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 the Chinese invention patent application filed on May 12, 2021, with international application number PCT / GB2021 / 051132, invention name “Methods and Modular Devices for Synthesizing RNA-Based Therapeutics”, and application number 202180046367.5 after entering the Chinese national phase. 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] Flow reactor (alternatively referred to as continuous flow reactor) provides continuous flow of material or reactant by being connected to form a conduit network of fluid channels. When assembling the mixing module, reaction module and filtering module of the flow system, by enabling or disabling the fluid channel with ports and valves, the various configurations of the conduit network are used to achieve fluid communication, to control the synthesis, purification and configuration conditions of RNA or nucleic acid therapeutic agents, so as to manufacture products in the form of continuous flow. The computer system of the software code constructed purposefully enables directional control of the entire process parameter set in the flow system. These parameters include mixing conditions, temperature, pH value, reagent concentration, monitoring, residence time, purity curve, output, etc. The flow reactor of the example prior art is usually formed as a component of a separate module, and these modules are connected face to face to form an integral block through which a fluid is guided. Example flow reactor is described in WO 2013 / 050764.

[0004] Flow technology provides more sustainable, flexible and efficient drug manufacturing production. Combined with microtechnology and precision engineering, flow systems can be constructed and configured as microfactories that integrate multiple unit operations (e.g., mixing, reaction synthesis, extraction, separation, filtration and purification). In addition, the typical small volume to surface area ratio in flow mixing and reaction systems allows precise control of process conditions related to heat and mass transfer, thereby accelerating the process and improving productivity under a small device footprint. The integrated flow system combined with these unit operations can be regarded as a microfactory or a reduced production system that reduces the use of factory floor space, reduces energy consumption, and improves resource utilization. Therefore, compared with the current manufacturing system currently used to produce substances for preventive vaccines, sustainable growth can be achieved with better environmental impact, cost-effectiveness and flexibility.

[0005] In molecular biology and biotechnology, there are some schemes that increasingly use lab-on-a-chip (LOC) devices. Such LOC devices use microfabrication technology to miniaturize and integrate laboratory analysis and carry out small-scale synthesis or filtration in small chips. These small devices consume less material, produce less waste, reduce costs, and also allow faster reaction times. By guiding the fluid to flow through a micromixer, microchannel, and filter, the various steps of a given scheme can be integrated into the LOC device, and the various steps of a given scheme allow cell sorting, mixing, and enable reactions to be generated for, for example, the synthesis of DNA or RNA. Importantly, the LOC device is formed of a reaction inert material and is preferably transparent so as to enable real-time visual inspection of the reaction in the chip. In addition, the transparent device also makes it possible to perform spectral analysis and research. The preferred material for rapid prototyping of such applications is PDMS (polydimethylsiloxane).

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

[0007] Therefore, there is an urgent need for equipment 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, 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 around the world, and with high productivity and cost-effective operation) is needed. Summary of the invention

[0008] It is therefore an object of the present invention to provide a modular manufacturing platform that enables scalable synthesis and / or filtration of chemical compounds as well as biomolecules and non-biomolecules, particularly including RNA-based vaccines or other nucleic acid therapeutics. It is a particular object of the present invention 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 manufacturing RNA and nucleic acid materials rapidly and ultimately at the point of use (e.g., within a hospital setting).

[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 preventive vaccine. The integrated flow system can include several optional analytical probes (including fiber optic probes), detectors and light sources for UV-Vis absorbance or fluorescence spectroscopy to perform in situ and online control of the synthesis or manufacture of the target substance in a continuous flow process.

[0011] A specific object is to provide microfluidic based devices and methods which integrate modules including fluid flow bioreactor modules and fluid flow filtration modules interconnected via respective conduits / channels to create a continuous fluid flow path which can be operated continuously and can be 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 appreciated.

[0012] A specific purpose is to provide a fluid flow system, which is used for reacting and synthesizing selected chemical compositions 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 purpose is to provide a modular system in which each module unit is interconnected according to a synthetic approach. Such a unit can include ports, valves and suitable connections to achieve fluid communication and interconnection between each module. Another specific purpose is to provide a reaction system for fluid, which can be configured to measure and respond to the characteristics of the system (e.g., the volume or ratio of one or more selected chemical compositions of the pressure, temperature, pH, fluid, flow rate of the fluid and the reaction state of the fluid).

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

[0014] Another object is to provide a method and apparatus for synthesizing RNA. Another specific object is to provide an apparatus and method for synthesizing RNA from DNA. Yet another object is to provide a form of synthetic RNA that can be used to prepare a vaccine.

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

[0016] A specific object of the present invention is to provide an apparatus and method for forming a fluid flow integrated system, which fluid flow integrated system includes component modular parts, the component modular parts including a fluid flow bioreactor module and a fluid flow filtration module, wherein the fluid flow bioreactor module and the fluid flow filtration module are 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, a method for RNA synthesis is provided, the method comprising: introducing a plurality of reactants into a first fluid flow module via a plurality of inlet ports, the plurality of reactants comprising 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 a reaction channel or a well within the first module of the flow system; retaining the DNA at the first reactor module or recycling the DNA at the first reactor module, and allowing the reaction products of the reactants to flow into the first fluid filtration module; and filtering the reaction products within the first filtration module.

[0018] Optionally, the 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 comprises any one of an enzyme mixture, a salt solution, an RNA polymerase or a combination of an enzyme mixture, a salt solution, and an 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 products from the first filtration module to the second fluid flow reactor module, and inputting the capping enzyme into the second reactor module. Optionally, the method includes: conveying the fluid output from the second reactor module to the 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 includes MgCl 2 Optionally, the RNA polymerase comprises T7 polymerase.

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

[0024] According to another aspect of the present invention, there is provided use of RNA or RNA-based compounds prepared according to the methods described herein in the preparation of a vaccine.

[0025] According to another aspect of the present invention, a fluid flow filtration device is provided, which includes: a first elongated fluid flow channel having an inlet; a second elongated fluid flow channel having an outlet; and a permeable membrane positioned to separate the first channel from the second channel along their respective lengths, so that permeate can enter the second channel from the fluid in the first channel through the membrane along the length of the first channel and the length of the second channel.

[0026] Optionally, a majority of the length of the first channel is positioned adjacent to a majority 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, 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.

[0028] Optionally, the device comprises a first plate in which a first channel is formed and a second plate in which a second channel is formed, and the membrane is sandwiched between the respective opposing faces of the first and second plates. Optionally, the first and second channels each comprise a series of straight sections and curved sections. Optionally, the first and second channels comprise respective serpentine profiles in their longitudinal directions. Optionally, the first and second channels are open along their lengths and are positioned to be 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 the RNA molecules.

[0029] According to another aspect of the present invention, a fluid flow system for treating fluid is provided, 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 area, at least one inlet and at least one outlet configured to be connected to the outlet fluid of the first reactor module; wherein the first filtration module comprises the fluid flow filtration device 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 in fluid communication with the first filtration module.

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

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

[0033] According to another aspect of the present invention, a method for filtering a fluid using a fluid flow filtration device is provided, the method comprising: driving a fluid from an inlet through a first elongated fluid flow channel; forcing a permeate component of the fluid to pass through a membrane extending along the first channel and enter a second elongated 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 their respective lengths, so that the permeate can enter the second channel from the first channel via the membrane along their respective lengths.

[0034] According to another aspect of the present invention, a flow system is provided, which includes: 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 the fluid to flow through the channel or well; a first sensor for measuring any one of the pressure, temperature or pH of the fluid in the system or a combination of the pressure, temperature or pH of the fluid in the system; a second sensor for measuring any one of the pressure, temperature, pH of the fluid in the system or a combination of the pressure, temperature and pH of the fluid in the system; a reaction state monitoring device for monitoring a property of the fluid in the system, the property indicating a reaction state of at least two chemical components of the fluid in the system; 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 property of the fluid in the system.

[0035] Optionally, the characteristic of the system is any one or a combination of the following: pressure of the fluid within the system; temperature of the fluid within the system; pH of the fluid within the system; volume or ratio of one or more chemical components of the fluid within the system; flow rate of the fluid within the system. Optionally, the step of driving the fluid includes: pressurizing the fluid in the first channel. Optionally, the control unit includes a CPU, a PCB, a PLC, a PC, a processor chip, a handheld electronic device. Optionally, the additional sensor includes 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, a method for treating a fluid using a fluid flow device is provided, 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 to flow through a reaction flow channel or a well or using at least one flow driver to drive the fluid within a reaction flow channel or a 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 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 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.

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

[0038] Optionally, the method of RNA synthesis comprises recycling any unreacted NTPs 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 according to the method described herein. According to another aspect of the present invention, there is provided the use of RNA prepared according to the method of any preceding claim in the preparation of a vaccine.

[0040] According to another aspect of the present invention, a fluid flow device for treating fluid is provided, which fluid flow device includes: a first reactor module, which has a reaction flow channel or well, at least one inlet and at least one outlet; a first filtration module, which has a fluid filtration area, at least one inlet and at least one outlet, and the inlet is configured to be connected to the outlet fluid of the first reactor module.

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

[0042] Optionally, the apparatus comprises a fluid injection port arranged to be in fluid communication with an inlet region of the second reactor module. Optionally, the apparatus comprises a first recirculation conduit extending between a region of an outlet of the first reactor module and a region of at least one inlet of the first reactor module.

[0043] Optionally, the device includes a third recirculation conduit extending between the region of the outlet of the second filter 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 fluid chemistry to be input into reaction channel or wells. Optionally, the device includes at least one pump coupled to at least one inlet of the first reactor module to drive fluid flow through reaction channel or drive fluid in the well. Optionally, the device includes at least one valve, fluid flow gate, fluid flow port, heating element, fluid storage device or storage container that is arranged to be communicated with the fluid fluid in the device. Optionally, the pump includes a syringe 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 provided on or within the plate-like structure. Optionally, the device comprises a plurality of sensors positioned at different fluid flow areas of the device.

[0045] Optionally, the sensor includes any one 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 spectral sensor, at least one optical sensor, at least one optical fiber or spectral optical fiber, or a 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 spectral sensor, at least one optical sensor, at least one optical fiber or spectral optical 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 holding container and sensor to control the characteristics of the fluid flow in the device in response to the state of the physical, chemical or mechanical characteristics of the fluid determined by the sensor. Optionally, the control unit includes a CPU, a processor, a PCB, a PLC, a handheld electronic device.

[0047] Optionally, the control unit includes a control module, which includes any one or a combination of the following: software; electronic components; data storage means; wired or wireless communication modules and / or ports; a visual display output; a user interface; 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 fluid, the fluid flow system comprising: a plurality of devices according to the claims herein, the final fluid flow outlet of each of the devices being coupled to a collection unit to combine the fluid output from each of the devices.

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

[0050] According to another aspect of the present invention, a method for treating a fluid using a fluid flow device is provided, 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 a reaction flow channel or well or allowing the fluid to flow 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 filter module having a fluid filtration area via at least one inlet, and outputting the fluid from the first filter module at the outlet; wherein, in the first reactor module, a reaction occurs between at least two chemical components of the fluid, and filtration of the fluid occurs in the first filter module.

[0051] Optionally, the method includes using at least one fluid pump to drive the fluid through the device. Optionally, the method includes: recirculating at least a portion of the fluid from the outlet of the reactor module and / or the filtration module to the area of ​​at least one inlet of the reactor module via at least one recirculation conduit. Optionally, the method includes: using at least one sensor to monitor the state of the chemical reaction between the chemical components in the fluid. Optionally, the method includes: controlling the fluid flow through the device in response to the state of the chemical reaction identified by the sensor. Optionally, the method includes: using at least one sensor to monitor the physical, chemical and / or mechanical properties of the fluid in the device. Optionally, the method includes: controlling the fluid flow in 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 communicate with the fluid in the device. Optionally, controlling the fluid flow includes: using a CPU, a processor, a PCB, a PLC or a 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, a fluid flow filtration device is provided, which includes: a first elongated fluid flow channel having an inlet; a second elongated fluid flow channel having an outlet; and a permeable membrane positioned to separate the first channel from the second channel along their respective lengths, so that permeate can enter the second channel from the fluid in the first channel through the membrane along the length of the first channel and the length of the second channel.

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

[0054] According to another aspect of the present application, a fluid flow system for treating fluid is provided, which fluid flow system includes: a first reactor module, which has a reaction flow channel or well, at least one inlet and at least one outlet; a first filtration module, which has a fluid filtration area, at least one inlet and at least one outlet configured to be connected to the outlet fluid of the first reactor module; wherein the first filtration module includes the fluid flow filtration device described herein.

[0055] According to another aspect of the present invention, a method for filtering a fluid using a fluid flow filtration device is provided, the method comprising: driving a fluid from an inlet through a first elongated fluid flow channel; forcing a permeate component of the fluid to pass through a membrane extending along the first channel and enter a second elongated 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 their respective lengths, so that the permeate can enter the second channel from the first channel via the membrane along their respective lengths.

[0056] According to another aspect of the present invention, a fluid flow system is provided, which includes: 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 the fluid to flow through the channel or well; a first sensor for measuring any one of the pressure, temperature or pH of the fluid in the system or a combination of the pressure, temperature or pH of the fluid in the system; a second sensor for measuring any one of the pressure, temperature, pH of the fluid in the system or a combination of the pressure, temperature and pH of the fluid in the system; a reaction state monitoring device for monitoring a property of the fluid in the system, the property indicating the reaction state of at least two chemical components of the fluid in 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 device or a combination of the first sensor, the second sensor and the reaction state monitoring device, and controlling at least one property of the fluid in the system.

[0057] According to another aspect of the present invention, a method for treating a fluid using a fluid flow device is provided, 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 to flow through a reaction flow channel or a well or using at least one flow driver to drive the fluid within a reaction flow channel or a 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 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 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. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0059] Figure 1 is a schematic diagram of a continuous reaction and filtration apparatus suitable for RNA production;

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

[0061] Figure 3 Is applicable to Figure 1 A floor plan of a well or batch bioreactor module of the equipment;

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

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

[0064] Figure 5B yes Figure 5A An image of the membrane within the filtration module;

[0065] Fig. 6A is a diagram of a portion of a filter module having a filter configured for use with Figure 1 The area of ​​the device for centrifugal separation of chemical components;

[0066] Figure 6B is an image of a portion of a filtration module having an area for centrifugal force separation of chemical components;

[0067] Figure 7 It is a vertical tangential flow filtration (VTFF) module suitable for this fluid flow device;

[0068] Figure 8 yes Figure 7 A magnified view of an area of ​​the filtration module;

[0069] Fig. 9 is a plan view of a spiral tangential flow filtration module;

[0070] Fig. 10A It is a component of the filtering module according to the specific implementation;

[0071] Fig. 10B yes Fig. 10A A schematic diagram of additional components of the filtration module;

[0072] Fig. 10C yes Fig. 10A and Fig. 10B Another schematic diagram of components of a filtration module;

[0073] Fig.11A is a plan view of another filtration module formed as a double-layer microchannel TFF;

[0074] Fig. 11B is a schematic diagram of a filtering mechanism and a filtering module according to a specific implementation method;

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

[0076] Fig.13 is a graph used in the design of computational fluid flow bioreactors and filtration systems;

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

[0078] Fig.15 This is a perspective view of the TFF module;

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

[0080] Fig.17 It is a schematic diagram of the outline of the prototype construction process;

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

[0082] Fig.18B is suitable as Fig.18A A schematic diagram of components of a reaction state monitoring arrangement for a portion of a fluid flow system;

[0083] Fig. 18C It is formed according to the specific implementation method Fig.18A A schematic diagram of a series of sensors, a reaction state monitoring arrangement and a control unit as part of a fluid flow system;

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

[0085] Fig. 20 It is a schematic diagram of the serial communication program of the control system;

[0086] Fig.21 RNA yield and NaCl, MgCl 2 , NTP, etc.;

[0087] Fig. 22 is a graph of data analysis (RNA yield vs. magnesium ion) and specific reaction time selection according to various aspects of the present invention;

[0088] Fig.23A It is a schematic diagram of the simulation of velocity distribution analysis results;

[0089] Fig. 23B is a graph of the velocity analysis graph;

[0090] Fig.24 is a schematic diagram of the velocity of the tangential flow filtration channel in the filtration module;

[0091] Fig.25A is a graph of the pressure drop in the bioreactor channel;

[0092] Fig.25B is a graph of the pressure drop as a multi-step descent;

[0093] Fig.26A is a photograph of a mask used to make a fluid flow reactor and filtration device;

[0094] Fig.26B is a photograph of the continuous microfluidic flow reactor mask;

[0095] Fig. 27 is an image of a continuous flow reactor sealed with Kapton tape;

[0096] Fig.28 Here is an image of the complete setup using two acrylic sheets;

[0097] Fig.29 It is an image of the 3D printed mold after use;

[0098] Fig. 30A is the first portion of a schematic diagram of an example embodiment of the present flow system integrated with a downstream formulation system using a modular flow reactor attached to a conventional fill and finish vaccine production line;

[0099] Fig. 30B is the second portion of a schematic diagram of an example embodiment of the present flow system integrated with a downstream formulation system using a modular flow reactor attached to a conventional fill and finish vaccine production line;

[0100] Fig. 30C is a third portion of a schematic diagram of an example embodiment of the present flow system integrated with a downstream formulation system using a modular flow reactor attached to a conventional fill and finish vaccine production line;

[0101] Fig.30Dwas evaluated using UV-Vis spectroscopy FIG. 30A to FIG. 30D A graph of the absorbance versus wavelength of the output solution of a flow reactor of an embodiment of the present invention compared to that evaluated using a conventional batch processing scheme. FIG. 30A to FIG. 30D A graph of absorbance versus wavelength for an output solution of a flow reactor according to an embodiment of the present invention;

[0102] Fig.31 This is a photo of an experimental fluid flow chemistry compound synthesis platform;

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

[0104] Fig.33 are images of samples with different concentrations (A: 25 mM / L, B: 50 mM / L, C: 75 mM / L, D: 100 mM / L) used in the microfluidic flow reactor module and the continuous filtration system;

[0105] Fig.34 It is a graph of UV-Vis spectral analysis;

[0106] Fig.35 This is an image of real-time plotting of pH data in Python;

[0107] Fig.36 is a graph of the prediction plot (left side is for the reaction with 1 mM dNTP, right side is for the reaction with 4 mM dNTP);

[0108] Fig.37 Graphs showing predicted profiles for the first and second experiments (upper for reactions with 1 mM dNTPs, lower for reactions with 4 mM dNTPs);

[0109] Fig.38 is a summary of various data for fluid flow bioreactor and filtration (the left side is the reaction for 1 mM dNTP, the right side is the reaction for 4 mM dNTP LogWorth=-log10(p value));

[0110] Fig.39 is a schematic diagram of an enlarged fluid flow reactor and apparatus of the present system;

[0111] Fig.40A is a graphic representation of the source code for absorbance data and real-time plot (absorbance vs. wavelength) in Python;

[0112] Fig.40B This is an illustration of the source code for plotting pH data in real time in Python. DETAILED DESCRIPTION

[0113] Principles of fluid mechanics in microporous channels

[0114] Advances in microfabrication have made it possible to construct microchannels with micron dimensions. Since microchannels are often integrated into these microsystems, determining the characteristics of fluid flow in microchannels is important for better designing various microfluidic devices. Due to technological limitations, the understanding of fluid behavior in porous media (especially microporous media designed in on-chip labs that can be used for synthetic RNA vaccine manufacturing) is very limited. The porous medium is considered to be saturated with the fluid of interest in the sense that a fluid-fluid interface is not formed, and a single fluid dominates the pore space. Let dp be the particle size and U be the velocity scale. It has been found that (1) applies to cases with Reynolds numbers up to 1, that is:

[0115]

[0116] The permeability of porous media is a property that depends on both pore size and pore structure. Dimensional analysis shows that permeability is a function of porosity e and particle diameter dp; each representing pore geometry and pore size, respectively. The Carman-Kozeny relationship relates these quantities empirically, but with dimensionally correctness:

[0117]

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

[0119]

[0120] It is called the Darcy number. In many applications, the diameter of the particles is about a fraction of a millimeter, while the length scale of the device is about a meter or longer. Equation 2 can be appropriately expressed as applicable within the limits of Da<<1, Re<1. Bahrami et al. (M.Bahrami, 2006) developed a general model for predicting the pressure drop in microchannels of arbitrary cross-sections. 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 of a laminar fully expanded flow can be obtained from the following:

[0121]

[0122] Where Ip*=Ip / A is the specific polar moment of inertia of the microchannel cross section, ΓΓ=4(W+H) is the circumference of the microchannel cross section, L is the fully expanded length, Q is the volume flow rate, and ε=the aspect ratio (width / height) of the rectangular microchannel.

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

[0124]

[0125] Minor losses ΔPmin: Other pressure losses associated with the measured pressure drop are inlet losses, outlet losses, and bend losses. These losses are usually obtained from traditional relationships used on a macro scale. Phillips (reference) showed that the minor pressure losses can be obtained from:

[0126]

[0127] Where A and A t are the channel cross-sectional area and the connecting pipe cross-sectional area respectively. b is the loss coefficient of the curve, K c and K e represents the coefficient of contraction and expansion losses due to area change. Phillips suggested that for a 90-degree bend, K b It is about 1.2. Assuming that the cross-sectional area of ​​the channel and the connecting pipe is equal, K c and K e is the maximum possible value, and the relatively small loss in relation to the measured pressure drop is negligible compared to the measured pressure drop. Hooman and Merrikh (M. Bahrami, 2010) have developed an analytical solution for flow and pressure drop in porous channels 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] Choosing the appropriate box depends on the total sample volume, the required processing time, and the desired final sample volume. Use the following formula to calculate the membrane area required to process the sample in a specified time:

[0132]

[0133] in,

[0134] A = membrane area (m2)

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

[0136] J = filtrate flow rate (liters / square meter / hour, LMH)

[0137] T = Processing time (hours)

[0138] Fluid flow simulation for prototype studies

[0139] The height of the channel and the type of liquid have an impact on the performance of the microchannel. Computational fluid dynamics (CFD) FLUENT software was used to simulate the microchannel. From CFD, the velocity distribution of the not fully developed and fully developed areas was analyzed to study the fluid flow behavior. The characteristics of the liquid affect the fluid flow in the microchannel. We need minimal kinematic viscosity and low surface tension, which depends on choosing the right liquid.

[0140] Fluid flows play an important role in guiding the integration of small-scale devices with many applications due to their potential in chemical and biochemical engineering. The mechanisms and fundamentals of single-phase fluid flows are reviewed using experimental data for different types of liquids and surface roughness. Therefore, at the microscale, the effects of liquid properties such as surface tension and viscosity are dominant. [Nawi, MNM, Manaf, AA, Arshad, MR, 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].

[0141] Reynolds number is the ratio of the inertial force and the viscous force of the fluid flowing in the channel (that is, the ratio of the momentum of the fluid to the friction force applied by the channel wall). Low Reynolds number flow is laminar or stratified flow, in which the fluid streams flow parallel to each other and are mixed only by convection and molecular diffusion. High Reynolds number flow is turbulent, in which "packets" of various sizes of fluid show random motion in both space and time, resulting in rapid mixing of the entire channel. The transition between laminar and turbulent flow usually occurs when Re=2000 in internal flow [Schulte, TH, Bardell, RL, and Weigl, BH (2002), Microfluidics in Clinical Diagnostics _2002_Clinical Chemistry Journal (Microfluidic-technologies-in-clinical-diagnostics_2002_Clinica-Chimica-Acta.), No. 321, Page 1 to Page 10].

[0142] Although turbulent flow field is more complicated than laminar flow field, there is significant second-order influence. One-dimensional model and two-dimensional model that describe the time-dependent evolution of distribution under such conditions have been improved. Confocal fluorescence microscopy experiment and three-dimensional numerical modeling can contribute to confirm the quantitative description [Schulte, TH, Bardell, RL, and Weigl, BH (2002), Microfluidics in Clinical Diagnostics _2002_Clinical Chemistry Journal (Microfluidic-technologies-in-clinical-diagnostics_2002_Clinica-Chimica-Acta.), the 321st phase, page 1 to page 10] of reaction-diffusion process near the wall.

[0143] Integration of sensors into flow systems

[0144] In the research, a sensor-integrated platform should be established to obtain and monitor data of the entire 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 to 2712] proposed 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 to 7]. In Wang's approach, complementary metal oxide semiconductor (CMOS) technology shows great potential in integration and detection. CMOS image sensors work as an effective reaction and detection platform, enabling them to monitor real-time photon changes according to the amplification process. The CMOS image sensor observes the photons and converts them into digital units. In addition, UV spectroscopy studies, optical color intensity detection, and pH analysis were performed 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 ISFET pH microsensor integrated in a CMOS standard process. The entire system is integrated in 1.12 mm. 2 It is in the silicon region; it exhibits a linearity of 59 mV / pH over a concentration range of pH levels 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 the cost and time. More realistically, the integration scheme of 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 uses an extended gate ion-sensitive field effect transistor (ISFET) with an integrated pseudo-reference electrode to monitor the pH value in a fluid flow reaction chamber. For the temperature sensor, a serpentine temperature sensor (line width: 50 mm) and a heater (line width: 400 mm) were 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, Progress of the Royal Society of Chemistry (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 to 7475] A new approach for in-plane digital microfluidic spectroscopy has been proposed. In this technique, a custom manifold aligns the optical fiber with the digital microfluidic device, allowing optical measurements to be made in the plane of the device, which provides a way for optical detection. The preferred analytical 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 the continuous RNA production process is shown. Figure 1 As 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 the 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 / inlet ports 16, the second bioreactor module includes respective inlet / input ports 17, and the second TFF module includes respective input / inlet ports 18. Each of the ports 16 to 18 may be provided with respective associated pumps or injection ports for introducing fluids to drive the fluid flow under pressure into the elongated reaction channel 24 of each reactor and the elongated filtration channel 25 within each respective module. A plurality of recirculation channels 19, 20, 21 interconnect the various fluid flow regions of the modules 10, 11, 12, 13 (at or toward the respective inlet / outlet end regions) to provide fluid communication and recirculation of reactants / reagents. Figure 1 The device also includes sensors, reaction state monitoring devices, control units and other associated electronic and actuator components (not shown) as described elsewhere herein, so as 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 to the inlet area of ​​the reactor 10 via the channel 19. In this way, selected reagents and reactants can be circulated to provide a continuous or semi-continuous process.

[0149] Reference Figure 1 and Figure 2 both, Figure 2 Shows Figure 1 A variation of the fluid system in which Figure 1 In contrast to the elongated reaction channel 24, the reactor 10 includes a reaction well 50. Figure 1 As described, multiple injection ports 14 are coupled to be in fluid communication with the well 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, so that selected species, reagents, solvents can be extracted from the filtration modules, and the final product, such as fully capped RNA, can also be collected at port 22, for example.

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

[0151] Reference Fig.39 , refer to Figure 1 and Figure 2 The multiple reactants and filtration systems described (and generally represented as plate-like units 10, 11, 12, 13) can be connected together as an expanded fluid flow reactor and device to form a fully integrated modular fluid flow reaction and filtration system for manufacturing RNA-based therapeutics and preventive vaccines at a scale sufficient for evaluation and clinical trials and ultimately production. In particular, appropriate connecting conduits provide fluid communication between the individual plate-like units 10, 11, 12, 13 so that the output end of each unit (at output end 22) can be combined to provide a total output. Fig.39 The collective system may include a single control unit, multiple control units and respective sensors and reactive state monitoring tools and units, as herein referred to FIG. 18A to FIG. 20 As described. With the parallel operation of multiple fluidic devices of the present invention, continuous production of RNA-based therapeutics and preventive vaccines can occur. Through this expansion strategy, one can achieve manufacturing output on a 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. Fig. 20 It is a schematic diagram of the serial communication program between Arduino and Python IDE control utility. In a possible implementation, an Arduino board can be used. In a preferred implementation, an industrial edge computing SBC (single board computer) node is used.

[0152] Take RNA synthesis as an example, in this process, segments of DNA are copied into RNA by the enzyme RNA polymerase. The transcription mixture consists of NTP (nucleoside triphosphate), DNA, MgCl 2 (with P 2 O 7 -4 The reaction produces precipitation, which affects the RNA productivity), T7 polymerase (catalysis), which are injected into the first bioreactor channel by a syringe pump. Hydroxy naphthol blue (HNB) is used to titrate Mg 2+ Colorimetric indicators of ions. Initially, HNB and Mg 2+ Ions combine to form 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 continues and RNA begins to be produced, accompanied by the precipitation of magnesium pyrophosphate (Mg 2 P 2 O 7 ). All of the above components except DNA flow through the first tangential flow filtration (TFF) module 11 with a large molecular weight (MW) cut-off membrane. 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 that is recycled into the inlet port 14 (4). Then m7G methyltransferase (5' cap) is injected into port 17 and flows into the second reactor 12 and channel 24, where the contents stay and react for 2 hours. The permeate enters the second TFF unit 13 with a medium molecular weight cut-off membrane. In this filtration stage, unreacted NTPs and 5' caps are retained and recycled (via 21 and 20) to the bioreactor 12 and / or 10. Finally, the purified RNA is extruded at outlet 22. At the same time, other components such as Mg 2 PP i , water, and salts are collected in another container or port 51 and optionally discarded or further processed. The output (capped RNA) can then be processed via additional processing procedures (not described herein but familiar to those skilled in the art) to produce the desired vaccine.

[0154] The goal is to achieve continuous synthesis and purification to obtain RNA material and achieve an increase in system yield. It is necessary to add raw materials to the reactor via 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 substantial increase in RNA yield output is possible.

[0155] Set-Based Concurrent Engineering (SBCE) Method

[0156] The Lean Product Development methodology has been chosen as the main framework for developing the design. The Lean Product Development methodology is Set-Based Concurrent Engineering (SBCE). SBCE is a process where the product to be developed is divided into different subsystems to allow for the development of a set of possible solutions for each of the subsystems in parallel. As the design evolves, the set of solutions for each subsystem is narrowed down through knowledge-based decision making using tools such as simulation, prototyping, testing, or other acquired knowledge. Following the SBCE methodology, the first basic step is to divide the product into different subsystems that can be developed separately. Considering the conceptual design of the system that needs to be developed, there are four main identified processes that can be divided into exactly two functions that will be developed: the bioreactor and the filtration process. At the same time, the system needs to be integrated with a control system that is able to control the pressure from the pump and have sensors in key parts of the product to measure key parameters. After drawing a conceptual design of the system, 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 combined appropriately in any desired order depending on the needs of the target therapeutic agent. For example, after the last filtration module, there can be additional reactors / mixing steps for formulating the RNA therapeutic substance into a therapeutic product.

[0158]

[0159] Subsystem Design Specification Methodology

[0160] Bioreactor

[0161] Regarding the bioreactor, two different designs have been developed. The first design was inspired by conventional batch processes used in chemistry for RNA synthesis. The design includes four input ports 14 for each of the raw materials, which are connected to a 1 mL main chamber where the reaction occurs by allowing sufficient residence time. The container can then be selectively vented by releasing a 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 continue to the next step of processing or is rejected. Figure 2 and Figure 3 The design of the bioreactor is shown.

[0162] This design requires a complex, well-coordinated control system to control the valves integrated with the unit. After the residence time is over, 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 that allows mixing of reactants and 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 inputs that are continuously fed into the flow reactor via a T-mixer. The reaction conditions and effects are controlled by the net flow entering the fluid conduit and the fluid velocity of the individual components through the T-mixer. These are controlled by setting the dispensing rate of the computer-controlled dispensing pump. When the mixture reaches the end of the serpentine flow bioreactor within a set residence time, the product is formed. At the output end, the device is characterized by: as a small fluid holding well, temporarily slowing the fluid flow to obtain online measurements of preferred analytical methods with specific chemical solutions, such as online UV-Vis spectroscopy. The signal from the spectrometer is parsed and analyzed in a specially built software by extracting certain features of the signal to determine whether the desired product has indeed been formed by comparing with a reference vector. Figure 4 is a schematic diagram of a serpentine flow bioreactor.

[0164] Filter module

[0165] There are two types of filtration: direct flow filtration (DFF) and tangential flow filtration (TFF). In DFF, the main flow is perpendicular to the filter, while in TFF, the flow is parallel to the filter. The risk of clogging is much higher in DFF, and it has been proven that TFF can provide a higher filtrate flow rate than DFF while increasing the filtration volume. In addition, typical applications of TFF are the concentration, diafiltration and fractionation of biomolecules and the purification and removal of cells; these applications are similar to those presented in this project. In other words, for the application of this project, TFF is the most effective option, and therefore, TFF is the type of filtration that was chosen.

[0166] For the filtration module, the TFF method was selected as a model. Several design schemes have been proposed in the research literature and commercial practice. For the present invention, several other factors have been considered, such as for this subsystem, manufacturability and the ability to be integrated with the entire system as a module have been considered. The first filtration module design includes a serpentine path with two channels at the same height, and the separation features specially patterned on the device are used as filter membranes. X. Chen et al. (2007) [Microfluidic Chip for Blood Cell Separation and Collection Based on Crossflow Filtration, Sensors and Actuator B 130 (2008) (pages 216 to 221), China], TFF filter designs have been used in the past, and compared with other solutions, TFF filter designs quickly and reproducibly separate biological cells and are less expensive.

[0167] In the present invention, membranes are used that are deliberately chosen to have dimensions suitable for separating biomolecules that are typically present in solution following nucleic acid synthesis. Figure 5A and Figure 5B A filtration module is shown, including filtration features within serpentine microchannels. The size and length of these channels are deliberately designed to allow selection of the correct biomolecules in the filtrate and retentate streams. Fabrication of the module employs microengineering techniques, including photolithography and deep reactive etching of glass or silicon substrates. These techniques are well understood, industrially available, comply with pharmaceutical regulations, and are low cost for large-scale device production.

[0168] The second filter module design does not require any filter membrane. It works on the principle of centrifugal force, which causes some particles to move faster than other particles if the fluid has a curved trajectory at a certain speed. Therefore, having a curved microchannel divided into two different channels makes it possible to allow particles of different sizes to 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 (for example, fluid speeds exceeding 1 [m / s]). Under appropriate conditions, this separation technology can achieve efficiencies of up to 90%, and since there is no membrane, the filtration process can be greatly simplified. Fig. 6A and Figure 6B A CAD design of this design solution is shown.

[0169] The third design is very similar to the first design module mentioned 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 Actuators B 180 (2013) (Page 122 to Page 129), China], this design adds a centrifugal effect to conventional serpentine filter modules, as described above. This centrifugal effect improves separation efficiency and reduces the risk of clogging. Figures 9 to 10C The design is shown.

[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 Progress] has used filter membranes for blood cell separation. However, in the present invention, the filtration module uses two microchannel layers, which sandwich the filter membrane in the middle to produce a filtrate flow and a retentate flow, and the retentate flow is recycled back to the reactor module, while the filtrate flow continues to be collected at the output end and is directed to the downstream module according to the 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, but 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. Fig.11A and Fig. 11B The design of such a solution is shown.

[0171] The four embodiments of the filtration module described above rely on the control of the pressure difference between the flow streams and the separation method used. However, in the fifth embodiment, the device combines digital microfluidics and digital macrofluidics with commonly used protocols in molecular biology to separate and extract nucleic acids, especially DNA. There are two types of these 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 DNA plasmids such as Invitrogen DNA binding beads (Thermofisher Scientific) can be held in a reaction vessel using a digitally encrypted magnetic field, such as in Figure 2 In the case of the bioreactor design of the present invention, the container is drained and refreshed with new solution, or magnetic beads attached to DNA plasmids, such as Invitrogen DNA binding beads, are forced through the filter membrane in the above-mentioned filter module design and routed back to the reaction module through the retentate channel.

[0172] Example Implementations

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

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

[0175] Fluid Flow Simulation Methods for Research Devices

[0176] As shown in Table 2, two different sizes of bioreactor channels are required. Computational fluid dynamics (CFD) software has been used to analyze hypothetical data in microchannels. Microchannels require steady-state flow rates.

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

[0178]

[0179] FLUENT: Fluent is a computational fluid dynamics (CFD) software that helps in solving fluid flow problems. It uses the finite volume method to solve the governing equations of the fluid and provides many different physical models like laminar or turbulent, viscous or inviscid, compressible or incompressible. The geometry and mesh generation is going on in GAMBIT, which is a preprocessor bundled with FLUENT.

[0180] The solution is obtained by following these steps: Geometry; Mesh; Setup; Solve; Results. The channel is represented by a 2D CAD design. Then, material properties and boundary conditions are set. Finally, the domain must be meshed. FLUENT converges the problem until a convergence limit is met or a specified number of iterations is reached.

[0181] a) Geometric shape

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

[0183] b) Grid

[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 grid types

[0186]

[0187] Fluid flow modeling using computational fluid dynamics:

[0188] Regarding the Reynolds number (Re=0.0269), laminar flow is 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 are specified in FLUENT.

[0190] Table 4 Boundary conditions specified in FLUENT

[0191]

[0192] c) Solution

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

[0194] Table 5 Input and output data of FLUENT

[0195]

[0196] Prototype building

[0197] To build the prototype, different steps are followed. First the mold is created and then the chip is cast from the mold. Different subsystems are built separately to test each design. Experiments will be conducted on each subsystem to validate the design. Different versions of the mold have been edited. The design evolves as Fig.14 shown.

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

[0199] ●The two templates have been recombined into one template, making all preparations for chip manufacturing process easier.

[0200] ●The cross-sectional area of ​​the channel has been increased from 0.4mm×0.8mm to 0.5mm×1mm, avoiding the collapse of the channel wall when testing the chip.

[0201] ● Additional space was added between channels (0.5mm to 1mm) and between the borders 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 were added to the edges of the mold to improve the quality of the entire PDMS chip when casting.

[0203] Regarding the vertical TFF device, the top and bottom layers have been edited separately to create two distinct layers that will be sealed together with the filter membrane in the middle. Fig.15 A two-layer design is shown. Table 6 illustrates the different features of the two designs.

[0204] Table 6 Description of the characteristics of the two designs

[0205]

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

[0207] Microfluidic chips require precise, accurate methods and techniques to make the reaction or filtration work as expected. The most common method used for microfluidic chips is soft lithography. In this case, soft lithography involves creating a mold from which the 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 based on the CAD design of the desired part. Although different technologies can be used for 3D printing, stereolithography was chosen in the case of this project.

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

[0209] Table 7 Microscope analysis values ​​of channel dimensions

[0210]

[0211]

[0212] Finally, the mold has been printed with two different materials. Both materials are photopolymer resins, and one of them is a high-temperature resin. The main difference between the two materials is the maximum temperature before heat deformation. The first material can reach 50°C, while the second material can go up to 250°C.

[0213] Selection of filter membranes

[0214] The selection of a specific filtration membrane is done in two steps. The first step involves finding the molecular cut-off size of two different membranes for the two filtration stages. In fact, each filtration stage is processing a different molecule of different molecular weight. 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 lists the different molecules and their sizes or molecular weights in detail.

[0215] Table 8 Molecular weight information

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

[0217] Regarding the two vertical TFF stages, the first vertical TFF is to process T7 polymerase, while the second vertical TFF is to process M7g+ methyltransferase. Taking into account the different molecular sizes, the selected filter membrane needs to have a molecular cut-off size that is three times smaller than the molecule that needs to be retained, in this case, it is RNA molecules [General Electric, 2014]. In addition, the filter needs to be large enough to allow the recovered 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 1 → molecular weight cut-off membrane is 500 kDa

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

[0221] PDMS masking and casting

[0222] With the mold printed and prepared, polydimethylsiloxane (PDMS) microfluidics can be cast from it. The product used to make the PDMS chip is Sylgard 184 silicone elastomer. This product consists of two elements, one is the silicone elastomer component and the other is the curing agent responsible for the cross-linking of the elastomer molecules, which will cure the elastomer while maintaining the casting properties. However, before applying PDMS on top of the mold, some preparation work is required. 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, you need to use a dryer to remove the bubbles in the mixture. Repeat this operation until all the bubbles disappear.

[0225] If there are still small bubbles, let the beaker sit. The bubbles will burst on their own.

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

[0227] ·surface

[0228] Table 9 Curing temperature and time

[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 properties and aspects. However, the first molds used in this process could not withstand high temperatures. That is why ambient temperature curing was also tested during a week. Finally, due to the different features cast on the PDMS mask, this mask needed to be sealed on the open side. Two different methods of sealing the mask have been used, allowing different test results ( Fig.17 is a summary of the different steps in the prototyping process to create a continuous flow bioreactor system):

[0231] The first method is to seal the PDMS mask with another flat layer of PDMS. This method allows adding extra thickness to the entire mask and facilitates the integration of the tube and its fittings.

[0232] The second method is to seal the PDMS mask on the open side with Kapton tape. 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 tubes and fittings will be more complicated.

[0233] Sensing method

[0234] The main sensing system was designed by combining the characteristics of the continuous flow microfluidic system and the reaction. First, the sensing system of the microfluidic chip is limited by the chip size and the characteristics of the factors that need to be detected. In addition, choosing 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+ micro-fiber spectrometer, Ocean Optics Inc., UK) can store 1000 full spectra per second, and its detection range is from 190nm to 1100nm wavelength. In addition, the official software is used, 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. In order to integrate the pH meter with the microfluidic chip, the size of the pH meter was limited due to design specifications. The diameter of the measuring cell on the microfluidic chip was 5 mm2, while the diameter of the body of the pH meter was 3 mm.

[0240] c) Pressure controller and sensor

[0241] CFD analysis of the microfluidic chip showed that the pressure in the bioreactor and TFF section was 0.27 Bar. The pressure sensor (MPS Microfluidics High Precision Pressure Sensor, ELVEFLOW, Paris, France) can provide 5 measurement ranges, from 70 mBar to 7 Bar. It also allows the detection of ultra-small internal volumes of 7.5 μL, which can be applied to microfluidic chips with a volume of 0.5 mL. The sensor has sensitive flow rate regulation and fast response, so it is suitable for real-time monitoring of small changes in flow rate. The matching sensor reader (Sensor Reader, ELVEFLOW, Paris, France) can provide high-speed capabilities and is easily integrated into the chip, which makes the measurement simple and feasible.

[0242] d) Pressure supply

[0243] A syringe pump (C3657 C Series Syringe Pump, Tricontinent, California, USA) was used as a pressure supplier to feed the reactant materials into the microfluidic chip. The stroke speed of the pump is 1.2 seconds to 100 minutes per stroke, and the resolution of the pump is 3000 steps in standard mode and 24,000 steps in high-resolution mode. The reaction time of the 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 ratio of the reactants to achieve an economical and effective yield of RNA vaccines.

[0244] method

[0245] The key parameters of the reaction that need to be monitored are RNA and Mg2+ concentrations, the pH value of the reaction, and the temperature. At the same time, the inlet pressure needs to be controlled in order to obtain the appropriate proportion of RNA yield and adjust the flow rate of the mixed solution in the continuous flow channel within the appropriate reaction time. There are three detection points on the microfluidic chip, two points for RNA and Mg2+ quantification, and the other point for pH measurement. In addition, the pressure sensor is manufactured outside the chip. 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 common method to achieve the quantification of RNA and Mg2+. UV absorbance measurement at 260nm is the gold standard for RNA quantification, and Mg2+ quantification can be obtained by absorbance measurement at 680nm. The absorbance measurements of RNA and Mg2+ can be converted into concentrations using the Beer-Lambert law. In the design, PDMS is used to make the bioreactor, which is transparent enough. This makes effective RNA detection possible.

[0246] In the reaction, the corresponding thermostable DNA polymerase is the main source of pH. When the pH value is 8.3 to 9.0, the results in the system are optimal. 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 of data collection.

[0247] Comprehensive experimental platform

[0248] 18A to 18C The schematic diagram of the present fluid flow reaction system is shown in FIG. The system includes a plurality of sensors, and the plurality of sensors are suitable for measuring various characteristics of the fluid flowing in the fluid flow system, including pressure, temperature, pH value, flow rate, flow rate, etc. The system also includes a plurality of valves, injection ports, pumps (especially, syringe pumps), gates, etc., to control the flow path of the fluid components in the device. For example, the components of the fluid can be recycled from the outlet port at the longitudinal end of the bioreactor and / or the filtration module to return to the inlet port of the upstream bioreactor and / or the filtration module as required. The system also includes a control unit, which generally includes a CPU, a microprocessor or other suitable electronic processor device. 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 a module, at least one analog-to-digital converter and / or a data source library. These components can be used in combination with sensors, injection ports, pumps, valves, etc. by the control unit to continuously and automatically control the fluid flow in the fluid flow system. Therefore, a continuous reactor system is provided, which is used for the continuous input of reagents to achieve the continuous output of the desired reactant product. The advantage of this system is 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 set up. If necessary, the PDMS chip should be fixed and clamped by the fixture. Then, the positions of other instruments are determined accordingly.

[0249] Initially, four syringe pumps were used to apply pressure to the four input containers, respectively. Tubing and fittings are generally used to deliver small volumes of samples to the PDMS chip. Another pump will be connected to the second container, which is located at the starting point of the second bioreactor (the end of the first TFF), allowing the injection of m7G methyltransferase and the regulation of pressure in the chip. The use of a pressure sensor with a feedback loop will significantly increase the responsiveness of the flow control. Pressure sensors are considered for measurement at the inlet and outlet of the channel. The goal is to keep the syringe pump working while still ensuring a constant pressure in the device.

[0250] To achieve concentration detection, UV / Vis light should be transmitted through the detection chamber, where is the end of the first bioreactor (serpentine channel), 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 the continuous bioreaction, it is planned to put the pH sensor in the buffer to measure the pH value, while the temperature sensor can be placed on the board with a heater on the other side to regulate the temperature. Both the pH sensor and the temperature sensor are equipped with BNC connectors that can be connected to the 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 analyte concentration and the absorbance of light at a specific wavelength:

[0254]

[0255] Among them, 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, in units of M -1 cm -1 , L is the path length, and C is the analyte concentration.

[0256] The expected spectrometer data is therefore exactly the absorption spectrum. Ocean Optics spectrometers have provided an easy way to access the data from Python. This is the Python-Seabreeze package, which wraps the Seabreeze library to communicate with the spectrometer. It provides a fully working and tested reference implementation of Ocean Optics' USB interface, which means that spectrometer data can be read and monitored using python. The connection between Python and the spectrometer is shown in the following example. Fig.19 shown.

[0257] Once the pH sensor is connected with the Arduino via the BNC connector, the sensor data is available and the live data can be checked on the serial monitor. The code for this is in Fig.19 The same mechanism was also used to obtain the temperature sensor data. Five full factorial experiments were conducted, focusing on the effects of different ratios of reagents as well as reaction time and magnesium ion ratio. The experiments were conducted based on a batch feed system with a volume of 500 μL. The background experimental data is shown below. Several conclusions can be drawn from the data. Fig.21Shown are the results from 10 mM NTP, 10 mM NaCl, 75 mM MgCl 2 The maximum yield of the mixture with acetate ion was about 900 mM RNA yield, which can be regarded as the best combination. The optimal reaction time for RNA yield is as follows Fig. 22 As shown. When the ratio of Mg(OAc)2 is about 80mmol, the RNA yield can reach the highest level. After four hours of reaction, the RNA yield reaches a stable level at around 1800mM. The best combination of reaction conditions depends on four main factors: MgCl 2 , NaCl and NTP ratios and reaction time. For other experiments, it is necessary to monitor the MgCl 2 The ratio of , NaCl and NTP was kept at a certain level to obtain maximum RNA yield. The most significant difference between the experiments at Imperial College and Cranfield University is the design of the reaction system. For manufacturing considerations, the continuous follower system was designed for bioreactor processes rather than batch feed reaction systems. To test the continuous flow system, the experiment was designed based on previous experiments conducted by the Shattock Group at Imperial College.

[0258] Three levels of four factors (Table 10) were selected to test the optimal ratio of RNA yield in the continuous flow system. The selected factors refer to the results of the batch feeding system experiment. In addition, due to the recycling reason, the values ​​of DNA and T7 polymerase are the same. Taking these factors into account, 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 trend of the reaction.

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

[0260]

[0261]

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

[0263]

[0264] Results and discussion

[0265] Fluid Mechanics and CFD Analysis

[0266] Appropriate governing equations are applied to the fluid mechanics studies in the bioreactor and TFF sections to calculate the filtrate volume with 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 arbitrary 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 performed at a width (μm) of 1000, a height (μm) of 500, a viscosity (cPa) of 3.5, and a density (g / cm 3 ) was 1.06.

[0268]

[0269] Table 13 Fluid properties at different flow rates. All measurements were made at a width (μm) of 1000, a height (μm) of 500, a viscosity (cPa) of 3.5, and a density (g / cm 3 ) was 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 for different numbers of bends can be obtained in Table 14.

[0272] Table 14 Calculation of the percentage of pressure loss in bends with different numbers of bends

[0273]

[0274] Fluid mechanics study of the TFF section

[0275] The Cerman-Kozeny relationship was used to obtain the permeability constant to solve for the flow parameters through the microporous channel under appropriate boundary conditions. Due to the variation of input particle size in Table 15, different particle diameters and pore sizes were used here.

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

[0277] <![CDATA[Particle diameter, d p (μm)]]> Pore ​​diameter, 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 for a given geometry can be predicted using Equation 10, which has the limitation that it will have mathematical errors if the unit of measurement is microns. The pressure drop over the length of a microporous channel is shown in Table 16.

[0279]

[0280] in,

[0281] μ=dynamic viscosity

[0282] Q = flow rate

[0283] h = height / depth of channel

[0284] K = Permeability

[0285] Table 16 Pressure loss along the length of microporous channel at different flow rates

[0286]

[0287] The results obtained in this study are only predictions and depend on geometry, fluid conditions, and boundary conditions. Based on the membrane selection parameters from the Pall Corporation article, the filtrate volumes can be estimated and are 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 a bioreactor channel

[0292] like Fig.23A and Fig. 23B As shown in the figure, the velocity in the X direction is across the entire bioreactor channel. There are two different colors showing the same velocity, which is between 0.0001ms^-1 and 0.0002ms^-1, because yellow represents the positive direction and green represents the negative direction. At the beginning, Fig. 23B The speed is shown to increase as the pump initially provides a higher pressure, then drops to a constant speed of approximately 0.00033ms^-1, which is approximately 2 times higher than predicted.

[0293] Tangential Flow Filtration Channel

[0294] Fig.24 The speed in the tangential flow filtration channel is shown. The colors show that the different speeds in the microchannel are between 0.0001327ms^-1 and 0.0001858ms^-1, which is close to the inlet speed. In addition, the data in Table 18 show that the outlet speed drops slightly from 0.000145ms^-1 to 0.000138ms^-1, as expected.

[0295] Table 18 Speed ​​size

[0296]

[0297] Isobars in a bioreactor

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

[0299] Experimental Results

[0300] PDMS mask test

[0301] Bioreactor

[0302] Since different versions of the die have been realized through the present work, the evolution of the results has been observed and will be illustrated.

[0303] a) First mold generation

[0304] First, the first version of both bioreactors has been tested. Different sealing options, Kapton tape and second PDMS layer have been tried and the results extracted. Both reactors have been cured at 35°C for more than two days. The two reactors were first sealed with another flat PDMS layer. Green water has been introduced into one of the inlets of each mask thanks to a syringe. Fig.26A and Fig.26B The first results on the fluid behavior for each mask are shown. 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 solidification, as in Fig.26A The blue part can be seen. In addition, Fig.26B The green water sample has passed through the T-junction 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 and collapsed during the curing process.

[0305] Secondly, a continuous flow reactor sealed with Kapton tape was tested. Fig. 27 The behavior of the fluid inside the new setup is shown. The fluid (dark green) follows the path correctly. The seal is better than before. However, due to the size of the channels, curing again does not work properly and some parts of the mask cannot be peeled off the mold properly, resulting in some defective areas in the mask.

[0306] According to all the observations of the different tests, the design has been changed. In addition, it has been decided to create a platform where the PDMS mask can be placed between two acrylic plates, thus increasing the pressure between the two PDMS layers and, therefore, avoiding the collapse of the channels when testing the mask. The two acrylic plates also allow the connection of fittings and tubing to the chip.

[0307] Fig.28 It is shown that the Kapton sealing mask has been placed between the two plates and has been connected with tubing, fittings and some sensing instrumentation to allow the first pH and absorbance testing and measurements to be carried out.

[0308] b) Second mold generation

[0309] The second mold was printed using a high temperature resistant material and the mask was cast at a curing temperature of 70°C for 4 hours. Fig.29 As shown, after the mold is used, the side that is in direct contact with the mold has bonded to the mold. Small parts of the PDMS mask cannot be peeled off and removed from the mask. Especially between the channels, a thin layer of PDMS adheres to the mask. Although the curing works fine, the peeling of the mask from the mold has failed. After trying to clean the mold as much as possible with isopropyl alcohol, another trial 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, which is in direct contact with the high temperature resistant material. The wall may also have an impact on destroying the uniformity of curing 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 followed for the bioreactor mask, and a sample of PDMS poured into a foil container was placed in the oven along with both masks in order to compare different aspects of the process and the results. Fig. 30A and Fig. 30B Aspects of the mask and samples cured in an oven are shown. For the bioreactor mask, the PDMS in contact with the mold has bonded to the mold. When the PDMS is peeled off the mold, the mask has cracked and broken, as shown in Figure 2. Fig. 30A However, the samples cured in foil were cured without any defects and nothing happened when the samples were peeled off the foil. Fig. 30B As shown, the sample has been perfectly cast into the shape of the foil.

[0312] Based on these different observations, some analysis can be done, and the results show that:

[0313] When the samples in the foil work, the chemistry and the protocol 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 for the first experiment should be kept and the curing parameters should be set to cure for 48 hours at ambient temperature to allow the PDMS mask to be cast properly and to avoid any thermal deformation of the mold.

[0316] Despite these results, new platforms were created, e.g. Fig.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. Fig.31 As shown, the PDMS chip is fixed and clamped by a fixture, and the pump and chip are connected by 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 a micro-spectrometer (Ocean Optics USB2000+).

[0318] Absorbance measurement and digital output

[0319] Accurate absorbance measurement

[0320] In order to make accurate absorbance measurements, a baseline needs to be set. First, record the spectrum of the background radiation source without the sample. Second, repeat the experiment by adding the sample in the absorption path. Third, subtract the first spectrum from the second spectrum to obtain a clean absorption spectrum of the sample. In order to eliminate the effect of noise, 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 with the light source turned off or blocked and no sample present. Ref is the reference light intensity.

[0323] UV-Vis Data Analysis

[0324] With the Python-Seabreeze API, it is easy to get real-time data from a UV-Vis spectrometer and plot intensity versus wavelength. Fig.32As shown, when the wavelength is around 580nm, the light intensity reaches a peak. In order to verify the relationship between absorbance and wavelength at different concentrations, vitamin B12 solutions of different concentrations were used, namely A (25mM / L), B (50mM / L), C (75mM / L), and D (100mM / L), see Fig.33 The UV-Vis spectrum results are as follows Fig.34 As shown, the absorbance increases with the increase of sample concentration, and the absorbance reaches a maximum value at a wavelength of 550 nm.

[0325] pH sensor calibration and digital output

[0326] pH sensor calibration

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

[0328] Real-time pH graph

[0329] The next step is to test the effectiveness of the pH sensor using acidic and alkaline solutions. Fig.35 The results in show that the dynamic changes of the real-time graph follow the changes of pH value, which proves its effectiveness.

[0330] discuss

[0331] Key results assessment

[0332] The work and results obtained are an effective attempt to implement a lab-on-a-chip technology for continuous flow reactions with maximum RNA output. Applying the SBCE approach allowed the construction of a complete design framework that was followed from the beginning to the end of the project. The modular design of the device with divided functional subsystems enabled the inclusion of different innovative solutions for mixing, reaction, filtration and purification unit operations. FIG. 30A to FIG. 30C A schematic diagram of an integrated system including downstream modules for formulation is shown. In particular, FIG. 30A to FIG. 30Cis an illustration of an example embodiment of the flow system 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 flow system is attached to a conventional fill and finish vaccine production line.

[0333] Fig.30D shows the synthetic performance of the device, which is FIG. 30A to FIG. 30D A graph showing the relationship between the absorbance of the output solution of the flow reactor and the wavelength according to an embodiment of the present invention. Fig.40A is a graphic representation of the source code for absorbance data and real-time graph (absorbance vs. wavelength) in Python. Fig.40B is an illustration of the source code for real-time graph PH data in Python.

[0334] The product was evaluated using a plot of the UV-Vis spectrum versus a conventional batch protocol, where the output solution of the flow reactor was evaluated using a plot of the UV-Vis spectrum versus a conventional batch protocol. It is envisioned that the present method has significantly higher productivity compared to the batch protocol, as indicated by higher levels of RNA concentration indicated by higher peaks in the graph. Fig.30D The UV-Vis spectra shown compare RNA material obtained using a conventional batch protocol and RNA material obtained using the continuous flow protocol of the present invention. The higher peaks of the flow protocol correspond to higher concentrations of nucleic acids present in solution compared to the batch protocol. The dashed lines correspond to samples of known concentrations of RNA and are included to aid in comparing samples obtained in the batch protocol with samples obtained in the flow protocol.

[0335] Two different design solutions have been developed for this reactor that allow the change of RNA manufacturing from a large 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 because it embodies developed equipment and processes and demonstrates its operation and performance in the rapid scale-up and productive manufacture of RNA-based materials 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 be integrated with a flow reactor, while being easy to manufacture and scale using common manufacturing methods. Although tangential flow filtration is a common technique that has been proven to be effective in RNA purification processes (A. Eon-Duval et al., 2002), the present invention includes a novel filtration device that has been specially designed and constructed. The purpose of the filtration device being developed is to be configured to be used as a continuous flow system, easy to manufacture, capable of being integrated in a continuous flow system, and allowing for scalable and cost-effective manufacture of the device. The present system can be integrated into a microfactory that allows for the automatic and compliant manufacture of therapeutic nucleic acid-based materials at the point of use. In addition, the modular design allows the use of the filtration system to recycle certain components (such as enzymes and plasmid DNA), thereby significantly reducing the total cost of the produced material.

[0337] Regarding the entire system, a new integrated platform has been developed for testing and experimentation. The system has been prototyped using an acrylic pressure plate, and the system also includes all ports for pumps, sensors, and fittings for inlets and outlets. The system allows testing of continuous flow reactions or filtrations while obtaining instant feedback from different elements and stages of each process. In addition, the system has been prototyped in a modular manner, which allows easy use of the framework for running experiments. 3D printing has become the tool of choice for making molds due to its high flexibility, low cost, and short lead time. Using techniques such as micromachining on glass, metal (such as stainless steel) and polymethyl methacrylate PMMA (acrylic) substrates, as well as photolithography and deep reactive ion etching on substrates including photosensitive glass and silicon, the construction of the module can be easily transferred to commercial manufacturing. In addition, for large-scale manufacturing of 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 online and in-situ analysis of the contents of the reaction solution in real time. This also provides a closed feedback loop to direct the fluid flow and adjust the temperature, pH and distribution of the reactants in the flow system to maintain process conditions throughout the process.

[0339] According to the implementation methods 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 into the first fluid flow module via a plurality of inlet ports, the plurality of reactants comprising: at least one nucleoside triphosphate (NTP), a reaction buffer, and a DNA, a DNA-based compound, or a DNA-based mixture;

[0342] allowing at least some of the reactants to react within a reaction channel or well within a first module of the flow system;

[0343] retaining the DNA at the first reactor module or recycling the DNA at the first reactor module and allowing the reaction products of the reactants to flow to the first fluid filtration module; and

[0344] The reaction product is filtered in a 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) 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), and uridine triphosphate (UTP).

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

[0348] (5) According to the method described in any of the preceding items, the multiple reactants also include any one of an enzyme mixture, a salt solution, and an RNA polymerase, or a combination of an enzyme mixture, a salt solution, and an RNA polymerase.

[0349] (6) The method according to any 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 area of ​​the first reactor module.

[0350] (7) The method according to any of the preceding items, comprising: transferring 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 area of ​​the first reactor module, and recycling any unreacted capping enzyme to the inlet area of ​​the second reactor module.

[0353] (10) The method according to any of the preceding items dependent on (5), wherein the salt solution comprises MgCl 2 .

[0354] (11) The method according to any of the preceding items dependent on (5), wherein the RNA polymerase comprises T7 polymerase.

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

[0356] (13) Use of RNA or an 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 filtering device comprising:

[0358] a first elongated fluid flow channel having an inlet;

[0359] a second elongated fluid flow channel having an outlet;

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

[0361] (15) The apparatus of (14), wherein a majority of the length of the first channel is positioned adjacent to a majority 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, in the range of 200 kDa to 600 kDa, in the range of 300 kDa to 10 MDa.

[0363] (17) The apparatus 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 respective opposing surfaces of the first plate and the second plate.

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

[0365] (19) The apparatus according to (18), wherein the first channel and the second channel include respective serpentine profiles in their longitudinal directions.

[0366] (20) An apparatus 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, which partially defines the longitudinal walls or faces 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 treating 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 area, at least one inlet disposed in fluid communication with an outlet of the first reactor module, and at least one outlet;

[0371] Wherein, the first filter module comprises the fluid flow filter device according to any one of (13) to (21).

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

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

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

[0375] (26) The system according to any one of (22) to (25), comprising a first recirculation conduit extending between the 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 the area of ​​the outlet of the first filtration module and the outlet of the first reactor module.

[0377] (28) The system of (27) as appended to (24), comprising a third recirculation conduit extending between the region of the outlet of the second filtration module and the inlet of the first reaction module.

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

[0379] driving a fluid from an inlet through a first elongated fluid flow channel;

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

[0381] retaining a retentate component of the fluid within the first passage;

[0382] wherein the membrane is positioned to separate the first channel from the second channel along their respective lengths such that permeate can pass from the first channel through the membrane into the second channel along their respective lengths.

[0383] (30) The method according to (29), wherein the step of driving the fluid includes: pressurizing the fluid in 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 actuator for driving a fluid flow through the channel or the well;

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

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

[0389] a reaction state monitoring device for monitoring a property of a fluid within the system, the property indicating a 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 property of the fluid within the system.

[0391] (32) The system according to (31), wherein the characteristic of the system is any one 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] pH of the fluid within the system;

[0395] ● the volume or ratio 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, or a handheld electronic device.

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

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

[0400] introducing at least one fluid into the reactor module via at least one inlet;

[0401] Using at least one flow actuator to drive the fluid through a reaction flow channel or a well or using at least one flow actuator 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 pressure, temperature, or pH of a fluid within the fluid flow device or a combination of pressure, temperature, or pH of a fluid within the fluid flow device;

[0403] monitoring a reaction state of chemical components within a fluid within the fluid flow device; and

[0404] 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 a reaction state of a chemical component 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 a reaction state of a chemical component of the fluid, at least one property of the fluid within the fluid flow device is controlled using a control unit.

[0405] (36) The system according to (35) includes a plurality of reactor modules and filtration modules coupled together in fluid communication.

[0406] (37) The system of (35), wherein the flow drive is at least one pump, and optionally, the flow drive is a syringe pump.

[0407] (38) The system of (35), wherein the fluid analysis sensor comprises 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, include: introducing a plurality of reactants into the first fluid flow module via a plurality of inlet ports, the plurality of reactants comprising: at least one nucleoside triphosphate (NTP), a reaction buffer, and a 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 recycling the DNA at the first reactor module and allowing the reaction products of the reactants to flow to the first fluid filtration module; and The reaction product is filtered in a first filtration module.

2. The method according to claim 1, in, 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, in, 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), and uridine triphosphate (UTP).

4. A method according to any preceding claim, in, The DNA is plasmid DNA.

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

6. A method according to any preceding claim, include: At least some of the plurality of reactants are recycled from the outlet of the first filtration module to the inlet region of the first reactor module.

7. A method according to any preceding claim, include: At least some of the filtered reaction product is transferred from the first filtration module to a second fluid flow reactor module, and a capping enzyme is input to the second reactor module.

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

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

10. A method according to any preceding claim as dependent upon claim 5, in, The salt solution includes MgCl 2 .

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