image of samples with different concentrations (A: 25 mM / L, B: 50 mM / L, C: 75 mM / L, D: 100 mM / L) for a microfluidic flow reactor module and a continuous filtration system;

[0105] Figure 34 is a graph of UV-Vis spectral analysis;

[0106] Figure 35 is an image of real-time plotting of pH value data in Python;

[0107] Figure 36 is a graph of a prediction plot (reaction with 1 mM dNTP on the left, reaction with 4 mM dNTP on the right);

[0108] Figure 37 shows a graph of the predicted distribution plots of the first experiment and the second experiment (reaction with 1 mM dNTP on the top, reaction with 4 mM dNTP on the bottom);

[0109] Figure 38 is a summary of various data on fluid flow bioreactors and filtration (reaction with 1 mM dNTP on the left, reaction with 4 mM dNTP LogWorth = -log10(p value) on the right);

[0110] Figure 39 is a schematic diagram of an enlarged fluid flow reactor and equipment of the present system;

[0111] Figure 40A is an illustration of the source code of absorbance data and a real-time graph (absorbance vs. wavelength) in Python;

[0112] Figure 40B is an illustration of the source code of real-time plotting of PH value data in Python. Detailed Description of the Invention

[0113] Hydrodynamics Principles in Microchannels

[0114] Advances in microfabrication have made it possible to construct microchannels with micron-sized dimensions. Since microchannels are typically integrated into these microsystems, it is important to determine the characteristics of fluid flow in microchannels for better design of various microfluidic devices. Due to technological limitations, the understanding of fluid behavior in porous media (specifically, microporous media for lab-on-a-chip designs that can be used in the manufacture of RNA vaccines) is very limited. In the sense that fluid-fluid interfaces do not form, porous media are considered to be saturated with the fluid of interest, and a single fluid predominates in the pore space. Let dp be the particle size and U be the velocity scale. It has been found that (1) is applicable for Reynolds numbers up to 1, i.e.:

[0115]

[0116] The permeability of a porous medium is a property that depends on the pore size and pore structure. Dimensional analysis shows that the permeability is a function of the porosity e and the particle diameter dp; each represents the pore geometry and pore size, respectively. The Carman-Kozeny relation empirically relates these quantities, but with dimensional correctness as:

[0117]

[0118] The particle diameter is expressed in meters, and the dimension of permeability is m2. In applications, we may expect to have the length scale of the device itself, say L, and the ratio is defined as:

[0119]

[0120] which is called the Darcy number (Da). In many applications, the particle diameter is on the order of a fraction of a millimeter, while the length scale of the device is about one meter or longer. Equation 2 can be appropriately formulated to be applicable within the limits of Da << 1 and Re < 1. Bahrami et al. (M. Bahrami, 2006) developed a general model for predicting the pressure drop in microchannels of arbitrary cross-section. The choice of characteristic length is an arbitrary choice and will not affect the final solution. According to the model of Bahrami et al., in a microchannel of arbitrary cross-section, the pressure drop for fully developed laminar flow can be obtained from:

[0121]

[0122] where Ip* = Ip / A is the specific polar moment of inertia of the microchannel cross-section, ΓГ = 4(W + H) is the perimeter of the microchannel cross-section, L is the fully developed length, Q is the volumetric flow rate, and ε = aspect ratio (width / height) of the rectangular microchannel.

[0123] Given the volumetric flow rate Q and cross-sectional area A, the Reynolds number is calculated as follows:

[0124]

[0125] Minor loss ΔPmin: Other pressure losses associated with the measured pressure drop are inlet loss, outlet loss, and bend loss. These losses are typically obtained from traditional relationships used at the macroscopic scale. Phillips (reference) shows that the minor pressure loss can be obtained from the following:

[0126]

[0127] where A and A t are the cross-sectional area of the channel and the cross-sectional area of the connecting pipe, respectively. K b is the loss coefficient of the bend, and K c and K e represent the contraction and expansion loss coefficients due to area changes. Phillips suggests that for a 90-degree bend, K b is approximately 1.2. Assuming that the cross-sectional areas of the channel and the connecting pipe are equal, K c and K e are at their maximum possible values, and the minor loss relative to the measured pressure drop can be neglected compared to the measured pressure drop. Hooman and Merrikh (M. Bahrami, 2010) have developed an analytical solution for the flow and pressure drop in a porous channel as follows:

[0128]

[0129] h, L, and W are the depth, length, and width of the porous channel, respectively.

[0130] In the samples tested in this study, the aspect ratio ε of the cross-section is 0.5. Therefore, instead of considering the entire rectangular cross-section, the sample can be modeled as a porous medium sandwiched between two parallel plates.

[0131] Selecting the appropriate cartridge depends on the total sample volume, the required processing time, and the desired final sample volume. The following equation is used to calculate the membrane area required to process the sample within a specified time:

[0132]

[0133] where,

[0134] A = membrane area (m2)

[0135] V = volume of filtrate produced (liters)

[0136] J = filtrate flux (liters per square meter per hour, LMH)

[0137] T = Processing time (hours)

[0138] Fluid flow simulation for prototype research

[0139] The height of the channel and the liquid type have an impact on the performance of the microchannel. Computational Fluid Dynamics (CFD) FLUENT software is used to simulate the microchannel. From the CFD, the velocity distributions in the non-fully developed and fully developed regions are analyzed to study the fluid flow behavior. The properties of the liquid affect the fluid flow inside the microchannel. We need the minimum kinematic viscosity and low surface tension, which depend on choosing the correct liquid.

[0140] Fluid flow plays an important role in guiding the integration of small-scale devices with many applications because of their potential in chemical and biochemical engineering. Using experimental data of different types of liquids and surface roughnesses, the mechanisms and fundamental principles of single-phase fluid flow are reviewed. Thus, at the microscale, the effects of liquid properties such as surface tension and viscosity are dominant. [Nawi, M.N.M., Manaf, A.A., Arshad, M.R., and 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, pp. 345–348].[[]END]]

[0141] The Reynolds number is the ratio of the inertial force to the viscous force of a fluid flowing in a channel (i.e., the ratio of the momentum of the fluid to the frictional force exerted by the channel walls). Low Reynolds number flow is laminar or stratified flow, in which the fluid streams flow parallel to each other and mix only by convection and molecular diffusion. High Reynolds number flow is turbulent flow, in which "packets" of various sizes of the fluid exhibit random motion in both space and time simultaneously, resulting in rapid mixing throughout the channel. The transition between laminar and turbulent flow typically occurs at Re = 2000 in internal flows [Schulte, T.H., Bardell, R.L., and Weigl, B.H. (2002), Microfluidic-technologies-in-clinical-diagnostics_2002_Clinica-Chimica-Acta., Vol. 321, pp. 1-10].

[0142] Although the turbulent field is more complex than the laminar field, there are significant second-order effects. One-dimensional and two-dimensional models that depict the time-dependent evolution of the analytical distribution under such conditions have been improved. Confocal fluorescence microscopy experiments and three-dimensional numerical modeling can help to confirm the quantitative description of the reaction-diffusion process near the wall [Schulte, T.H., Bardell, R.L., and Weigl, B.H. (2002), Microfluidic-technologies-in-clinical-diagnostics_2002_Clinica-Chimica-Acta., Vol. 321, pp. 1-10].

[0143] Integration of the sensor with the flow system

[0144] In the research, a platform for sensor integration should be established to obtain 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] presented a flowchart describing the path to achieve robust sensor integration in a fluid flow device, which can serve as a guide for integration research. 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, Vol. 133, pp. 1 - 7]. In Wang's method, complementary metal oxide semiconductor (CMOS) technology shows great potential in integration and detection. The CMOS image sensor works as an effective reaction and detection platform, enabling it to monitor real-time photon changes according to the amplification process. The CMOS image sensor observes photons and converts them into digital units. In addition, UV spectroscopy studies, optical color intensity detection, and pH value analysis were also carried out 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].López-Huerta [López-Huerta, F., Woo-Garcia, R., Lara-Castro, M., Estrada-López, 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] has proposed an integrated ISFET pH microsensor in a CMOS standard process. The whole system is integrated in a silicon area of 1.12 mm. 2 It presents a linearity of 59 mV / pH in the concentration range of pH values from 2 to 12, making it a good alternative for biological or medical applications.

[0145] CMOS is a relevant option, but it is difficult to implement considering cost and time. More realistically, the integration scheme for each sensor has been reviewed. For pH sensors, research on real-time feedback control of pH within microfluidics using integrated sensing and actuation has been found [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 utilizes an extended-gate ion-sensitive field-effect transistor (ISFET) and an integrated pseudo-reference electrode to monitor the pH value in a fluid flow reaction chamber. For temperature sensors, a serpentine temperature sensor (line width: 50 mm) and a heater (line width: 400 mm) are integrated below 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 fibre waveguides, Analytical and Bioanalytical Chemistry, 407(24), pp. 7467 - 7475] have proposed a new method for in-plane digital microfluidic spectroscopy. In this technique, a custom manifold aligns optical fibres with the digital microfluidic device, allowing optical measurements to be made in the plane of the device, which provides an idea for optical detection. The preferred analysis wavelength range of the present invention is between 190 nm and 1000 nm. The peak obtained is in the region of 260 nm.

[0146] Method

[0147] Continuous flow synthesis and scale-up

[0148] Figure 1 A schematic diagram of a continuous RNA production process is shown. As Figure 1 shown, the fluid flow device includes a first bioreactor module 10, a first tangential flow filtration (TFF) module 11, a second bioreactor module 12, and a second tangential flow filtration (TFF) module 13. Each of modules 10 to 13 is interconnected via respective channels or conduits. A plurality of fluid delivery input ports 14 are provided at the first reactor module 10 for introducing respective reactants into the first bioreactor 10. Each port 14 includes a respective injection pump 15. The first TFF includes respective input / entry ports 16, the second bioreactor module includes respective entry / input ports 17, and the second TFF module includes corresponding input / entry ports 18. Each of ports 16 to 18 may be provided with a respective associated pump or injection port for introducing fluid to drive a fluid stream under pressure into the elongated reaction channels 24 of each reactor and the elongated filtration channels 25 within each respective module. A plurality of recirculation channels 19, 20, 21 interconnect the respective fluid flow regions of modules 10, 11, 12, 13 (at or towards their respective inlet / outlet end regions) to provide fluid communication and reflux of reactants / reagents. Figure 1 The device further includes sensors, reaction state monitoring means, a control unit, and other associated electronic and actuator components (not shown) as described elsewhere herein to provide a fully automated and continuous fluid flow synthesis platform for manufacturing chemical compounds including biological and non-biological species. The first bioreactor 10 at the outlet end of the elongated reaction channel 24 includes a gate 23. The gate 23 is configured to filter large molecular weight chemical components such as DNA from the fluid based on molecular size and / or molecular weight. Then, the required filtered fluid is recirculated via channel 19 to the inlet region of the reactor 10. Thus, selected reagents and reactants can be cycled to provide a continuous or semi-continuous process.

[0149] Referring Figure 1 and Figure 2 both, Figure 2 shows Figure 1 a variant of the fluid system of Figure 1 wherein, in contrast to the elongated reaction channel 24 of Figure 1 the reactor 10 includes reaction wells 50. As described with reference to Figure 1 a plurality of injection ports 14 are coupled in fluid communication with the wells 50 for delivering chemical compounds, reagents, solvents, etc. required for the reaction. The filtration modules 11, 12 may include a series of extraction ports 51, 22 such that selected species, reagents, solvents can be extracted from the filtration modules and also the final product, such as fully capped RNA, can be collected, for example, at port 22.

[0150] As can be appreciated, the specific details of the channel profile, ports, gates, inlets, outlets, pumps, and use of fluid flow control actuators can be implemented with the arrangements described herein.

[0151] Referring Figure 39 , referring Figure 1 and Figure 2 The multiple reactant and filtration systems described (and generally represented as plate units 10, 11, 12, 13) can be connected together as an expanded fluid flow reactor and apparatus to form a fully integrated modular fluid flow reaction and filtration system for manufacturing RNA-based therapeutics and prophylactic vaccines at a scale sufficient for evaluation and clinical trials and ultimately production. In particular, appropriate connecting conduits provide fluid communication between the respective plate units 10, 11, 12, 13 such that the output ends (at output 22) of each unit can be combined to provide a total output. Figure 39 The collective system of Figures 18A to 20 can include a single control unit, multiple control units, and respective sensors and reaction status monitoring tools and units as described herein with reference to Figures 18A to 20 . With the parallel operation of the multiple fluid devices of the present invention, continuous production of RNA-based therapeutics and prophylactic vaccines can occur. Through this expansion strategy, one can achieve a manufacturing output at the scale required by the target market, which can be a cluster of local hospitals, or the unit can be attached to a conventional filling and finishing line at the manufacturer's site. Figure 20 is a schematic diagram of a serial communication program between an Arduino and a Python IDE control utility. In a possible embodiment, an Arduino board can be used. In a preferred embodiment, an industrial edge computing SBC (single board computer) node is used.

[0152] Taking RNA synthesis as an example, in this process, a DNA fragment is replicated by the enzyme RNA polymerase into RNA. The transcription mixture consists of NTP (nucleoside triphosphate), DNA, MgCl 2 (which reacts with P 2 O 7 -4 to produce a precipitate, affecting the productivity of RNA), and T7 polymerase (catalytic action), which are respectively injected into the first bioreactor channel by syringe pumps. Hydroxynaphthol blue (HNB) is used as a colorimetric indicator for titrating Mg 2+ ions. Initially, HNB and Mg 2+ ions combine to form the HNB-Mg 2+ complex. Mg 2+The reduction of ions causes the color to change from purple to sky blue. The mixture stays in the first reactor 10 for about 6 hours, during which the RNA transcription reaction has been ongoing, and RNA begins to be produced, accompanied by the precipitation of magnesium pyrophosphate (Mg 2 P 2 O 7 ). Except for DNA, all the above components flow through the first tangential flow filtration (TFF) module 11 with a large molecular weight (MW) cut-off membrane. The DNA is filtered back to the original inlet port 14(2) via the recirculation channel 19 and is retained in the first reactor, while the other components are filtered to the next stage.

[0153] After the first filtration module, the above components permeate downstream through the T7 polymerase in the inlet port 14(4) via recirculation. Then the m7G methyltransferase (5’ cap) is injected into the port 17 and flows into the second reactor 12 and the channel 24, where the contents stay and react for 2 hours. The permeate enters the second TFF unit 13 with a medium molecular weight cut-off membrane. At this filtration stage, the unreacted NTPs and 5’ caps are retained and (via 21 and 20) recycled to the bioreactor 12 and / or 10. Finally, the purified RNA is extruded at the outlet 22. Meanwhile, other components such as Mg 2 PP i O, water, and salts are collected in another container or port 51 and optionally discarded or further processed. Then, the output (capped RNA) can be processed via additional processing protocols (not described herein but familiar to those skilled in the art) to produce the desired vaccine.

[0154] The aim is to achieve continuous synthesis and purification to obtain RNA substances and increase the system yield. It is necessary to add raw materials to the reactor via the component 14 to compensate for the consumed molecules to maintain the continuous reaction of the process, which can be achieved by adjusting the actuator injection pump and the feedback loop of the material flow. By optimizing the reaction conditions (such as magnesium concentration, pH, temperature, reaction time), a significant increase in the RNA yield output is possible.

[0155] Set-based concurrent engineering (SBCE) method

[0156] The lean product development method has been selected as the main framework for the development design. The lean product development method is based on set-based concurrent engineering (SBCE). SBCE is a process in which the product to be developed is divided into different subsystems to allow for the parallel development of a set of possible solutions for each of the subsystems. As the design evolves, the set of solutions for each subsystem is narrowed through knowledge-based decision-making using tools such as simulation, prototyping, testing, or other acquired knowledge. Following the SBCE method, the first basic step is to divide the product into different subsystems that can be developed separately. Considering the conceptual design of the system to be developed, there are four main defined processes, and these four main defined processes can be divided into exactly two functions to be developed: the bioreactor and the filtration process. At the same time, the system needs to be integrated with a control system that can control the pressure from the pump and has sensors in the critical parts of the product to measure critical parameters. After the conceptual design of the system is drawn, the different subsystems that can be developed in parallel can be defined. Table 1 shows the different subsystems, with a brief description and the corresponding innovation level defined for each subsystem.

[0157] Table 1: Modules of the flow system. These modules can be appropriately combined in any required order according to the needs of the target therapeutic agent. For example, after the last filtration module, there may be additional reactor / mixing steps for formulating the RNA therapeutic substance into a therapeutic product.

[0158]

[0159] Subsystem Design Specification Method

[0160] Bioreactor

[0161] Regarding the bioreactor, two different designs have been developed. The first design is inspired by the conventional batch process used in chemistry for RNA synthesis. This design includes four input ports 14 for each of the raw materials, and these four input ports 14 are connected to a 1 mL main chamber where the reaction occurs by allowing sufficient residence time. Then, the container can be selectively deflated by releasing the microfluidic valve at the outlet and introducing fresh reagents at the inlet. This means that the device operates in a continuous (perfusion) mode, comparable to a conventional batch system. Once the reaction is complete, the product is released into the output channel. A sensor evaluates whether it can proceed to the next step of processing or be rejected. Figure 2 and Figure 3 shows the design of the bioreactor.

[0162] This design requires a complex and well-coordinated control system to control the valves integrated with the device. After the residence time ends, the product needs to be fed into the rest of the system.

[0163] The second design of the bioreactor is a microfluidic reactor in the shape of a serpentine fluid conduit, which allows for the mixing of reactants and the continuous synthesis of target products, as described in WO 2019 / 193346 A1, the details of which are incorporated herein by reference. This embodiment achieves continuous synthesis by controlling fluid flow. In this design, there are four reagent input ports that are continuously fed into the flow reactor via a T-shaped mixer. The reaction conditions and effects are controlled by the net flow rate into the fluid conduit and the fluid velocities of the individual components through the T-shaped mixer. These are controlled by setting the dispensing rates of computer-controlled dispensing pumps. The product is formed when the mixture reaches the end of the serpentine flow bioreactor within a set residence time. At the output, the device is characterized by a small fluid holding well that temporarily slows down the fluid flow to obtain an on-line measurement of a preferred analytical method with a specific chemical solution, such as on-line UV-Vis spectroscopy. By extracting certain features of the signal, the signal from the spectrometer is parsed and analyzed in specially constructed software to determine whether the desired product has indeed been formed by comparison with a reference vector. Figure 4 is a schematic diagram of a serpentine flow bioreactor.

[0164] Filtration module

[0165] There are two types of filtration: direct flow filtration (DFF) and tangential flow filtration (TFF). The main flow of DFF is perpendicular to the filter, while the flow of TFF is parallel to the filter. The risk of blockage is much higher for DFF, and it has been shown that the filtrate flow rate of TFF is higher than that of DFF while increasing the filtration volume. In addition, typical applications of TFF are the concentration, diafiltration, and fractionation of biomolecules as well as the purification and removal of cells; these applications are similar to those that occur in this project. In other words, for the applications of this project, TFF is the most effective choice, and therefore, TFF is the filtration type that is selected.

[0166] For the filtration module, the TFF method is selected as the model. Several design solutions have been proposed in the research literature and commercial practice. For the present invention, several other factors have been considered, such as manufacturability and the ability to integrate as a module with the entire system for this subsystem. The first filtration module design includes a serpentine path with two channels at the same height, and the separation features formed as patterns on the device are used as the filtration membrane. X. Chen et al. (2007) [Microfluidic Chip for Blood Cell Separation and Collection Based on Crossflow Filtration, Sensors and Actuators B 130 (2008) (pp. 216 - 221), China], have used the TFF filter design in the past, which separates biological cells quickly and reproducibly and at a lower cost compared to other solutions.

[0167] In the present invention, a specifically selected membrane is used, the size of which is suitable for separating biomolecules that are typically present in solution after nucleic acid synthesis. Figure 5A and Figure 5B The filtration module is shown, including the filtration features within the serpentine microchannels. The size and length of these channels are specifically designed to allow the correct selection of biomolecules in the filtrate and retentate streams. The module is fabricated using micro - engineering techniques, including photolithography and deep reactive etching of glass or silicon substrates. These techniques are well - understood, industrially available, compliant with drug regulations, and low - cost for high - volume device production.

[0168] The second filtration module design does not require any filter membrane. It works on the principle of centrifugal force. If a fluid has a curved trajectory at a certain speed, the centrifugal force will cause some particles to move faster than others. Therefore, with a curved microchannel that divides into two different channels, it is possible to make particles of different sizes enter different outlet channels [Blatter, C., Jurischka, R., Tahhan, I., Schoth, A., Kerth, P., Menz, W. (2005), Microfluidic Blood / Plasma Separation Unit Based on Microchannel Bend Structures, Proceedings of the 3rd Annual International IEEE EMBS, Hawaii]. It is worth mentioning that this filtration method is only effective under appropriate conditions (such as the fluid velocity exceeding 1 [m / s]). Under suitable conditions, this separation technique can achieve an efficiency of up to 90%, and since there is no membrane, the filtration process can be greatly simplified. Figure 6A and Figure 6B shows the CAD design of this design solution.

[0169] 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 WO 2019 / 193346 A1, the details of which are incorporated herein by reference. As described by Z. Geng et al. (2012) [Continuous Blood Separation Utilizing Spiral Filtration Microchannel with Gradually Varied Width and Micro-Pillar Array, Sensors and Actuator B 180 (2013) (pp. 122 - 129), China], this design adds a centrifugal effect to the conventional serpentine filtration module, as mentioned above. This centrifugal effect improves the separation efficiency and reduces the risk of clogging. Figures 9 to 10C shows this design.

[0170] The fourth design of the filtration module uses a membrane sandwiched between two fluid 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] have used filtration membranes for blood cell separation. However, in the present invention, the filtration module uses two microchannel layers that sandwich the filtration membrane to produce a filtrate stream and a retentate stream, and the retentate stream is recycled back to the reactor module, while the filtrate stream continues to be collected at the output and directed to downstream modules according to processing requirements. The fluid comes from the reactor and enters the TFF process through the top channel. Due to the pressure difference, some molecules in the fluid pass through the membrane and enter the bottom channel, while molecules that are too large to pass through the membrane remain on the top channel. This design solution provides good efficiency, and most importantly, it provides a new perspective in terms of manufacturability. Figure 11A and Figure 11B shows the design of this solution.

[0171] The four embodiments of the filtration module described above rely on the control of the pressure difference between the flowing streams and the separation methods used. However, in the fifth embodiment, the device combines digital microfluidics technology and digital macrofluidics technology with protocols commonly used in molecular biology to separate and extract nucleic acids, particularly DNA. There are two such protocols. One is charge-based, and the other is to attach magnetic beads to DNA or RNA for purification. For example, in the present invention, magnetic beads attached to a DNA plasmid such as Invitrogen DNA Binding Beads (Thermofisher Scientific) can be held in a reaction vessel using a digitally encrypted magnetic field, as in the case of the bioreactor design of the present invention in Figure 2 , at which time the vessel is emptied and updated with a new solution, or magnetic beads attached to a DNA plasmid such as Invitrogen DNA Binding Beads are forced through the filtration membrane in the above-described filtration module design and routed back to the reaction module through the retentate channel.

[0172] Example Embodiment

[0173] Although all the designs are modules in various configurations of a flow system for the manufacture of nucleic acid-based therapies, the serpentine filtration module represents a preferred example due to its easy and low-cost fabrication. The entire process of fabricating a prototype for testing the device will be further described herein.

[0174] Figure 7 and Figure 8 shows a flow preferential filtration module, which preferably includes a first plate-like layer 30 and a second plate-like layer 31. The main surfaces of the first plate-like layer 30 and the second plate-like layer 31 face each other and sandwich the membrane 36 therebetween to provide a laminate structure. Each of the plates 30, 31 includes a serpentine channel having a straight section 34 and a bent or curved section 35 to form corresponding fluid flow channels 37, 38 that are in direct contact with and at least partially defined by the intermediate membrane 36. A collection, input or buffer container 33 is provided at or towards a corresponding longitudinal end of either the first channel 37 or the second channel 38. Thus, fluid can flow through the channel 37 of the upper plate, and selected chemical components in the fluid having a smaller molecular size can diffuse through the membrane 36 into the adjacent fluid flow channel 38 of the second plate 31. Thus, it can be understood that Figure 7 and Figure 8 the TFF module is configured to separate permeate and retentate, which differ in particle size (particle diameter) and / or molecular weight.

[0175] Fluid flow simulation method for research device

[0176] As shown in Table 2, two different sizes of bioreactor channels are required. Computational Fluid Dynamics (CFD) software has been used to analyze the hypothetical data in the microchannels. The microchannels require a steady-state flow rate.

[0177] Table 2 Calculation results of bioreactor channels of different sizes

[0178]

[0179] FLUENT: Fluent is Computational Fluid Dynamics (CFD) software that helps solve fluid flow problems. It uses the finite volume method to solve the governing equations of the fluid and provides many different physical models, such as laminar or turbulent, viscous or inviscid, compressible or incompressible. Geometry and mesh generation are being carried out in GAMBIT, which is a preprocessor bundled with FLUENT.

[0180] The solution can be obtained through the following steps: geometry; mesh; setup; solve; results. The channel is represented by a 2D CAD design. Thereafter, the material properties and boundary conditions are set. Finally, the domain must be meshed. FLUENT converges on the problem until the convergence limit is met or a specified number of iterations is reached.

[0181] a) Geometry

[0182] The geometry consists of walls, inlet and outlet boundaries, which are in Figure 12is shown in

[0183] b) Mesh

[0184] Coarse and fine mesh types are available. The mesh density varies according to the refinement factor, as shown in Table 3.

[0185] Table 3 Coarse and fine mesh types

[0186]

[0187] Fluid flow modeling using computational fluid dynamics:

[0188] Regarding the Reynolds number (Re = 0.0269), laminar flow was selected. Air and water-liquid need to be selected as materials. The following material properties can be specified: density and viscosity.

[0189] As shown in Table 4, the following boundary conditions were specified in FLUENT.

[0190] Table 4 Boundary conditions specified in FLUENT

[0191]

[0192] c) Solving

[0193] The mesh is exported to FLUENT together with the physical properties and the specified initial conditions. When the solution converges or reaches the specified number of iterations, FLUENT exports the data. As shown in Table 5, the total flow time is 14400 seconds, which is 4 hours. So the CFD software needs to record the data repeated 240 times every 60 seconds, and then the CFD software analyzes the velocity, energy, and continuity in the X and Y directions. Finally, after 38 iterations, the results have converged, as Figure 13 shown.

[0194] Table 5 Input and output data of FLUENT

[0195]

[0196] Prototype construction

[0197] To construct the prototype, different steps were followed. First, the mold was created, and then the chip was cast from the mold. Different subsystems were built separately to test each design. Experiments will be conducted on each subsystem to verify the design. Different versions of the mold have been edited. The design evolution is as Figure 14 shown.

[0198] After experimenting on each mold, some improvements were made between the two versions.

[0199] · Two templates have been recombined into one template to make all the processes for preparing chip manufacturing easier.

[0200] · The cross-sectional area of the channel has been increased from 0.4 mm × 0.8 mm to 0.5 mm × 1 mm to avoid the collapse of the channel walls when testing the chip.

[0201] · Additional space has been added between the channels (0.5 mm to 1 mm) and between the boundaries and features of the mold to improve the quality of the chip and avoid any accidents when removing the PDMS mask from the mold.

[0202] · Finally, some walls have been added to the edges of the mold to improve the quality of the entire PDMS chip during casting.

[0203] Regarding the vertical TFF device, the top layer and the bottom layer have been edited separately to create two different layers that will be sealed together with the middle filtration membrane. Figure 15 The designs of the two layers are shown. Table 6 illustrates the different features of the two designs.

[0204] Table 6 Feature descriptions of the two designs

[0205]

[0206] Soft lithography technology and SLA 3D printing mold

[0207] Microfluidic chips require precise and accurate methods and technologies to make the reactions or filtrations work as expected. The most common method used for microfluidic chips is soft lithography technology. In this case, soft lithography technology involves creating a mold from which PDMS will be cast [Kamei, K.ichiro, Mashimo, Y., Koyama, Y., Fockenberg, C., Nakashima, M., Nakajima, M.,…Chen, Y. (2015), 3D printing of soft lithography mold for rapid production of polydimethylsiloxane-based microfluidic devices for cell stimulation with concentration gradients, Biomedical Microdevices]. The mold is 3D printed according to the CAD design of the required parts. Although different technologies can be used for 3D printing, stereolithography technology has been selected in the case of this project.

[0208] After designing different molds, the parts were sent for 3D printing using SLA. SLA printing allows the construction of complex shapes with a precision of 10 μm in about 24 hours. Regarding the required utilization rate of the molds and different shapes and features, the optimal resolution was set on the SLA machine to optimize the printing resolution and accuracy. The first version of the mask was analyzed by a microscope to compare the sizes between the CAD file and the 3D printed parts. Figure 16 Two microscopic analyses of the channel dimensions are shown.

[0209] Microscopic analysis values of the channel dimensions in Table 7

[0210]

[0211]

[0212] Finally, the molds have been printed with two different materials. Both materials are photopolymer resins, and one of the materials is a high-temperature resin. The main difference between the two materials is the maximum temperature before thermal deformation. The first material can reach 50 °C, while the second material can be as high as 250 °C.

[0213] Selection of the filter membrane

[0214] The selection of a specific filter membrane is carried out in two steps. The first step involves finding the molecular cut-off sizes of two different membranes for two filtration stages. In fact, each filtration stage is dealing with different molecules of different molecular weights. As shown in the conceptual process of this article, due to their high cost, some reagent molecules need to be filtered and recycled back to their respective bioreactors. Table 8 details the different molecules and their sizes or molecular weights.

[0215] Table 8 Molecular weight information

[0216] Molecule Molecular weight and spherical size Filtration stage DNA 10 MDa → 30 nm End of bioreactor 1 RNA 5 MDa → 23 nm Final purified product T7 polymerase 99 kDa → 6.3 nm Vertical TFF 1 M7g+ methyltransferase 300 kDa → 9 nm Vertical TFF 2

[0217] Regarding the two vertical TFF stages, the first vertical TFF is dealing with T7 polymerase, while the second vertical TFF is dealing with M7g+ methyltransferase. Considering the different molecular sizes, the selected filter membrane needs to have a molecular cut-off size three times smaller than the molecules that need to be retained. In this case, it is the RNA molecule [General Electric, 2014]. In addition, the filter needs to be large enough to allow the recycled molecules to pass through it. Given all this information, the molecular cut-off size of each membrane is assigned as follows:

[0218] ● Vertical TFF 1 → Molecular weight cut-off membrane is 300 kDa

[0219] ● Vertical TFF 2 → Molecular weight cut-off membrane is 500 kDa

[0220] The second step of the process is to select a suitable membrane supplier. Thus, due to time constraints, a 1m x 1m flat sheet membrane from Synder Filtration was selected. Membranes with three different pore sizes: 300 kDa, 400 kDa, and 500 kDa have been ordered in order to conduct different tests and experiments and analyze the filtration performance of each membrane.

[0221] PDMS Mask and Casting

[0222] With the printing and preparation of the mold, polydimethylsiloxane (PDMS) microfluidics can be cast from it. The product used to fabricate the PDMS chips is Sylgard 184 silicone elastomer. This product consists of two components, one is the silicone elastomer part and the other is the curing agent responsible for cross-linking the elastomer molecules, which will cure the elastomer while maintaining the casting properties. However, some preparatory work needs to be done before applying PDMS over the mold. Table 9 shows the curing temperatures and their curing times.

[0223] ● Mix the silicone elastomer with the curing agent at a mass ratio of 10:1 (silicone / curing agent) in a beaker for 10 minutes.

[0224] ● After mixing, the bubbles in the mixture need to be removed with the help of a desiccator. Repeat this operation until all the bubbles disappear.

[0225] ● If there are still small bubbles, let the beaker stand still. The bubbles will burst by themselves.

[0226] · After the mixture is ready, apply it on the 3D printed mold and let it stand for 10 to 20 minutes. Place the mask with PDMS on top in the oven or let it stand at ambient temperature.

[0227] ● Table

[0228] Table 9 Curing Temperatures and Times

[0229] Curing temperature Curing time 22℃ 48 hours 60℃ 5 hours 100℃ 35 minutes 125℃ 20 minutes 150℃ 10 minutes

[0230] Different temperatures have been tested to compare PDMS curing performance and aspects. However, the first mold used in this process could not withstand high temperatures. That's why ambient temperature curing was also tested within a week. Finally, since there are different features cast on the PDMS mask, this mask needs to be sealed on the open side. Two different methods have been used to seal the mask to allow for different test results ( Figure 17 is a summary of the different steps of the prototype construction process for creating a continuous flow bioreactor system):

[0231] · The first method is to seal the PDMS mask with another flat layer of PDMS. This method allows for an additional thickness of the entire mask and helps with the integration of the tubes and their fittings.

[0232] ● The second method is to seal the PDMS mask with Kapton tape on the open side. This method allows for a perfect seal between the features of the PDMS mask and the tape. However, as the thickness of the mask decreases, the integration of the tubes and fittings will be more complex.

[0233] Sensing method

[0234] The main sensing system is designed by combining the characteristics of continuous flow microfluidic systems and reactions. First, the sensing system of the microfluidic chip is limited by the chip size and the characteristics of the factors to be detected. In addition, selecting a suitable sensor to obtain accurate and real-time detection results of the chip is another key point. The sensing system of the microfactory includes a spectrometer, a pH sensor, and a pressure controller and sensor. L. Zhu (2005) [Nuno Miguel Matos Pires (2014) and Kihwan Choi (2015)] discussed the theory behind the spectrometer. Yung-Shin Sun (2016) also explored the theory behind the pressure controller and sensor.

[0235] Material selection

[0236] a) Spectrometer

[0237] The spectrometer (USB2000+ miniature fiber optic spectrometer, Ocean Optics inc, UK) can store 1000 full spectra per second, and its detection range is from 190 nm to 1100 nm in wavelength. In addition, to use the official software, it is mainly programmed with open-source packages in Python according to the characteristics of the sensing system. This spectrometer is suitable for monitoring continuous reactions.

[0238] b) pH meter

[0239] The pH meter (HI-1093B pH electrode, Hanna Instruments Ltd, Bedfordshire, UK) has a measurement range of 0 to 13. To integrate the pH meter with the microfluidic chip, due to design specifications, the size of the pH meter is limited. The diameter of the measurement unit on the microfluidic chip is 5 mm², while the diameter of the main body of the pH meter is 3 mm.

[0240] c) Pressure controller and sensor

[0241] CFD analysis of the microfluidic chip shows that the pressure in the bioreactor and TFF section is 0.27 Bar. The pressure sensor (MPS microfluidic high-precision pressure sensor, ELVEFLOW, Paris, France) can provide 5 measurement ranges, from 70 mBar to 7 Bar. It also allows the detection of an ultra-small internal volume of 7.5 μL, which can be applicable to a microfluidic chip with a volume of 0.5 mL. The flow rate regulation of this sensor is sensitive and the response is rapid, so it is suitable for real-time monitoring of minute changes in the flow rate. The accompanying sensor reader (Sensor Reader, ELVEFLOW, Paris, France) can provide high-speed capabilities and is easily integrated into the chip, making the measurement simple and feasible.

[0242] d) Pressure supplier

[0243] The syringe pump (C3657 C series syringe pump, Tricontinent, California, USA) is used as the pressure supplier to supply the reactant materials into the microfluidic chip. The stroke speed of the pump is from 1.2 seconds to 100 minutes per stroke, and the resolution of the pump is 3000 steps in the standard mode and 24,000 steps in the high-resolution mode. The reaction time of this system is 4 to 6 hours. At the same time, these syringe pumps can provide a stable flow rate in the microfluidic system. The pressure of the 5 inlets for injecting the reaction components can control the proportion of the reactants to achieve an economical and effective production of RNA vaccines.

[0244] Method

[0245] The key parameters of the reaction to be monitored are the RNA and Mg2+ concentrations, the pH value and temperature of the reaction. At the same time, the inlet pressure needs to be controlled in order to obtain an appropriate proportion of RNA production and regulate the flow rate of the mixed solution in the continuous flow channel within an appropriate reaction time. There are 3 detection points on the microfluidic chip, two points for the quantification of RNA and Mg2+, and another point for pH value measurement. In addition, the pressure sensor is fabricated outside the chip. The changes in the concentration levels of Mg2+ and RNA can be used to detect the reaction rate. The method of converting absorbance to concentration is a commonly used method for the quantification of RNA and Mg2+. The ultraviolet absorbance measurement at 260 nm is the gold standard for RNA quantification, and the quantification of Mg2+ can be obtained through the absorbance measurement at 680 nm. The absorbance measurement values of RNA and Mg2+ can be converted into concentrations using the Beer-Lambert law. In the design, PDMS is used to fabricate the bioreactor, which is sufficiently transparent. This makes effective RNA detection possible.

[0246] In the reaction, the corresponding heat-resistant DNA polymerase is the main source of the pH value. The best results in the system are obtained when the pH value is between 8.3 and 9.0. In addition, increasing the pH value in the reaction helps to stabilize the DNA template and enhance the transcription results. The activity of the recombinase is significantly affected by the pH value of the experiment. For the integrated device, an Arduino microcontroller (ARDUINO UNO REV3, Arduino, UK) has been used as the core for data collection.

[0247] Integrated experimental platform

[0248] Figures 18A to 18C A schematic diagram of the present fluid flow reaction system is shown. The system includes a plurality of sensors adapted to measure various characteristics of the fluid flowing in the fluid flow system, including pressure, temperature, pH value, flow rate, flow volume, etc. The system also includes a plurality of valves, injection ports, pumps (specifically, syringe pumps), gates, etc. to control the flow path of the fluid components within the device. For example, the components of the fluid can be recycled from the outlet ports at the longitudinal ends of the bioreactor and / or filtration module and returned to the inlet ports of the upstream bioreactor and / or filtration module as needed. The system further includes a control unit, which generally includes a CPU, a microprocessor or other suitable electronic processor devices. According to a specific embodiment, the processor is integrated into a PCB or a PLC. The control unit is provided with a user interface, a sensing data storage tool, software, a visual display device, a communication port and / or module, at least one analog-to-digital converter and / or a data source library. These components can be used in combination with the control unit, sensors, injection ports, pumps, valves, etc. to continuously and automatically control the fluid flow within the fluid flow system. Thus, a continuous reactor system is provided for the continuous input of reagents to enable the continuous output of the desired reaction products. The advantages of the present system are that reagents, solvents, etc. can be recycled and reused to minimize waste and maximize efficiency. Once the facilities are ready, their layout will be designed and the experimental environment will be established. When necessary, the PDMS chip should be fixed and clamped by a fixture. Then, the positions of other instruments will also be determined accordingly.

[0249] At the beginning, 4 syringe pumps are used to apply pressure to 4 input containers respectively. Pipes and fittings are generally used to transport small-volume samples to the PDMS chip. Another pump will be connected to the second container, which is located at the starting point (the end of the first TFF) of the second bioreactor, so that m7G methyltransferase can be injected and the pressure in the chip can be regulated. Using a pressure sensor with a feedback loop will significantly increase the responsiveness of the flow control. The pressure sensor is considered to be measured at the inlet and outlet of the channel. The goal is to ensure a constant pressure in the device while keeping the syringe pumps working.

[0250] To achieve concentration detection, UV / Vis light should pass through the detection chamber, where at the end of the first bioreactor (serpentine channel), it is transmitted through an optical fiber. Two micro-optical fibers matching the specific wavelength requirements are installed perpendicular to each other: the source fiber connected to the light source and the collection fiber connected to the micro-spectrometer. During continuous biological reactions, it is planned to place the pH sensor in the buffer to measure the pH value, while the temperature sensor can be placed on the plate, with a heater on the other side to adjust the temperature. Both the pH sensor and the temperature sensor are equipped with BNC connectors that can be connected to Arduino. Therefore, data can be obtained from these sensors.

[0251] Data Acquisition and Monitoring

[0252] Spectrometer Data

[0253] The Lambert-Beer law describes the linear relationship between the analyte concentration and the absorbance of light at a specific wavelength:

[0254]

[0255] where I 0 is the initial intensity of the light source, I 1 is the light intensity after passing through the sample, ∈ represents the wavelength-dependent molar absorptivity, with the unit M -1 cm -1 , L is the path length, and C is the analyte concentration.

[0256] Therefore, the expected spectrometer data is exactly the absorption spectrum. The Ocean Optics spectrometer has provided a simple method to access data from Python. This is the Python-Seabreeze package, which wraps the Seabreeze library to communicate with the spectrometer. It provides a complete working and test reference implementation for the USB interface of Ocean Optics, meaning that the spectrometer data can be read and monitored using python. The connection between Python and the spectrometer is as Figure 19 shown.

[0257] Once the pH sensor is connected to Arduino through the BNC connector, the sensor data can be obtained, and the real-time data can be checked on the serial monitor. The code for this is shown in Figure 19 . The same mechanism is also used to obtain the temperature sensor data. Five full-factor experiments were conducted, focusing on the effects of different ratios of reagents, reaction time, and magnesium ion ratio. The experiments were carried out based on a 500 μL volume batch-feed system. The background experimental data is shown below. Several conclusions can be drawn from the data. Figure 21shows the maximum yield from a mixture of 10 mM NTP, 10 mM NaCl, 75 mM MgCl 2 with acetate ions, approximately 900 mM RNA yield, which can be considered the optimal combination. The optimal reaction time for RNA yield is as Figure 22 shown. When the proportion of Mg(OAc)2 is approximately 80 mmol, the RNA yield can reach the highest level. After a four-hour reaction, the RNA yield stabilizes at around 1800 mM. The optimal combination of reaction conditions depends on four main factors: MgCl 2 , NaCl, and the proportions of NTP, as well as the reaction time. For additional experiments, the proportions of MgCl 2 , NaCl, and NTP need to be monitored and maintained at a certain level to obtain the maximum RNA yield. The most significant difference in the experiments between Imperial College London and Cranfield University is the design of the reaction system. For manufacturing considerations, a continuous follow-up system was designed for the bioreactor process instead of a batch-fed reaction system. To test the continuous flow system, the experiments were designed based on previous experiments conducted by the Shattock Group at Imperial College London.

[0258] Three levels of the four factors (Table 10) were selected to test the optimal proportions for RNA yield in the continuous flow system. The factors selected refer to the results of the batch-fed system experiments. Additionally, due to recycling, the values of DNA and T7 polymerase are the same. Considering these factors, the new experimental design is shown in Table 11. This experiment aims to find the relationship between RNA yield and the four factors to identify the trends of the reaction.

[0259] Table 10 Factors and levels selected for the flow synthesis experiment.

[0260]

[0261]

[0262] Table 11 Experimental design table for the flow synthesis experiment.

[0263]

[0264] Results and Discussion

[0265] Hydrodynamics and CFD Analysis

[0266] Apply appropriate control equations to the hydrodynamic studies in the bioreactor and TFF section to calculate the filtrate volume with a constant fluid velocity and appropriate pressure gradient under steady-state flow conditions. Equation (6) has been used to predict the pressure drop in the microchannel. The choice of characteristic length is an arbitrary choice and will not affect the final solution, as shown in Tables 12 and 13.

[0267] Table 12 Fluid properties under different channels. All measurements were carried out under the conditions of width (μm) of 1000, height (μm) of 500, viscosity (cPa) of 3.5, and density (g / cm 3 ) of 1.06.

[0268]

[0269] Table 13 Fluid properties under different flow rates. All measurements were carried out under the conditions of width (μm) of 1000, height (μm) of 500, viscosity (cPa) of 3.5, and density (g / cm 3 ) of 1.06.

[0270]

[0271] Other pressure losses associated with the measured pressure drop are the bend losses at the inlet and outlet. The bend pressure losses within different numbers of bends can be obtained in Table 14.

[0272] Table 14 Calculation of bend pressure loss percentages for different numbers of bends

[0273]

[0274] Hydrodynamic studies of the TFF section

[0275] The Cerman-Kozeny relationship is used to obtain the permeability constant to solve the fluid parameters passing through the microporous channels under appropriate boundary conditions. Different particle diameters and pore sizes are adopted here due to the variation of the input particle sizes in Table 15.

[0276] Table 15 Permeation constants for different particle diameters and pore sizes

[0277] <![CDATA[Particle diameter, d p (μm)]]> Pore size, ε (μm) <![CDATA[Permeability, K ( / μm 3 )]]> Darcy number, Da 0.020 0.060 5.4323E-10 2.122E-22 0.021 0.063 6.9776E-10 2.726E-22 0.022 0.066 8.8616E-10 3.462E-22 0.023 0.069 1.1139E-09 4.351E-22 0.024 0.072 1.3869E-09 5.418E-22 0.025 0.075 1.7120E-09 6.688E-22

[0278] The pressure drop of the geometry can be predicted by Equation 10, and its limitation is that it will produce a mathematical error if the measurement unit is in microns. Shown in Table 16 is the pressure drop along the length in the microporous channels.

[0279]

[0280] Where

[0281] μ = Dynamic viscosity

[0282] Q = Flow rate

[0283] h = Height / depth of the channel

[0284] K = Permeability

[0285] Table 16 Pressure loss along the length in the microchannel at different flow rates

[0286]

[0287] The results obtained in this study are only predictions and depend on the geometry, fluid conditions, and boundary conditions. Based on the membrane selection parameters in the Pall Corporation article, the filtrate volume can be estimated, which is given in Table 17.

[0288] Table 17 Prediction of filtrate volume in different filter length regions

[0289]

[0290] Computational fluid dynamics analysis

[0291] Velocity distribution in the bioreactor channel

[0292] As Figure 23A and Figure 23B shown, the velocity in the X direction passes through the entire bioreactor channel. There are two different colors showing the same velocity, which is between 0.0001 m / s and 0.0002 m / s, because yellow represents the positive direction and green represents the negative direction. At the beginning, Figure 23B shows an increase in velocity because the initial pump provides a higher pressure, and then it drops to a constant velocity of about 0.00033 m / s, which is approximately 2 times higher than the predicted result.

[0293] Tangential flow filtration channel

[0294] Figure 24 shows the velocity in the tangential flow filtration channel. The colors indicate that the different velocities in the microchannel are between 0.0001327 m / s and 0.0001858 m / s, which is close to the inlet velocity. In addition, the data in Table 18 show that the outlet velocity slightly decreases from 0.000145 m / s to 0.000138 m / s, as expected.

[0295] Table 18 Velocity magnitude

[0296]

[0297] Isobars in the bioreactor

[0298] Figure 25 shows the pressure drop in the bioreactor channels. The colors represent different pressure values. Figure 25B It is shown that the pressure drops due to the many bends in the bioreactor channels. These different results have been taken into account in the design evolution.

[0299] Experimental results

[0300] PDMS mask tests

[0301] Bioreactor

[0302] Since different versions of the mold have been achieved through the current work, the evolution of the results has been observed and will be illustrated.

[0303] a) First mold generation

[0304] First, the first versions of both bioreactors have been tested. Different sealing options, Kapton tapes, and a second PDMS layer have been tried, and the results have been extracted. Both reactors were cured at 35 °C for more than two days. First, the two reactors were sealed with another flat PDMS layer. Due to the syringe, green water was introduced into one of the multiple inlets of each mask. Figure 26A and Figure 26B shows the first results regarding the fluid behavior of each mask. These two pictures show that in the batch bioreactor, when green water was introduced into the mask, the water formed a contour in the middle of the reactor. This has been explained by the fact that the middle of the reactor has collapsed during curing, as can be seen in the blue part of Figure 26A In addition,[[]] Figure 26B shows that the green water sample has passed through the T-joints of the mask and followed the main channel path. However, the sample has avoided some areas. This can be explained by the size of the channel walls, which could not withstand the pressure during the curing process and collapsed.

[0305] Secondly, the continuous flow reactor sealed with Kapton tape was tested. Figure 27 shows the fluid behavior inside the new device. The fluid (dark green) follows the path correctly. The sealing effect is better than before. However, due to the size of the channels, the curing could not proceed properly again, and some parts of the mask could not be peeled off the mold properly, resulting in some defective areas in the mask.

[0306] Based on all the observations of different tests, the design has been changed. Additionally, it has been decided to create the following platform where a PDMS mask can be placed between two acrylic plates to increase the pressure between the two PDMS layers, thus avoiding channel collapse when testing the mask. These two acrylic plates also allow the connection of fittings and pipes to the chip.

[0307] Figure 28 It is shown that the Kapton-sealed mask has been placed between two plates and has been connected to pipes, fittings, and some sensing instruments to allow for the first pH and absorbance tests and measurements.

[0308] b) Second mold generation

[0309] A high-temperature-resistant material is used to print the second mold generation, and the mask is cast for 4 hours at a curing temperature of 70 °C. As Figure 29 shown, after the mold is used, the side that was in direct contact with the mold has bonded to the mold. A small part of the PDMS mask could not be peeled off and removed from the mask. Especially between the channels, a thin layer of PDMS adhered to the mask. Although the curing worked properly, the peeling of the mask from the mold has failed. After trying to clean the mold as much as possible with isopropyl alcohol, another test was given with a curing temperature of 100 °C for 35 minutes. Unfortunately, the same problem occurred. The explanation for such a problem is that the high-temperature-resistant material from the 3D printer has bonded to the PDMS layer that was in direct contact with it. The walls may also have an impact on disrupting the curing uniformity of the entire mask.

[0310] Vertical flow filtration module

[0311] Only one version of the vertical TFF mold was printed using a high-temperature-resistant material. The same process was also followed for the bioreactor mask, and the PDMS samples poured into foil containers were placed in the oven with the two masks to compare different aspects and results of the process. Figure 30A and Figure 30B show the aspects of the mask and the samples cured in the oven. For the bioreactor mask, the PDMS in contact with the mold has bonded to the mold. When the PDMS was peeled off the mold, the mask had cracked and broken, as Figure 30A shown. However, the samples cured in the foil have cured without any defects, and nothing happened when the samples were peeled off the foil. Additionally, as Figure 30B shown, the samples have perfectly cast the shape of the foil.

[0312] Based on these different observations, some analyses can be made and it is found that:

[0313] · When the samples in the foil are functional, the chemistry and protocols are not questioned.

[0314] · The same default situation has occurred several times on different masks. Therefore, high-temperature materials have a negative impact on PDMS mask casting and should not be used for such experiments.

[0315] · The first material used in the first experiment should be retained, and the curing parameters should be set to cure at ambient temperature for 48 hours to enable normal casting of the PDMS mask and avoid any thermal deformation of the mold.

[0316] Despite these results, new platforms have been created, as Figure 31 shown.

[0317] Based on the integrated experimental design, all facilities have been collected in the laboratory, and an experimental platform has been built to verify the effectiveness of the system. The experimental platform constructed in the laboratory is as Figure 31 shown. The PDMS chip is fixed and clamped by a fixture and connected to the pump and the chip through pipes and fittings. Two optical fibers are installed perpendicular to each other: the source fiber is connected to the light source, and the collection fiber is connected to the micro-spectrometer (Ocean Optics USB2000+).

[0318] Absorbance measurement and digital output

[0319] Accurate absorbance measurement

[0320] To perform accurate absorbance measurements, a baseline needs to be set. First, the spectrum of the background radiation source is recorded without a sample. Second, the experiment is repeated by adding a sample in the absorption path. Third, the first spectrum is subtracted from the second spectrum to obtain the clean absorption spectrum of the sample. To eliminate the noise effect, the background spectrum should be subtracted from both the sample and the reference spectrum. Therefore, Equation (9) should become:

[0321]

[0322] where: I sample is the light intensity after passing through the sample, I B is the background light intensity recorded by the spectrometer when the light source is off or blocked and there is no sample. I Ref is the reference light intensity.

[0323] UV-Vis data analysis

[0324] Through the Python-Seabreeze API, real-time data can be easily obtained from the UV-Vis spectrometer, and a graph of intensity versus wavelength can be plotted. As Figure 32As shown, when the wavelength is around 580 nm, the light intensity reaches its peak. To verify the relationship between absorbance and wavelength at different concentrations, vitamin B12 solutions with different concentrations were used, namely A (25 mM / L), B (50 mM / L), C (75 mM / L), and D (100 mM / L), see Figure 33 . The UV-Vis spectral results are as Figure 34 shown, and the absorbance increases with the increase in sample concentration, and the absorbance reaches its maximum at a wavelength of 550 nm.

[0325] PH Sensor Calibration and Digital Output

[0326] PH Sensor Calibration

[0327] To ensure accuracy, the PH detector used for the first time needs to be calibrated. Two standard buffer solutions were used to calibrate the PH sensor, which are 4.0 and 7.0 respectively. According to the calibration steps provided by DFROBOT, the calibration has been completed.

[0328] Real-time PH Value Graph

[0329] The next step is to test the effectiveness of the PH sensor with acidic and alkaline solutions. Figure 35 The results in

[0330] show that the dynamic changes in the real-time graph follow the changes in the PH value, which proves its effectiveness.

[0331] Discussion

[0332] The work and results obtained are an effective attempt to implement lab-on-a-chip technology for continuous flow reactions to achieve maximum RNA output. Applying the SBCE method allows for the construction of a complete design framework that follows from the start to the end of the project. The modular design of the device has divided functional subsystems enabling the inclusion of different innovative solutions for unit operations such as mixing, reaction, filtration, and purification. Figures 30A to 30C shows a schematic diagram of an integrated system including downstream modules for formulation. In particular, Figures 30A to 30CIt is a diagram of an exemplary embodiment of the present flow system, which is integrated with a downstream formulation system (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) using a modular flow reactor (Module 2) and an integrated UV-Vis detector, and the present flow system is attached to a conventional filling and finishing vaccine production line.

[0333] Figure 30D The synthetic performance of the device is shown, which is Figures 30A to 30D a graph showing the relationship between the absorbance and wavelength of the output solution of the flow reactor of the embodiment of Figure 40A It is a diagram of the source code of the absorbance data and real-time graph (relationship between absorbance and wavelength) in Python, Figure 40B It is a diagram of the source code of the real-time graph of pH data in Python.

[0334] The product is evaluated using a graph showing the relationship between UV-Vis spectroscopy and a conventional batch process, where the output solution of the flow reactor is evaluated using a graph showing the relationship between UV-Vis spectroscopy and a conventional batch process. It can be envisioned that the present method has a significantly higher productivity compared to the batch process, as indicated by higher peaks in the graph indicating higher levels of RNA concentration. Figure 30D The UV-Vis spectra shown compare the RNA species obtained using a conventional batch process and the RNA species obtained using the continuous flow process of the present invention. Compared to the batch process, the higher peaks in the flow process correspond to higher concentrations of nucleic acids present in the solution. The dashed line corresponds to a sample of known concentration RNA and is included to assist in comparing the samples obtained in the batch process and the samples obtained in the flow process.

[0335] Two different design solutions have been developed for this reactor, which allow the manufacture of RNA to be changed from a large-scale manual batch process (conventional RNA synthesis) to a continuous flow format. In fact, the continuous flow reactor of the present invention is innovative and proven to be feasible. The present invention represents a step change in the prior art as it embodies advanced equipment and processes and demonstrates its operation and performance in rapidly scaling up and productively manufacturing RNA-based substances in a continuous flow format using automated computer control.

[0336] The filtration module has been studied and analyzed in depth to meet the requirements of continuous flow processing and the ability to integrate with a flow reactor, while being easy to manufacture and scale using common manufacturing methods. Although tangential flow filtration is a commonly used technique proven effective in RNA purification processes (A. Eon-Duval et al., 2002), the present invention includes a novel filtration device that has been specifically designed and constructed, developed to be configured as a continuous flow system, easy to manufacture, capable of being integrated into a continuous flow system, and allowing for scalable and cost-effective manufacture of the device. This system can be integrated as a microfactory that can allow for the automated and compliant manufacture of therapeutic nucleic acid-based substances at the point of use. Additionally, the modular design allows for the recycling of certain components (such as enzymes and plasmid DNA) using this filtration system, thereby significantly reducing the total cost of the substances produced.

[0337] Regarding the overall system, a new integration platform has been developed for testing and experimentation. The system has been prototyped using acrylic pressure plates and also includes all ports for pumps, sensors, and fittings for inlets and outlets. The system allows for testing of continuous flow reactions or filtration while obtaining immediate feedback from different elements and stages of each process. Additionally, the system has been prototyped in a modular manner, which allows for easy use of a framework for running experiments. 3D printing has become the tool of choice for manufacturing molds due to its high flexibility, low cost, and short delivery cycle. Using techniques such as micromachining on substrates such as glass, metals (such as stainless steel), and polymethyl methacrylate PMMA (acrylic), and photolithography and deep reactive ion etching on substrates including photosensitive glass and silicon, the construction of the modules can be easily transferred to commercial manufacturing. Additionally, for large-scale manufacture of the modules, injection molding can be used for PMMA and polycarbonate structural materials.

[0338] UV-Vis spectroscopy demonstrates the ability of the present invention to provide reliable on-line and in-situ analysis of the reaction solution contents in real time. This also provides a closed feedback loop to direct fluid flow and regulate the temperature, pH, and distribution of reactants in the flow system, thereby maintaining process conditions throughout the process.

[0339] According to the embodiments including the above embodiments, the following technical solutions are also disclosed:

[0340] (1) A method for RNA synthesis, comprising:

[0341] introducing a plurality of reactants via a plurality of inlet ports into a first fluid flow module, the plurality of reactants including: at least one nucleoside triphosphate (NTP), a reaction buffer, and DNA, a DNA-based compound, or a DNA-based mixture;

[0342] Allow at least some of the reactants to react within reaction channels or wells in a first module of a flow system;

[0343] Retain the DNA at or recycle the NDA at the first reactor module, and allow the reaction product stream of the reactants to flow into a first fluid filtration module; and

[0344] Filter the reaction product within the first filtration module.

[0345] (2) The method according to (1), wherein the at least one nucleoside triphosphate (NTP) is a solution of at least one nucleoside triphosphate (NTP).

[0346] (3) The method according to (1) or (2), wherein 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 of adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), uridine triphosphate (UTP).

[0347] (4) The method according to any one of the preceding items, wherein the DNA is plasmid DNA.

[0348] (5) The method according to any one of the preceding items, wherein the plurality of reactants further comprises any one of an enzyme mixture, a salt solution, an RNA polymerase or a combination of an enzyme mixture, a salt solution, an RNA polymerase.

[0349] (6) The method according to any one of the preceding items, comprising: 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.

[0350] (7) The method according to any one of the preceding items, comprising: conveying at least some of the filtered reaction product from the first filtration module to a second fluid flow reactor module, and inputting a capping enzyme into the second reactor module.

[0351] (8) The method according to (7), comprising: conveying the fluid output from the second reactor module to a second fluid flow filtration module.

[0352] (9) The method according to (8), comprising: recycling any unreacted NTP to the inlet region of the first reactor module, and recycling any unreacted capping enzyme to the inlet region of the second reactor module.

[0353] (10) According to any one of the preceding items subordinate to (5), wherein the salt solution comprises MgCl 2 .

[0354] (11) According to any one of the preceding items subordinate to (5), wherein the RNA polymerase comprises T7 polymerase.

[0355] (12) An RNA or RNA-based compound prepared by the method according to any one of the preceding items.

[0356] (13) Use of an RNA or RNA-based compound prepared by the method according to any one of (1) to (11) in the preparation of a vaccine.

[0357] (14) A fluid flow filtration device, comprising:

[0358] A first elongate fluid flow channel having an inlet;

[0359] A second elongate fluid flow channel having an outlet;

[0360] A permeable membrane, the permeable membrane being positioned to separate the first channel from the second channel along respective lengths of the first channel and the second channel, such that permeate can pass from the fluid within the first channel through the membrane into the second channel along the lengths of the first channel and the second channel.

[0361] (15) The device according to (14), wherein most of the length of the first channel is positioned to be adjacent to most of the length of the second channel via the membrane.

[0362] (16) The device according to (14) and (15), wherein the pore size of the membrane is in the range of 100 kDa to 1000 kDa, in the range of 100 kDa to 800 kDa, in the range of 200 kDa to 800 kDa, or in the range of 200 kDa to 600 kDa, in the range of 300 kDa to 10 MDa.

[0363] (17) The device according to any one of (14) to (16), comprising a first plate in which the first channel is formed and a second plate in which the second channel is formed, the membrane being sandwiched between opposite faces of the first plate and the second plate respectively.

[0364] (18) The device according to any one of (14) to (17), wherein each of the first channel and the second channel comprises a series of straight portions and curved portions.

[0365] (19) The device according to (18), wherein the first channel and the second channel each include a serpentine profile in their longitudinal directions.

[0366] (20) The device according to any one of (14) to (19), wherein the first channel and the second channel are open along their lengths and are positioned in direct contact with the membrane, and the membrane partially defines the longitudinal walls or surfaces of the first channel and the second channel.

[0367] (21) The device according to any one of (14) to (20), wherein the pore size of the membrane is smaller than the average molecular size of RNA molecules.

[0368] (22) A fluid flow system for processing a fluid, comprising:

[0369] A first reactor module having a reaction flow channel or well, at least one inlet, and at least one outlet;

[0370] A first filtration module having a fluid filtration region, at least one inlet and at least one outlet configured to be in fluid communication with the outlet of the first reactor module;

[0371] wherein the first filtration module includes a fluid flow filtration device according to any one of (13) to (21).

[0372] (23) The system according to (22), including a second reactor module having a reaction flow channel or well, at least one inlet and an outlet, and the inlet is configured to be in fluid communication with the first filtration module.

[0373] (24) The system according to (23), further including a second filtration module having a fluid filtration region, at least one inlet and an outlet, and the inlet is configured to be in fluid communication with the outlet of the second reactor module.

[0374] (25) The system according to any one of (22) to (24), including a fluid injection port configured to be in fluid communication with the inlet region of the first filtration module.

[0375] (26) The system according to any one of (22) to (25), including a first recirculation conduit extending between a region of the outlet of the first reactor module and at least one inlet of the first reactor module.

[0376] (27) The system according to any one of (22) to (26), comprising a second recirculation conduit extending between an area at the outlet of the first filtration module and the outlet of the first reactor module.

[0377] (28) The system according to (27) which depends on (24), comprising a third recirculation conduit extending between an area at the outlet of the second filtration module and the inlet of the first reaction module.

[0378] (29) A method of filtering a fluid using a fluid flow filtration device, comprising:

[0379] Driving the fluid from an inlet through a first elongate fluid flow channel;

[0380] Forcing a permeate component of the fluid through a membrane extending along the first channel and into a second elongate fluid flow channel; and

[0381] Retaining a retentate component of the fluid within the first channel;

[0382] wherein the membrane is positioned to separate the first channel from the second channel along respective lengths of the first channel and the second channel such that the permeate can enter the second channel from the first channel via the membrane along the respective lengths of the first channel and the second channel.

[0383] (30) The method according to (29), wherein the step of driving the fluid comprises pressurizing the fluid within the first channel.

[0384] (31) A flow system, comprising:

[0385] At least one reactor module having a reaction fluid flow channel or well, at least one fluid inlet, and at least one fluid outlet;

[0386] At least one flow driver for driving fluid through the channel or the well;

[0387] A first sensor for measuring any one of pressure, temperature, or pH of the fluid within the system or a combination of pressure, temperature, or pH of the fluid within the system;

[0388] A second sensor for measuring any one of pressure, temperature, and pH of the fluid within the system or a combination of pressure, temperature, and pH of the fluid within the system;

[0389] A reaction state monitoring device for monitoring the characteristics of a fluid within the system, where the characteristics indicate the reaction state of at least two chemical components of the fluid within the system;

[0390] A control unit for receiving data from at least one of the first sensor, the second sensor, and the reaction state monitoring device or a combination of the first sensor, the second sensor, and the reaction state monitoring device and controlling at least one characteristic of the fluid within the system.

[0391] (32) The system according to (31), wherein the characteristics of the system are any one of the following or a combination of the following:

[0392] · The pressure of the fluid within the system;

[0393] · The temperature of the fluid within the system;

[0394] ● The pH of the fluid within the system;

[0395] ● The volume or proportion of one or more chemical components of the fluid within the system;

[0396] ● The flow rate of the fluid within the system.

[0397] (33) The system according to (32), wherein the control unit includes a CPU, a PCB, a PLC, a PC, a processor chip, a handheld electronic device.

[0398] (34) The system according to (33), wherein the additional sensors include any one of a temperature sensor, a pH sensor, a pressure sensor, a flow rate sensor, a flow sensor, or a spectral sensor or a combination of a temperature sensor, a pH sensor, a pressure sensor, a flow rate sensor, a flow sensor, or a spectral sensor.

[0399] (35) A method of processing a fluid using a fluid flow device, comprising:

[0400] Introducing at least one fluid into a reactor module via at least one inlet;

[0401] Using at least one flow driver to drive the fluid through a reaction flow channel or well or using at least one flow driver to drive the fluid within the reaction flow channel or the well and outputting the fluid at an outlet;

[0402] Measuring at least one of the pressure, temperature, or pH of the fluid within the fluid flow device or a combination of the pressure, temperature, or pH of the fluid within the fluid flow device;

[0403] Monitor the reaction state of the chemical components in the fluid within the fluid flow device; and

[0404] In response to at least one of the measurement of the pressure of the fluid, the measurement of the pH of the fluid, the measurement of the temperature of the fluid, and / or the measurement of the reaction state of the chemical components of the fluid, or a combination of the measurement of the pressure of the fluid, the measurement of the pH of the fluid, the measurement of the temperature of the fluid, and / or the measurement of the reaction state of the chemical components of the fluid, use a control unit to control at least one characteristic of the fluid within the fluid flow device.

[0405] (36) The system according to (35), comprising a plurality of reactor modules and filtration modules fluidly connected to each other.

[0406] (37) The system according to (35), wherein the flow driver is at least one pump, and optionally, the flow driver is an injection pump.

[0407] (38) The system according to (35), wherein the fluid analysis sensor includes a UV-Vis spectrometer for absorbance or fluorescence analysis in the range of 190 nm to 1000 nm.

Claims

1. A method for RNA synthesis, comprising: introducing a plurality of reactants via a plurality of inlet ports into a first fluid flow module, the plurality of reactants including: at least one nucleoside triphosphate (NTP), a reaction buffer, and DNA, a DNA-based compound, or a DNA-based mixture; allowing at least some of the reactants to react within reaction channels or wells in the first module of the flow system; retaining the DNA at the first reactor module or recycling the NDA at the first reactor module, and allowing the reaction product stream of the reactants to flow into a first fluid filtration module; and filtering the reaction product within the first filtration module.

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

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

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

5. The method according to any one of the preceding claims, the plurality of reactants further includes any one of an enzyme mixture, a salt solution, an RNA polymerase or a combination of an enzyme mixture, a salt solution, an RNA polymerase.

6. The method according to any one of the preceding claims, comprising: 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.

7. The method according to any one of the preceding claims, comprising: transporting at least some of the filtered reaction product from the first filtration module to a second fluid flow reactor module, and inputting a capping enzyme into the second reactor module.

8. The method according to claim 7, comprising: transporting the fluid output from the second reactor module to a second fluid flow filtration module.

9. The method according to claim 8, comprising: recycling any unreacted NTP to the inlet region of the first reactor module, and recycling any unreacted capping enzyme to the inlet region of the second reactor module.

10. The method according to any one of the preceding claims dependent on claim 5, wherein, The salt solution includes MgCl 2 .

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