Systems and methods for producing RNA

Abstract

The present application relates to a system for producing RNA comprising an in vitro transcription (IVT) unit and a treatment unit for downstream treatment of the compound wherein the IVT unit comprises a plurality of chambers wherein each chamber of the plurality of chambers is configured to receive one or more IVT reagents and to conduct an IVT reaction according to a DNA template, thereby obtaining a plurality of RNA molecules; and wherein the processing unit comprises a purification device for carrying out at least one downstream purification step of the plurality of RNA molecules, the purification device comprising one or more sample holders adapted to receive a container containing a liquid medium comprising RNA molecules and magnetic particles, and wherein a magnet is located at each sample, the magnet unit is configured to capture the magnetic particles or to direct movement of the magnetic particles, and wherein the sample holder is configured to perform mechanical movement such that the magnetic particles are mixed with the liquid. The present application also discloses a method for producing RNA, more preferably mRNA.

Description

Technical Field

[0001] The present application relates to a system and method for producing RNA, which enables efficient large-scale RNA production for further downstream applications, such as the production of RNA vaccines. Background Art

[0002] Therapeutic nucleic acids, including RNA molecules, represent an emerging class of drugs. RNA-based therapies include mRNA molecules encoding antigens used as vaccines. In addition, RNA molecules are also envisioned for replacement therapy, such as providing patients with missing proteins, such as growth factors or enzymes. In addition, RNA molecules are also considered for therapeutic use in genome editing (e.g., CRISPR / Cas9 guide RNA). Therefore, RNA-based therapies for immunotherapy, gene therapy, and vaccination are among the most promising and fastest-growing therapeutic areas in modern medicine.

[0003] The currently established RNA molecule manufacturing process implements many separate manufacturing steps. In particular, each manufacturing step is performed by several different devices. In addition, the manufacture of RNA requires a lot of manual work by well-trained technicians in GMP-regulated laboratories. Therefore, the currently established manufacturing process is time-consuming, costly, and requires a lot of laboratory space and laboratory equipment.

[0004] Automated analyzer systems are known from US9372156B2, CN211402409 and CN113504380, however, these systems are not suitable for in vitro transcription. A system for IVT using 2D purification of mRNA is known from WO2021030271. However, 2D purification has major drawbacks, and alternative purification methods using magnetic particles, such as those disclosed in WO2016077294A1 and US2013302810A, can circumvent these problems.

[0005] The present invention provides solutions to at least some of these problems by providing an efficient automated RNA production and purification system. Summary of the invention

[0006] The present disclosure and its embodiments are intended to provide solutions to one or more of the above-mentioned shortcomings. To this end, the present disclosure provides an automated system and method for producing RNA. The system and method allow rapid, efficient and automated mass production of RNA that meets the quality requirements for therapeutic use. Conventionally, in order to manage the production of a large number of therapeutic products (such as RNA), a large number of large instruments are required. However, the compactness of the design and the number of supporting resources have become an important issue. The COVID-19 epidemic and the ensuing shortage of COVID-19 RNA vaccines show that for the production of RNA and RNA-based therapies, mobile, easy-to-transport and easy-to-operate solutions are highly needed.

[0007] The footprint of the current system is kept low by combining certain steps in an efficient and clever way, thereby making optimal use of the available space. As a result, the system can be easily transported, even to remote areas. Furthermore, due to the highly automated approach, the system is easy to operate and does not require highly trained or skilled staff. The present method and system (almost) eliminates the need for manual handling, thus also significantly reducing the risk of contamination and the risk of degradation of the final product. Due to the (semi-)continuous process, large amounts of RNA can be produced in a short time. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 A schematic diagram of a system according to an embodiment of the present disclosure is shown.

[0009] Figure 2 A schematic diagram of an IVT unit according to an embodiment of the present disclosure is shown.

[0010] Figure 3A and 3B A schematic diagram of a purification device according to an embodiment of the present disclosure is shown.

[0011] FIG. 4 shows the principle of capturing magnetic beads using a magnetic unit. Figure 4A The middle bead is in a free state. Figure 4B The middle bead is captured.

[0012] Figure 5 A schematic diagram of a sample holder according to an embodiment of the present disclosure is shown.

[0013] Figure 6 A schematic diagram of a sample holder according to another embodiment of the present disclosure is shown.

[0014] Figure 7 A schematic diagram of an embodiment of a downstream processing unit with two adjacent purification devices is shown.

[0015] Figure 8 A schematic diagram of an embodiment of a downstream processing unit having two nested devices is shown.

[0016] Fig. 9 A robotic arm according to an embodiment of the present disclosure is schematically shown.

[0017] Fig.10 A schematic diagram of a cabinet according to one embodiment of the present disclosure is shown.

[0018] Fig.11 A schematic diagram of a cabinet adapted in a transport container is shown according to one embodiment of the present disclosure. DETAILED DESCRIPTION

[0019] US2021 / 0261897 describes a bioreactor for in vitro transcription. However, the system is only suitable for the first step of the RNA production chain and does not provide a fully integrated system for RNA production. In addition, the bioreactor described here cannot (semi-)continuously produce RNA.

[0020] The present disclosure aims to address at least some of the above-mentioned problems and disadvantages.In addition, the present disclosure also describes systems and methods for large-scale, (semi-)continuous RNA production.

[0021] definition

[0022] Unless otherwise defined, all terms (including technical and scientific terms) used have the meanings commonly understood by those of ordinary skill in the art. By way of further guidance, terminology modifiers are included in this application to better understand the teachings of this application.

[0023] As used herein, the following terms have the following meanings:

[0024] Unless the context clearly indicates otherwise, "a", "an" and "the" as used herein refer to both singular and plural referents. For example, "a compartment" refers to one or more than one compartment.

[0025] As used herein, "about" refers to a measurable value, such as a parameter, quantity, duration, etc., and is intended to include variations of + / -20% or less, preferably + / -10% or less, more preferably + / -5% or less, even more preferably + / -1% or less, and still more preferably + / -0.1% or less from the specific value, as long as these variations are suitable for implementation in the disclosed application. However, it should be understood that the value referred to by the modifier "about" itself is also specifically disclosed.

[0026] As used herein, "comprising" is synonymous with "including" and is an inclusive or open term that specifies the presence of subsequent contents (e.g., components) but does not exclude the presence of other additional, unlisted components, features, elements, elements, steps known or disclosed in the art.

[0027] In addition, the terms first, second, third, etc. in the specification and claims are used to distinguish similar elements, and are not necessarily used to describe a sequential or chronological order unless otherwise indicated. It should be understood that the terms used in this way can be used interchangeably under appropriate conditions, and the embodiments described herein are capable of operating in a sequence different from that described or illustrated herein.

[0028] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

[0029] Unless otherwise defined, the expressions "weight %", "weight percent", "% wt" or "wt %" herein and throughout the specification refer to the relative weight of the corresponding component based on the total weight of the formulation.

[0030] As used herein, the term "magnetic particle" and variations thereof are intended to mean a particle having a magnetic (eg, paramagnetic or superparamagnetic) core coated with at least one material having a surface capable of reversibly binding to a target compound.

[0031] Although the term "one or more" or "at least one", such as one or more or at least one of a group of elements, is clear in itself, by further example, the term specifically includes reference to any one of the elements, or to any two or more of the elements, such as any ≥3, ≥4, ≥5, ≥6 or ≥7, etc., of the elements, as well as to all the elements.

[0032] Unless otherwise defined, all terms used (including technical terms and scientific terms) have the meanings commonly understood by those of ordinary skill in the art. By way of further guidance, the present application includes definitions of the terms used in the specification to better understand the teachings of the present application. The terms or definitions used herein are only used to help understand the present application.

[0033] "In one embodiment" and "in one embodiment" mentioned throughout this specification refer to specific features, structures or characteristics related to the embodiment that are included in at least one embodiment of the present application. Therefore, the phrases "in one embodiment" and "in one embodiment" that appear in various places throughout this specification do not necessarily refer to the same embodiment, but may do so. In addition, as will be apparent to those skilled in the art in this disclosure, in one or more embodiments, specific features, structures or characteristics may be combined in any suitable manner. In addition, those skilled in the art will understand that although some embodiments described herein include some features included in other embodiments and do not include other features, the combination of features of different embodiments should also fall within the scope of this application and form different embodiments. For example, in the appended claims, any claimed embodiments may be used in any combination.

[0034] System and components of said system

[0035] In the first aspect, the present disclosure discusses a system for producing RNA. The term RNA or RNA molecule covers long-chain RNA, coding RNA, non-coding RNA, long non-coding RNA, single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), linear RNA (linRNA), circular RNA (circRNA), messenger RNA (mRNA), self-amplification mRNA (SAM), trans-amplification mRNA, RNA oligonucleotides, antisense oligonucleotides, small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA (asRNA), CRISPR / Cas9 guide RNA, riboswitch, immunostimulatory RNA (isRNA), ribozyme, aptamer, ribosomal RNA (rRNA), transfer RNA (tRNA), viral RNA (vRNA), retroviral RNA or replicon RNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA) and Piwi interaction RNA (piRNA). In some embodiments, the RNA comprises a modified RNA molecule. In some embodiments, the modification of the RNA molecule includes chemical modification, and the chemical modification includes backbone modification and sugar modification or base modification. In this article, the RNA molecule of modification defined herein comprises nucleotide analogs / modifications, such as backbone modifications, sugar modifications or base modifications. The backbone modification associated with the present disclosure is a modification in which the phosphate of the nucleotide backbone contained in the RNA molecule is chemically modified. The sugar modification associated with the present disclosure is a chemical modification of the sugar of the nucleotide of the RNA molecule. In addition, the base modification associated with the present disclosure is a chemical modification of the base portion of the nucleotide of the RNA molecule. In this article, nucleotide analogs or modifications are selected from nucleotide analogs suitable for transcription and / or translation. In a further embodiment, the modified RNA includes a nucleoside modification selected from the group consisting of 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudoisocytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxyuridine, deoxy-thymidine, 5-methyl-uridine, pyrrolocytidine, inosine, α-thio-guanosine, 6-methyl-guanosine, 5-methylcytosine, 8-oxoguanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudoisocytidine, 6-chloropurine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, and 7-deaza-adenosine.

[0036] In one embodiment, a system for producing RNA is disclosed, the system comprising an in vitro transcription (IVT) unit and a processing unit for downstream processing of the RNA molecules, wherein the IVT unit comprises a plurality of chambers, wherein each of the plurality of chambers is configured to receive one or more IVT reagents and perform an IVT reaction according to a DNA template, thereby obtaining a plurality of RNA molecules; and wherein the processing unit comprises a purification device for performing at least one downstream purification step of the plurality of RNA molecules, the purification device comprising one or more sample holders, the sample holders being adapted to receive containers containing a liquid medium containing RNA molecules and magnetic particles, and wherein a magnet unit is located at each sample holder, the magnet unit being configured to capture or guide the movement of the magnetic particles, and wherein the sample container is configured to perform mechanical movement so that the magnetic particles are mixed with the liquid. Advantageously, the system disclosed herein allows automation of IVT of RNA molecules and downstream purification of the RNA molecules, while performing the procedure continuously and with high precision without manual intervention. Since the device simulates small-scale manual operations performed in a laboratory, the production protocol can be directly performed on the system disclosed herein without any process scale amplification.

[0037] The IVT unit is to be understood as a part of the system that accommodates the IVT reaction initiated from a DNA template by a DNA-dependent RNA polymerase. In some embodiments, the DNA-dependent RNA polymerase comprises at least one of T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, RNA polymerase V, and a single-subunit RNA polymerase. The DNA template for RNA in vitro RNA transcription can be obtained by cloning nucleic acids, in particular cDNAs corresponding to the respective RNAs to be transcribed in vitro, and introducing them into a suitable vector for RNA in vitro transcription, such as a circular plasmid DNA in a plasmid. The cDNA can be obtained by reverse transcription or chemical synthesis of mRNA. In addition, the DNA template for in vitro RNA synthesis can also be obtained by gene synthesis.

[0038] In some embodiments, the DNA template relates to a DNA molecule comprising a nucleic acid sequence encoding an RNA sequence. The template DNA is used as a template for RNA in vitro transcription to produce RNA encoded by the template DNA. Therefore, the template DNA comprises all elements required for RNA in vitro transcription, particularly promoter elements for binding to DNA-dependent RNA polymerases (e.g., T3, T7, and SP6 RNA polymerases), which are located at the 5' end of the DNA sequence encoding the target RNA sequence. The poly (A) tail can be encoded into the DNA template, or can be enzymatically added to the RNA in a separate step after in vitro transcription. In some embodiments, the template DNA comprises primer binding sites at the 5' end and / or 3' end of the DNA sequence encoding the target RNA sequence, so as to determine the identity of the DNA sequence encoding the target RNA sequence by means of, for example, PCR or DNA sequencing. In some embodiments, the DNA template comprises a DNA vector, such as a plasmid DNA, which comprises a nucleic acid sequence encoding an RNA sequence. In some embodiments, the DNA template comprises a linear or circular DNA molecule.

[0039] In some embodiments, the chambers of the IVT unit are disposed within a cartridge. In further embodiments, the cartridge contains 2 to 48 chambers.

[0040] In some embodiments, the cartridge comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 chambers. In some embodiments, the barrel comprises at least about 60 chambers, about 70 chambers, about 80 chambers, about 90 chambers, about 100 chambers, about 110 chambers, about 120 chambers, about 130 chambers, about 140 chambers, about 150 chambers, about 160 chambers, about 170 chambers, about 180 chambers, about 190 chambers, or about 200 chambers.

[0041] In one embodiment, the IVT unit comprises 1 cartridge, 2 cartridges, 3 cartridges, 4 cartridges, 5 cartridges, 6 cartridges, 7 cartridges, 8 cartridges, 9 cartridges, or 10 cartridges. In another embodiment, the IVT unit comprises 1 to 10 cartridges, 1 to 20 cartridges, 1 to 30 cartridges, or 1 to 40 cartridges, 1 to 50 cartridges, 1 to 60 cartridges, 1 to 70 cartridges, 1 to 80 cartridges, 1 to 90 cartridges, or 1 to 100 cartridges.

[0042] In one embodiment, the chamber of the IVT unit comprises at least three planar polygonal surfaces. In another embodiment, the chamber comprises one, two, three, four, five, six, seven or eight planar polygonal surfaces, preferably six planar polygonal surfaces. Advantageously, the design of the chamber allows it to be equipped with sensors and / or probes.

[0043] In one embodiment, the cross section of the chamber is a polygon, such as a triangle, quadrilateral, pentagon or hexagon. In a preferred embodiment, the cross section of the chamber is a hexagon. A plurality of hexagonal chambers (e.g., combined in a barrel) can form a honeycomb structure. Without wishing to be bound by theory, it is known that in geometry the hexagonal shape is best suited for filling a plane with equal-sized units without leaving unused space. In addition, since the hexagonal filling has a 120-degree angle, the perimeter of a given area is minimized, so the hexagonal chamber ensures that less raw materials are used.

[0044] In one embodiment, the open end of chamber is configured to receive a detachable cover to at least partially seal the open end. The cover prevents unwanted components from entering the chamber (e.g., RNase, microbial contamination or other degradation compounds or organisms), and separates the contents of the chamber from the external environment. In one embodiment, the cover also prevents the contents of the chamber from evaporating. In some embodiments, chamber comprises a cover to limit the exchange with the environment. In some embodiments, the cover of chamber is detachable. In some embodiments, the cover of chamber is non-detachable. Alternatively, chamber is not covered.

[0045] In some embodiments, the cover can prevent excessive evaporation of water and loss of other key volatile components. In some embodiments, the cover can prevent oxidation of reagents or any components. In some embodiments, the cover can provide light protection (if necessary). In some embodiments, the cover prevents contamination of any other potential chemical compounds.

[0046] In some embodiments, the cover comprises at least one opening for filling, draining and sampling. In some embodiments, at least one opening is located at the top of the cover.

[0047] In some embodiments, the bottom of the chamber is rounded. In other embodiments, the bottom of the chamber is flat or pointed.

[0048] In some embodiments, the chamber is removable from the cartridge. In some embodiments, the chamber is not removable from the cartridge.

[0049] A system according to any preceding claim, wherein the volume of the chamber is about 0.1 mL to 500 mL.

[0050] In some embodiments, the volume of the chamber is at least about 0.1 ml, about 0.3 ml, about 0.5 ml, about 1 ml, about 1.5 ml, about 2 ml, about 2.5 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, about 10 ml, about 12 ml, about 15 ml, about 17 ml, about 20 ml, about 25 ml, about 30 ml, about 35 ml, about 40 ml, about 45 ml, about 50 ml, about 55 ml, about 60 ml, about 65 ml, about 70 ml, about 75 ml, about 80 ml, about 85 ml, about 90 ml, about 95 ml or about 100 ml. In some embodiments, the volume of the chamber is no more than about 100 ml, no more than about 95 ml, no more than about 90 ml, no more than about 85 ml, no more than about 80 ml, no more than about 75 ml, no more than about 70 ml, no more than about 65 ml, no more than about 60 ml, no more than about 55 ml, no more than about 50 ml, no more than about 45 ml, no more than about 40 ml, no more than about 35 ml, no more than about 30 ml, no more than about 25 ml, no more than about 20 ml, no more than about 15 ml, no more than about 10 ml, no more than about 9 ml, no more than about 8 ml, no more than about 7 ml, no more than about 6 ml, no more than about 5 ml, no more than about 4 ml, no more than about 3 ml, no more than about 2 ml, no more than about 1 ml, no more than about 0.5 ml, no more than about 0.3 ml, or no more than about 0.1 ml. In some embodiments, the volume of the chamber is about 1 ml to about 100 ml, about 10 ml to 90 ml, about 15 ml to about 80 ml, about 20 ml to about 70 ml, about 25 ml to about 60 ml, or about 30 ml to about 50 ml.

[0051] In some embodiments, the volume of the chamber is at least about 150 ml, about 200 ml, about 250 ml, about 300 ml, about 350 ml, about 400 ml, about 450 ml, about 500 ml, about 550 ml, about 600 ml, about 650 ml, about 700 ml, about 750 ml, about 800 ml, about 850 ml, about 900 ml, about 950 ml, about 1000 ml, about 2000ml, about 3000ml, about 4000ml, about 5000ml, about 6000ml, about 7000ml, about 8000ml, about 9000ml, about 10000ml, about 15000ml, about 20000ml, about 25000ml, about 30000ml, about 35000ml, about 40000ml, about 45000ml or about 50000ml. In some embodiments, the volume of the chamber is no more than about 50,000 ml, no more than about 45,000 ml, no more than about 40,000 ml, no more than about 35,000 ml, no more than about 30,000 ml, no more than about 25,000 ml, no more than about 20,000 ml, no more than about 15,000 ml, no more than about 10,000 ml, no more than about 9,000 ml, no more than about 8,000 ml, no more than about 7,000 ml, no more than about 6,000 ml, no more than about 5,000 ml, no more than about 4,000 ml, no more than about 300,000 ml, 0ml, no more than about 2000ml, no more than about 1000ml, no more than about 950ml, no more than about 900ml, no more than about 850ml, no more than about 800ml, no more than about 750ml, no more than about 700ml, no more than about 650ml, no more than about 600ml, no more than about 550ml, no more than about 500ml, no more than about 450ml, no more than about 400ml, no more than about 350ml, no more than about 300ml, no more than about 250ml, no more than about 200ml, no more than about 150ml. In some embodiments, the volume of the chamber is about 150 ml to about 50,000 ml, about 200 ml to about 45,000 ml, about 250 ml to about 40,000 ml, about 300 ml to about 35,000 ml, about 350 ml to about 30,000 ml, about 400 ml to about 25,000 ml, about 450 ml to about 20,000 ml, about 500 ml to about 15,000 ml, about 550 ml to about 10,000 ml, about 600 ml to about 9,000 ml, about 650 ml to 8,000 ml, about 700 ml to about 7,000 ml, about 750 ml to about 6,000 ml, about 800 ml to about 5,000 ml, about 850 ml to about 4,000 ml, about 900 ml to about 3,000 ml, about 950 ml to about 2,000 ml, or about 1000 ml to about 1500 ml.

[0052] In some embodiments, the IVT unit comprises at least one carrier configured to dispense and / or remove reagents, reagent mixtures, or liquids in the chamber. Without limitation, the liquid dispensed and / or removed can be deionized water, purified water, a buffer, a cleaning solution, a reagent, an enzyme, a DNA template, a transcription buffer, a solution containing nucleoside triphosphates (NTPs), an RNase inhibitor, or an RNA polymerase, or a combination thereof or waste. Non-limiting examples of buffers are magnesium acetate, HEPES, Tris, or Tris HCl. In a preferred embodiment, the liquid dispensed and / or removed is an IVT premix comprising water, an RNA polymerase buffer, a linear DNA template, and dNTPs. In a further preferred embodiment, the IVT premix comprises water, a T7 polymerase buffer, a linear DNA template, and dNTPs.

[0053] The carrier device of the IVT unit can be a syringe or a mechanical arm equipped with one or more nozzles, needles and / or tips. Any syringe and mechanical arm known in the art and capable of dispensing and / or removing components or liquids can be used with the system disclosed herein. Non-limiting examples of carrier devices are syringe pumps, vacuum pumps, peristaltic pumps, centrifugal pumps or combinations thereof. In some embodiments, a pipette, micropipette, needle or tip is detachable and / or disposable. In other embodiments, a pipette, micropipette, needle or tip is non-detachable.

[0054] Alternatively, the distribution and / or removal of the components is carried out by the carrier directly through a conduit with which it is equipped.

[0055] In one embodiment, the IVT unit disclosed herein comprises a robotic arm. In another embodiment, the IVT unit comprises one or more syringes. In yet another embodiment, the IVT unit comprises one or more pumps. In yet another embodiment, the IVT unit comprises a robotic arm and one or more syringes. In yet another embodiment, the IVT unit comprises a robotic arm and one or more pumps. In a preferred embodiment, the IVT unit comprises a robotic arm, one or more syringes, and one or more pumps.

[0056] The vehicle of the IVT unit is configured to move. In some embodiments of the IVT unit, the movement is vertical, horizontal, centrifugal or 3D movement. In one embodiment, the vehicle is controlled by a motor unit, preferably an electric motor.

[0057] In some embodiments, the IVT unit comprises a control system arranged and adapted to control the dispensing and / or removal of reagents, reagent mixtures or liquids by the vehicle. Based on input information, such as sensor data and predefined algorithms, the control system adjusts the performance of the vehicle.

[0058] In one embodiment of the IVT unit, the delivery device is adapted to dispense an amount of the IVT premix and / or one or more enzymes in said chamber.

[0059] In one embodiment, the system comprises a storage unit for storing one or more reagents, and the storage unit can be cooled to a temperature below 10° C. In one embodiment, the storage unit stores one reagent, two reagents, three reagents, four reagents, five reagents, six reagents, seven reagents, eight reagents, nine reagents or ten reagents, preferably four reagents. The reagents can be selected from water, T7 reaction buffer, linear DNA template and NTP, but are not limited to these.

[0060] In some embodiments, the storage unit may be cooled to less than 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, 4°C, 3°C or 1°C, with a preferred cooled storage temperature of 4°C.

[0061] The storage unit is fluidly connected to a pump system, such as a peristaltic pump or a syringe pump. In other embodiments, the pump comprises a vacuum pump, a peristaltic pump, a centrifugal pump, or a combination thereof.

[0062] In one embodiment, the fluid connection is carried out through at least one pipeline. In some embodiments, the pipeline includes and is preferably made of a material selected from the group consisting of polyethylene (PE), nylon, polyurethane, copper, stainless steel, aluminum, polyvinyl chloride (PVC), polypropylene (PP), polyurethane (PU), vinyl, polyvinylidene fluoride (PVDF), fiberglass, glass, rubber and combinations thereof. In some embodiments, the pipeline has chemical resistance.

[0063] In one embodiment, the pump system is configured to provide the IVT premix container with the desired amount of one or more reagents for preparing the IVT premix.

[0064] In one embodiment, the system disclosed herein comprises a heating device for heating the IVT premix container. In one embodiment, the heating temperature of the IVT premix container is 35°C to 55°C, preferably 35°C to 50°C, 35°C to 45°C, 35°C to 40°C, or 35°C to 37°C. Alternatively, the heating temperature of the IVT premix container is 37°C to 55°C, preferably 40°C and 45°C to 55°C, or 40°C to 55°C. In a preferred embodiment, the heating temperature of the premix container is 37°C.

[0065] In some embodiments, the system comprises an intermediate vessel for collecting the contents of a final IVT reaction from multiple chambers. In some embodiments, the intermediate storage container is in fluid communication with the chamber. In other embodiments, the intermediate storage container is not in fluid communication with the chamber. In some embodiments, the intermediate storage container is configured to store RNA molecules produced by IVT. In some embodiments, the intermediate storage container is configured to detect contamination of RNA molecules. In some embodiments, the intermediate storage container comprises a device for performing some chemical, physical, or mechanical treatment of the RNA molecules and / or unwanted residues - for example, binding products, partially eliminating residues, or adjusting pH.

[0066] In one embodiment, the purification device of the processing unit disclosed herein comprises a sample plate, wherein the sample plate comprises a base portion, and the base portion is provided with a sample holder. The purification device separates and / or purifies RNA molecules produced by the IVT reaction, thereby performing purification of downstream RNA molecules in an automated manner.

[0067] In one embodiment, each sample holder of the purification device is configured to be mechanically moved independently of the remaining sample holders. In this way, a different step of the separation and / or purification process is performed in each sample holder. In other embodiments, the sample holders are moved simultaneously.

[0068] In one embodiment, the mechanical movement of the sample holder is driven by a motor unit or an electromagnetic unit. In a preferred embodiment, a motor is used. In another embodiment, a plurality of electromagnets are energized in sequence to produce the movement of the sample holder. In another embodiment, the mechanical movement of the sample holder is driven by an oscillator unit. The mechanical movement of the sample holder can be a rotation or shaking or vibration movement around the axis of the sample holder. The mechanical movement of the sample holder can be activated and turned off. The purpose of the mechanical movement includes, but is not limited to: homogenizing the mixture formed by the sample and the buffer for separation and / or purification, and / or promoting contact between the magnetic particles and the sample.

[0069] In one embodiment, the sample holder may be located on top of a sample plate or in a groove or recess in the sample plate.

[0070] In another embodiment, the purification device does not contain a sample plate, but comprises one or more sample holders present in the system.

[0071] In one embodiment, the magnet unit of each sample holder comprises a permanent magnet, a temporary magnet or an electromagnet, preferably a permanent magnet. When an electromagnet is used, the magnetic field can be quickly changed by controlling the amount of current. In another embodiment, each magnet unit comprises a magnet array, wherein each magnet can be a permanent magnet, a temporary magnet or an electromagnet.

[0072] In a preferred embodiment, the magnet unit is arranged along the side of the sample holder and extends above the sample holder. In another embodiment, the magnet unit is arranged around the sample holder.

[0073] In another embodiment, the sample holder is equipped with at least two, three, four, five, six, seven, eight, nine or ten magnet units. In one embodiment, the magnet units are arranged along different sides of the sample holder. Alternatively, all magnets are arranged along the same side of the sample holder.

[0074] In one embodiment, the length of the magnet unit is the same as the length of the sample holder, or is shorter than the length of the sample holder. In a particularly preferred embodiment, the magnet unit is rod-shaped, and at least 1 / 5 of the length of the magnet unit extends above the sample holder. In another embodiment, at least 1 / 4, 1 / 3, 1 / 2, 2 / 5, 2 / 4, 2 / 3, 2 / 1, 3 / 5, 3 / 4, 3 / 2, 3 / 1, 4 / 5, 4 / 3, 4 / 2, 4 / 1, 5 / 4, 5 / 3, 5 / 2 or 5 / 1 of the length of the magnet unit extends above the sample holder. In a preferred embodiment, the height of the magnet unit is directly proportional to the liquid level in the sample container. In a preferred embodiment, the height and thickness of the magnet unit are directly proportional to the surface of the sample container to ensure that there is a sufficiently large magnetic field to capture magnetic particles. In another embodiment, the magnet unit extends at least to the entire length of the sample container, preferably extends to the bottom of the sample container.

[0075] In yet another embodiment, the magnet is in the shape of a bar, a horseshoe, a disk, a sphere, a cylinder, or a ring.

[0076] In one embodiment, the magnet unit is arranged in a housing, which is preferably open on the side facing the sample holder. Alternatively, the housing is located around the sample holder and contains a plurality of magnets. In another embodiment, the magnet unit is annular and completely surrounds the sample holder.

[0077] The housing may be made of any suitable material known in the art, such as, but not limited to, a polymer, a thermoplastic, a metal, or a metal alloy.

[0078] The position of the magnet unit can be fixed or mobile. For example, the magnet unit can move vertically or laterally. These movements can affect whether the magnetic particles will be attracted to the magnet. If the position is too far away, the magnetic particles will remain in the liquid.

[0079] In one embodiment, an adapter is present in the sample holder to adjust the size of the sample holder. The use of adapters allows the device to be used with sample containers of various shapes and sizes. The adapter can be made of any suitable material known in the art, such as PC (polycarbonate), PP (polypropylene), PAI (polyamideimide) (e.g., Torlon), PI (polyimide) (e.g., Tecasint), PPS (polyphenylene sulfide) (e.g., Tecatron), PPSU (polyphenylene sulfone) (e.g., Tecason P), PSU (polysulfone) (e.g., Tecason S), PEI (polyetherimide) (e.g., Tecapei), glass (e.g., borosilicate glass), technical ceramics (e.g., ), polyaryletherketones (e.g. polyetheretherketone (PEEK)), thermoplastics (e.g. Pa or GF), metal or metal alloy.

[0080] The sample plate of the purification device disclosed herein is configured to rotate around an axis, preferably around the central axis of the base portion. In a preferred embodiment, the sample plate is configured to rotate clockwise and counterclockwise. In another embodiment, the sample plate is configured to perform linear motion. The rotation of the sample plate is driven by a motor unit, preferably by a motor. The rotation of the sample plate transports the sample container located in the sample holder to different fixed distributors, wherein components or liquids can be added or removed from the sample container.

[0081] In another embodiment, the sample plate of the purification device disclosed herein is fixed or immovable and cannot rotate about an axis.

[0082] The base portion of the sample plate can be rectangular, polygonal, circular, oval or annular. In one embodiment, the sample plate is circular with a diameter of 20 cm to 50 cm, more preferably 20 cm to 40 cm, such as 35 cm or 30 cm. In another embodiment, the sample plate is rectangular. The sample plate can be made of any suitable material known in the art, such as a polymer, a metal, a metal alloy, a resin or any non-magnetic or paramagnetic material. Polymers include but are not limited to polystyrene, PVC, Perspex or Lucite.

[0083] In one embodiment, the sample plate comprises a plurality of sample holders, and the sample holders are placed at regular intervals along the circumference of the sample plate. In another embodiment, the plurality of sample holders are placed at irregular intervals along the circumference of the sample plate. Alternatively, the sample holders are placed along the sides of the sample plate.

[0084] In one embodiment, the plate may include one sample holder or multiple sample holders. The sample plate may include 1 to 100, more preferably 1 to 50 sample holders, more preferably 1 to 20 sample holders, more preferably 1 to 16 sample holders, more preferably 12 sample holders. For example, the sample plate may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 sample holders.

[0085] In one embodiment, the sample holder has a shape that is compatible with the shape of the sample container. In a preferred embodiment, the cross-section of the sample holder is hexagonal. However, it is obvious to those skilled in the art that, as described above, the sample holder can have any shape that is compatible with the sample container.

[0086] In some embodiments, the sample holder is removable from the sample plate. In some embodiments, the sample holder is a fixed part of the sample plate.

[0087] In one embodiment, the sample holder may include an identification (ID) tag for identifying the sample container when the sample container is located in the sample holder. The ID tag includes an RFID tag, a smart tag, or a reader for reading an RFID tag or a smart tag. Smart tags include, but are not limited to, QR codes and bar codes.

[0088] In some embodiments, the processing unit of the system comprises at least one transport device configured to distribute and / or remove components or liquids from the sample container in the sample holder of the purification device. The particle components can be magnetic particles, and the liquid can be deionized water, purified water, buffer, wash buffer, elution buffer, reagent or a combination thereof, or waste. Obviously, the liquid is not limited to these.

[0089] The carrier used in combination with the purification device can include any suitable equipment in the art, such as a syringe or a mechanical arm, for example, equipped with one or more nozzles, needles and / or tips. Alternatively, the distribution by the carrier is carried out directly through the pipeline of the carrier. Any syringe, pump or mechanical arm for distributing and / or removing components or liquids known in the art can be used together with the system disclosed herein. The non-limiting example of the carrier is a syringe pump, a vacuum pump, a peristaltic pump, a centrifugal pump or a combination thereof.

[0090] In one embodiment, the syringe and pump are fixed dispensers located near the sample plate and the sample containers are brought to them by the rotation of the sample plate. In one embodiment, the robotic arm moves along three independent axes and can access the sample containers in a manner independent of the rotation of the sample plate. In another embodiment, the robotic arm is located on or in the center of the sample plate.

[0091] In embodiments where the sample plate is fixed, handling of the sample, dispensing and / or removal of components or liquids into and from the sample is performed by a robotic arm.

[0092] In one embodiment, at least one robotic arm is used in combination with a purification device. In another embodiment, one or more syringes are used in combination with a purification device. In yet another embodiment, one or more pumps are used in combination with a purification device. In yet another embodiment, a robotic arm and one or more syringes are used in combination with a purification device. In yet another embodiment, a robotic arm and one or more pumps are used in combination with a purification device. In a preferred embodiment, a robotic arm, one or more syringes, and one or more pumps are used in combination with a purification device.

[0093] The carrier used in combination with the purification device is connected to one or more reagent storage containers, waste containers and / or harvesting vessels. Any reagent storage container, waste container and harvesting vessel known in the art can be used in combination with this system. Non-limiting examples include bags, vials, tubes, bottles, cans or barrels.

[0094] In one embodiment, the carrier used with the purification device is controlled by a motor unit (preferably an electric motor) and is configured to move. In some embodiments, the movement is vertical, horizontal, centrifugal or 3D movement. In a preferred embodiment, the carrier is operably connected to at least one computer processor to control the carrier.

[0095] The system may also include a control system arranged and adapted to control the dispensing and / or removal of components or liquids by the carrier used in conjunction with the purification device. Based on input information (e.g., sample ID and sensor data) and predefined algorithms, the control system adjusts the performance of the carrier.

[0096] In one embodiment, the system disclosed herein for producing RNA molecules comprises a plurality of the above-mentioned purification devices. In another embodiment, the system comprises two of the above-mentioned purification devices. In another embodiment, the purification devices are placed adjacent to each other. Alternatively, the purification devices are in a nested configuration. A multi-device system allows multiple purification steps to be performed simultaneously, so that different stages of separation and / or purification can be performed simultaneously. In addition, a multi-device system allows the continuous operation of the system and the processing of a large number of samples.

[0097] The system may also include at least one harvesting vessel for harvesting the target compound. In one embodiment, the system may include multiple harvesting vessels. In another embodiment, the harvesting vessel is located in a well of a sample plate of the device. In another embodiment, the harvesting vessel is located near a purification device in the system. In another embodiment, it is expected that all purification devices are equipped with at least one harvesting vessel. In another embodiment, one purification device is equipped with a harvesting vessel, while other purification devices do not have a harvesting vessel.

[0098] The system comprises one or more sample containers, wherein the sample containers are located in a sample holder of a purification device.

[0099] In one embodiment, the sample container is disposable. In another embodiment, the container can be reused multiple times.

[0100] In some embodiments, the material of the harvest vessel and / or sample container of the purification device comprises, for example, a material that is resistant to cleaning procedures (chemical resistance), extreme temperatures (e.g., nucleic acid denaturation), extreme pH values ​​(sterilization of the reactor with bases and acids, e.g., with NaOH), mechanical forces (e.g., friction caused by magnetic particles), and / or corrosion. In other embodiments, the material of the harvest vessel and / or sample container comprises a material with appropriate light transmittance (transparent, translucent, or opaque) for the respective purpose. In some embodiments, the material of the harvest vessel and / or sample container comprises a material with appropriate gas permeability for the respective purpose. In further embodiments, the thermal conductivity of the material of the harvest vessel and / or sample container at an operating temperature between 37° C. and 65° C. (e.g., W / (mK) should be at least 10, preferably at least 15). In some embodiments, the inner surface of the harvest vessel and / or sample container contains a surface material that does not release unwanted compounds that can contaminate the final product. In a further embodiment, the material of the harvesting vessel and / or the sample container and / or its inner surface is PC (polycarbonate), PP (polypropylene), PAI (polyamide-imide) (e.g. Torlon), PI (polyimide) (e.g. Tecasint), PPS (polyphenylene sulfide) (e.g. Tecatron), PPSU (polyphenylene sulfone) (e.g. Tecason P), PSU (polysulfone) (e.g. Tecason S), PEI (polyetherimide) (e.g. Tecapei), glass (e.g. borosilicate glass), technical ceramics (e.g. ), polyaryletherketones (e.g. polyetheretherketone (PEEK)), thermoplastics (e.g. Pa or GF), all of which have chemical resistance, pH resistance and high temperature resistance. In other embodiments, the material of the harvest vessel and / or sample container comprises a disposable material, including but not limited to polyethylene terephthalate and other polyethylene, polyvinyl acetate, polyvinyl chloride. In some embodiments, the material of the harvest vessel and / or sample container comprises a material that is resistant to sterilization processes (including steam treatment or ethylene oxide (EtO) exposure / gamma radiation) even before adding any reaction-related reagents. In some embodiments, the material of the harvest vessel and / or sample container provides light protection (if necessary) for the medium contained in the harvest vessel and / or sample container. In some embodiments, the harvest vessel and / or sample container are made of any non-magnetic material or paramagnetic material known in the art.

[0101] In some embodiments, the harvest vessel and / or sample container of the purification device is in the shape of a pie wedge, a regular or irregular polygon, a concave polygon, a convex polygon, a triangle, a quadrilateral, a pentagon, a hexagon, an equilateral polygon, an equiangular polygon, a heptagon, an octagon, a nonagon, a decagon, an eleventh polygon, a dodecagon, a thirteenth polygon, a tetragon, a pentagon, a hexadecagon, a heptagon, an octadecagon, a nineteenth polygon, a pentagon, a hexadecagon, a heptagon, an octadecagon, a nineteenth polygon, an icosagon, an N-gon or an ellipse, preferably a circle.

[0102] In some embodiments, the volume of the harvest vessel and / or sample container of the purification device is at least about 0.1 ml, about 0.3 ml, about 0.5 ml, about 1 ml, about 1.5 ml, about 2 ml, about 2.5 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, about 10 ml, about 12 ml, about 15 ml, about 17 ml, about 20 ml, about 25 ml, about 30 ml, about 35 ml, about 40 ml, about 45 ml, about 50 ml, about 55 ml, about 60 ml, about 65 ml, about 70 ml, about 75 ml, about 80 ml, about 85 ml, about 90 ml, about 95 ml or about 100 ml. In some embodiments, the volume of the harvest vessel and / or sample container of the purification device is no more than about 100 ml, no more than about 95 ml, no more than about 90 ml, no more than about 85 ml, no more than about 80 ml, no more than about 75 ml, no more than about 70 ml, no more than about 65 ml, no more than about 60 ml, no more than about 55 ml, no more than about 50 ml, no more than about 45 ml, no more than about 40 ml, no more than about 35 ml, no more than about 30 ml, no more than about 25 ml, no more than about 20 ml, no more than about 15 ml, no more than about 10 ml, no more than about 9 ml, no more than about 8 ml, no more than about 7 ml, no more than about 6 ml, no more than about 5 ml, no more than about 4 ml, no more than about 3 ml, no more than about 2 ml, no more than about 1 ml, no more than about 0.5 ml, no more than about 0.3 ml, or no more than about 0.1 ml. In some embodiments, the volume of the harvest vessel and / or sample container is about 1 ml to about 100 ml, about 10 ml to 90 ml, about 15 ml to about 80 ml, about 20 ml to about 70 ml, about 25 ml to about 60 ml, or about 30 ml to about 50 ml.

[0103] In some embodiments, the volume of the harvesting vessel and / or sample container of the purification device is about 50 ml. In some embodiments, the volume of the harvesting vessel and / or sample container of the purification device is about 150 ml. In some embodiments, the volume of the harvesting vessel and / or sample container of the purification device is about 200 ml.

[0104] In some embodiments, the volume of the harvest vessel and / or sample container of the purification device is at least about 150 ml, about 200 ml, about 250 ml, about 300 ml, about 350 ml, about 400 ml, about 450 ml, about 500 ml, about 550 ml, about 600 ml, about 650 ml, about 700 ml, about 750 ml, about 800 ml, about 850 ml, about 900 ml, about 950 ml, about 10 ... 1000ml, about 2000ml, about 3000ml, about 4000ml, about 5000ml, about 6000ml, about 7000ml, about 8000ml, about 9000ml, about 10000ml, about 15000ml, about 20000ml, about 25000ml, about 30000ml, about 35000ml, about 40000ml, about 45000ml or about 50000ml. In some embodiments, the volume of the harvest vessel and / or sample container is no more than about 50,000 ml, no more than about 45,000 ml, no more than about 40,000 ml, no more than about 35,000 ml, no more than about 30,000 ml, no more than about 25,000 ml, no more than about 20,000 ml, no more than about 15,000 ml, no more than about 10,000 ml, no more than about 9,000 ml, no more than about 8,000 ml, no more than about 7,000 ml, no more than about 6,000 ml, no more than about 5,000 ml, no more than about 4,000 ml, no more than about 8,000 ml, no more than about 9,000 ml, no more than about 10,000 ml, no more than about 11,000 ml, no more than about 12,000 ml, no more than about 13,000 ml, no more than about 14,000 ml, no more than about 15,000 ml, no more than about 16,000 ml, no more than about 17,000 ml, no more than about 18,000 ml, no more than about 19,000 ml, no more than about 20,000 ml, no more than about 21,000 ml, no more than about 24,000 ml, no more than about 26,000 ml, no more than about 28,000 ml, no more than about 29,000 ml, no more than about 30,000 ml, no more than about 31,000 ml, no more than about 36,000 ml, no more than about 37,000 ml, no more than about 38,000 ml, no more than about 39,0 Not more than about 3000ml, not more than about 2000ml, not more than about 1000ml, not more than about 950ml, not more than about 900ml, not more than about 850ml, not more than about 800ml, not more than about 750ml, not more than about 700ml, not more than about 650ml, not more than about 600ml, not more than about 550ml, not more than about 500ml, not more than about 450ml, not more than about 400ml, not more than about 350ml, not more than about 300ml, not more than about 250ml, not more than about 200ml, not more than about 150ml. In some embodiments, the volume of the harvest vessel and / or sample container is the volume of the chamber from about 150 ml to about 50,000 ml, from about 200 ml to about 45,000 ml, from about 250 ml to about 40,000 ml, from about 300 ml to about 35,000 ml, from about 350 ml to about 30,000 ml, from about 400 ml to about 25,000 ml, from about 450 ml to about 20,000 ml, from about 500 ml to about 15,000 ml.From about 550 ml to about 10000 ml, from about 600 ml to about 9000 ml, from about 650 ml to about 8000 ml, from about 700 ml to about 7000 ml, from about 750 ml to about 6000 ml, from about 800 ml to about 5000 ml, from about 850 ml to about 4000 ml, from about 900 ml to about 3000 ml, from about 950 ml to about 2000 ml, or from about 1000 ml to about 1500 ml.

[0105] Preferably, the sample container of the purification device is configured to have a volume of 0.25 mL to 100 mL, more preferably 0.25 mL to 500 mL, more preferably 0.25 mL to 1000 mL, more preferably 0.25 mL to 1500 mL.

[0106] Alternatively, the sample container of the purification device is configured to have a volume of at least 50ml, 100ml, 150ml, 200ml, 250ml, 300ml, 350ml, 400ml, 450ml or 500ml. In some embodiments, the sample container of the purification device has a volume of at least 200ml.

[0107] In some embodiments, harvest vessel and / or sample container comprise capping or lid, to prevent unwanted components from entering the harvest vessel and / or sample container (for example, RNase, microbial contamination or other degradation compounds or biology) and prevent the content of harvest vessel and / or sample container from being isolated from external environment. In some embodiments, harvest vessel and / or sample container comprise lid to limit the exchange with environment. In some embodiments, the lid of harvest vessel and / or sample container is detachable. In some embodiments, the lid of harvest vessel and / or sample container is non-detachable. As a selection, harvest vessel and / or sample container are not covered.

[0108] In some embodiments, the cover can prevent excessive evaporation of water and loss of other key volatile components. In some embodiments, the cover can prevent oxidation of reagents or any components. In some embodiments, the cover can provide light protection (if necessary). In some embodiments, the cover prevents contamination from any other potential chemical compounds.

[0109] In some embodiments, the cover comprises at least one opening for filling, draining and sampling. In some embodiments, at least one opening is located at the top of the cover.

[0110] In one embodiment, the sample container of the purification device contains a liquid containing the target compound and magnetic particles.

[0111] In one embodiment, the liquid is from an IVT reaction. In another or further embodiment, the liquid comprises DNA, RNA, modified RNA, polypeptides, proteins and / or modified proteins. In yet another embodiment, the liquid contains at least one reagent. In yet another embodiment, the liquid contains one or more reagents for IVT. In yet another embodiment, the liquid contains impurities. In further embodiments, the impurities are nucleotides, enzymes, proteins, proteins, DNA templates, dsRNA, or any other IVT byproduct known in the art.

[0112] The magnetic particles can be of any size suitable for binding nucleic acids, including commercially available sizes, such as about 0.3 μm to about 10 μm in diameter, such as about 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 μm in diameter, including all ranges and subranges therebetween.

[0113] In one embodiment, the magnetic particles are silica-based magnetic particles, paramagnetic particles coated with carboxyl groups, silica-based paramagnetic particles or combinations thereof. In some embodiments, silica-based magnetic particles may include a paramagnetic core coated with silica, thereby providing a hydrated silica adsorption surface (e.g., a surface comprising silanol groups) to which nucleic acids can bind. In other embodiments, the magnetic particles may be surface-modified to produce functionalized surfaces, such as with a weak or strong positive charge, a weak or strong negative charge or a hydrophobic surface, to name a few. Alternatively, the magnetic particles may be polystyrene divinylbenzene particles, polymethacrylate particles, cross-linked agarose particles or allyl dextran with NN-bisacrylamide particles. It will be apparent to those skilled in the art that any material suitable for binding nucleic acids, proteins or other biomolecules may be used together with the device or system disclosed herein.

[0114] The magnetic particles of the system can be dispensed into a sample container via one of the delivery devices.

[0115] The target compound can bind to the magnetic particles by affinity binding. In one embodiment, the target compound binds to the magnetic particles in the presence of a binding buffer (preferably a buffer containing a chaotropic agent). In the presence of a chaotropic agent, the target compound reversibly binds to the magnetic particles.

[0116] In another embodiment, the magnetic particles are coated with ligands that interact with the compound of interest.

[0117] The system disclosed herein may be arranged in a cabinet, preferably with a laminar flow generating unit. In one embodiment, a storage unit for storing ingredients is located outside the cabinet.

[0118] In one embodiment, the cabinet is designed to provide filtered sterile air that is circulated within the unit. The air filtration device may include, for example, an HVAC system with a HEPA filter.

[0119] The housing of the cabinet can be made of any suitable material in the art, such as metal alloy, metal or plastic. In one embodiment, the cabinet is made of a material comprising aluminum or stainless steel. In a particularly preferred embodiment, the cabinet is made of a material comprising stainless steel.

[0120] Preferably, the system and cabinet are designed and operated to require only limited operator intervention. This is to avoid contamination and interference with process conditions. If an abnormal situation is observed, the operator can manipulate the process via one or more control devices inside or outside the cabinet. These control devices control (part of) the process occurring in the cabinet. The cabinet may be connected to one or more control devices configured to perform multivariate analysis, automatically control the operation of the process, and optionally, remotely communicate with the components (e.g. using a network protocol) to control the operations in the unit.

[0121] Each cabinet is preferably movable and equipped with a transport device. The transport device may include any suitable, manually and / or electronically controlled device known in the art, including but not limited to wheels, tracks or rollers.

[0122] In one embodiment, the system disclosed herein comprises at least one computer processor operably connected to an IVT unit and / or a processing unit. In one embodiment, the system comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 computer processors. In another embodiment, the IVT unit and the processing unit are operably connected to the same computer processor. In another embodiment, the IVT unit and the processing unit are operably connected to different computer processors. In another embodiment, multiple chambers and carriers of the IVT unit are operably connected to different computer processors. In another embodiment, multiple chambers and carriers of the IVT unit are operably connected to the same computer processor. In another embodiment, the purification device and carrier of the device are operably connected to different computer processors. In another embodiment, the purification device and carrier of the device are operably connected to the same computer processor. In another embodiment, all carriers are connected to the same computer processor.

[0123] In some embodiments, the system disclosed herein produces RNA without the need for scale-up and ensures rapid availability in the clinic. In some embodiments, the system has a small footprint while integrating all process steps required for the production of high-quality RNA. In some embodiments, the process is optimal and suitable for custom mRNA constructs.

[0124] method

[0125] The present disclosure also relates to a method for producing RNA. In a particularly preferred embodiment, the method is performed by a system as described in one of the above embodiments.

[0126] In one embodiment, the IVT reaction is performed in multiple chambers and includes an in vitro transcription step starting from a DNA template. The steps of IVT can be:

[0127] (i) providing one or more IVT reagents in a first chamber of the plurality of chambers;

[0128] (ii) repeating step (i) until at least a portion of the plurality of chambers are provided with one or more IVT agents;

[0129] (iii) performing the IVT reaction in a chamber equipped with one or more IVT reagents; and

[0130] Therein, the resulting IVT reaction is subsequently subjected to at least one purification step.

[0131] Without wishing to be bound by theory, the reaction conditions that need to be met for the IVT reaction include: providing linear DNA as a template (to be replicated into RNA by a polymerase), dNTPs, an RNA polymerase that incorporates the dNTPs; a magnesium-containing buffer to catalyze the reaction, and a specific reaction temperature (usually 37° C.) It will be apparent to those skilled in the art that any reaction conditions that produce mRNA can be used with the methods described herein.

[0132] In one embodiment of the method disclosed herein, at least a portion of the IVT reagents are combined into an IVT premix. In another embodiment, the IVT premix comprises at least dNTPs, template DNA, and one or more RNA polymerase buffer components. In one embodiment, the IVT reagents are stored in a storage unit, wherein the storage unit is fluidically connected to a pump system, and wherein the pump system is configured to provide the required amount of one or more IVT reagents for preparing the IVT premix to the IVT premix container. As previously described, the storage unit can be cooled to a temperature below 10°C, preferably 4°C.

[0133] In one embodiment, the total volume of the premix is ​​equal to or greater than the sum of the volumes of the individual IVT reactions in the chamber.

[0134] In one embodiment, the IVT premix is ​​heated to a temperature of 35° C. to 55° C. prior to providing a portion of the premix to one of the plurality of chambers. In one embodiment, the IVT premix is ​​heated to a temperature of 35° C. to 55° C., preferably 35° C. to 50° C., 35° C. to 45° C., 35° C. to 40° C., or 35° C. to 37° C. prior to providing a portion of the premix to one of the plurality of chambers. Alternatively, the IVT premix is ​​heated to a temperature of 37° C. to 55° C., preferably 40° C. and 45° C. to 55° C., or 40 to 55° C. prior to providing a portion of the premix to one of the plurality of chambers.

[0135] In one embodiment, the heating temperature of the IVT premix container is 35° C. to 55° C., preferably 35° C. to 50° C., 35° C. to 45° C., 35° C. to 40° C., or 35° C. to 37° C. Alternatively, the heating temperature of the IVT premix container is 37° C. to 55° C., preferably 40° C. and 45° C. to 55° C., or 40° C. to 55° C. In a preferred embodiment, the heating temperature of the premix container is 37° C.

[0136] In one embodiment of the methods disclosed herein, an RNA polymerase is added to at least one of the multiple chambers. In other embodiments, multiple RNA polymerases are added to the chamber, preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10 RNA polymerases. In a further embodiment, the RNA polymerase is added before or after the chamber receives a portion of the IVT premix. Combining the IVT premix with the RNA polymerase initiates the start of the reaction. In some embodiments, it is desirable to heat the premix in the absence of the RNA polymerase so that the start of the reaction is delayed until the premix and the RNA polymerase are combined in multiple chambers. In other embodiments, the RNA polymerase is part of the IVT premix. The RNA polymerase used is as described above.

[0137] In some embodiments of the method, the IVT reagent comprises a capping agent for co-transcriptional capping. Non-limiting examples of co-transcriptional capping agents include CleanCap (TriLink) or ARCA (CellScript). Alternatively, capping is performed post-transcriptionally using any cap analog structure known in the art.

[0138] Preferably, the system disclosed herein is used to purify mRNA molecules from an IVT process, or to purify before and / or after capping. In one embodiment, when the mRNA molecule is co-transcriptionally capped, the mRNA is purified after capping. In another embodiment, when the mRNA is capped post-transcriptionally, the purification step is performed before capping. In another or other embodiment, the purification is performed after capping. In another embodiment, the purification step is performed before capping and the second purification step is performed after capping. In addition to the desired mRNA product, the product obtained by IVT also contains a series of reaction by-products, such as salts, nucleotides, enzymes, proteins, DNA templates or dsRNA. These can interfere with the capping process and reduce the conversion efficiency and overall purity of the final product. Enzymatic capping is performed immediately after IVT without intermediate treatment of the reaction product, and the amount of capped mRNA molecules produced is reduced or close to 0%. The system disclosed herein is intended to purify mRNA in a high-precision, automated manner under GMP-compliant conditions, and is suitable for upstream and / or downstream capping purification. The system allows continuous production of small or moderate amounts of target compounds.

[0139] In some embodiments of the method, the final IVT reaction from one of the plurality of chambers is transferred to an intermediate vessel. In further embodiments, the IVT reactions of the plurality of chambers are pooled in the intermediate vessel. In yet another embodiment, the transfer and pooling of the IVT reactions is performed by a robotic arm.

[0140] In one embodiment of the method, the IVT reaction is terminated by adding at least one DNase. In another embodiment, the IVT reaction is terminated by adding EDTA. In yet another embodiment, the IVT reaction is terminated by adding DNase and / or EDTA simultaneously. DNase enzymatically digests the DNA template, thereby terminating the IVT reaction, while EDTA is a chelating agent that consumes Mg. 2+ ions, which also leads to the termination of the IVT reaction.

[0141] In one embodiment, DNase and / or EDTA are added in a plurality of chambers. In another embodiment, said DNase and / or EDTA are added in an intermediate vessel. In a further embodiment, DNase and / or EDTA are added by means of a pump, a syringe or a mechanical arm.

[0142] In one embodiment, at least one purification step after IVT uses magnetic particles that are capable of binding to the RNA molecules. In a further embodiment, the IVT reaction or a portion of the IVT reaction containing the RNA molecules is provided to one or more sample containers located in a sample holder. In a further embodiment, the magnetic particles are added to the sample containers. Alternatively, the sample containers contain magnetic particles and the IVT reaction or a portion of the IVT reaction containing the RNA molecules is provided to the containers.

[0143] In a preferred embodiment of at least one purification step, a portion of the IVT reaction is provided to a container by a delivery means and purified.

[0144] In some embodiments of the method, RNA is bound to magnetic particles, which are then captured or induced to move toward a magnet unit near a sample holder, thereby causing the RNA molecules bound to the magnetic particles to be separated from any remaining IVT liquid. In some embodiments, a binding buffer is used to mediate reversible binding between the target compound and the magnetic particles. In further embodiments, the binding buffer may comprise a chaotrope, an alcohol, PEG, a salt, or a mixture thereof. The chaotrope may be selected from guanidine salts, such as hydrochloride (GuHCl) and guanidine thiocyanate (GuSCN); lithium salts, such as lithium acetate and lithium perchlorate; or sodium salts, such as NaCl, and combinations thereof. In one embodiment, the binding buffer does not contain a chaotrope.

[0145] The alcohol may be selected from isopropanol, ethanol, methanol, butanol and combinations thereof. In one embodiment, the concentration of the alcohol is 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 15% to 20% v / v, including all ranges and sub-ranges therebetween.

[0146] In one embodiment, the binding buffer may include PEG as a substitute for or in combination with an alcohol. The concentration range of PEG in the binding buffer may be 10% to 40%, 20% to 40%, 20% to 35%, 20% to 30% or 25% to 35%, including all ranges and subranges therebetween. In one embodiment, 30% PEG is used. In one embodiment, the PEG used in the binding buffer is selected from PEG 600, PEG 1000, PEG 2000, PEG 3000, PEG 4000, PEG 6000, PEG 8000, PEG 10.000, PEG 20.000. In one embodiment, the PEG used is PEG 8000.

[0147] The binding buffer may contain at least one salt in a concentration range of 0.1 M to 5 M, such as 0.1 M to 4 M, 0.1 M to 3 M, 0.1 M to 2 M, 0.1 M to 1 M, 0.5 M to 1 M, 0.5 M to 2 M, 1 M to 2 M, 2 M to 3 M, and 3 M to 5 M, including all ranges and subranges therebetween. According to various embodiments, the at least one salt may be sodium chloride (NaCl).

[0148] In one embodiment, the binding buffer comprises Tris-HCl, NaCl, EDTA, and ethanol.

[0149] In an embodiment, the binding buffer has a pH range of 5 to 10, such as 5 to 9, 5.5 to 8.5, 6 to 8, or 6.4 to 7.5, and all ranges and subranges therebetween. In an embodiment, the binding buffer comprises at least one first alcohol and / or PEG, at least one salt, and at least one selective chelating agent, such as EDTA.

[0150] The magnetic particles can be present in the binding buffer at a concentration range of, e.g., about 0.1 μg / μl to about 60 μg / μl, e.g., about 0.5 μg to about 60 μg / μl, about 0.75 μg / μl to about 55 μg / μl, about 1 μg / μl to about 50 μg / μl, about 2 μg / μl to about 45 μg / μl, about 3 μg / μl to about 40 μg / μl, about 4 μg / μl to about 35 μg / μl, about 5 μg / μl to about 30 μg / μl, about 6 μg / μl to about 25 μg / μl, about 7 μg / μl to about 20 μg / μl, about 8 μg / μl to about 15 μg / μl, or about 9 μg / μl to about 10 μg / μl, including all ranges and subranges therebetween. As a non-limiting embodiment, at least one magnetic particle can be selected from Qbeads and can be present in the binding buffer B1 at a concentration ranging from about 0.5 μg / μl to about 5 μg / μl. In an alternative embodiment, at least one magnetic particle can be selected from Grace beads and can be present in the binding buffer B1 at a concentration ranging from about 2 μg / μl to about 60 μg / μl.

[0151] In embodiments of the methods taught herein, the volume ratio between the sample and the binding buffer may range, for example, from 1:1 to 1:3, such as from 1:1 to 1:1.5, or from 1:1.5 to about 1:2.5, including all ranges and subranges therebetween. The incubation time of the mixed solution comprising a sample containing at least one target nucleic acid, a binding buffer, and silica-based magnetic particles may range from 0.1 minute to 30 minutes, 0.1 minute to 25 minutes, 0.1 minute to 20 minutes, 0.1 minute to 10 minutes, or 0.1 minute to 5 minutes, 0.1 minute to 2 minutes, including all ranges and subranges therebetween.

[0152] In further embodiments, after isolating the RNA molecules from the remaining IVT fluid, the remaining IVT fluid is removed.

[0153] In yet another embodiment, after removing the remaining liquid, additional liquid is added to the RNA bound to the magnetic beads and the separation step is repeated.

[0154] In some embodiments of the method, the sample holder is mechanically moved to mix the IVT reaction or other fluid with the magnetic particles.

[0155] In some embodiments of the method, the sample holder with the sample container is rotated to at least one subsequent position between or after the capturing steps.

[0156] The sample holder with the containers is located on the base part of the sample plate, which can be rotated. The rotation of the sample plate transports the sample containers to different dispensers, such as syringes, robotic arms or pumps, where components or liquids can be added or removed from the sample containers.

[0157] In other embodiments of the method, the sample plate is rotated between or after capture method steps, thereby moving the sample holder with the sample containers to a subsequent position.

[0158] In alternative embodiments of the methods disclosed herein, the sample plate is not moved between capture steps, thereby maintaining the sample holder in a fixed position.

[0159] In some embodiments of the method, as described above, the magnetic particles are added to the sample container by a syringe or a robotic arm equipped with one or more nozzles, needles and / or tips. In further embodiments, as described above, the addition and / or removal of liquid is performed by one or more syringes or a robotic arm equipped with one or more nozzles, needles and / or tips.

[0160] In one embodiment, the mechanical movement of the sample holder that mixes the RNA with the magnetic particles is shaking or stirring. The shaking or stirring of the sample holder causes the movement of the magnetic particles, which are suspended in the liquid medium and come into contact with the target compound.

[0161] In an embodiment of the method disclosed herein, the magnet unit comprises a permanent magnet.The mechanical movement prevents the capture of magnetic particles or guides the movement of magnetic particles towards the magnet unit.

[0162] In a particularly preferred embodiment, as described above, the magnetic particles used in the methods disclosed herein are silica-based magnetic particles. In one embodiment of the method, the above-mentioned binding buffer is used to mediate the binding of RNA to the magnetic particles.

[0163] After the nucleic acid is bound to the magnetic particles and the modified magnetic particles are separated using a magnet, in some embodiments, the magnetic particles can be combined, rinsed or washed with one or more wash buffers. The wash buffer can contain, for example, at least one alcohol and optionally at least one salt. The modified magnetic particles can be rinsed with wash buffer once or multiple times, and any additional washes can use the same or different ingredients, concentrations and / or volumes. According to various embodiments, at least one alcohol can be selected from isopropanol, methanol, ethanol, butanol and combinations thereof. According to various embodiments, at least one salt can be selected from ammonium sulfate ((NH4)2SO4), ammonium acetate (NH4Ac), lithium acetate (LiAc), potassium acetate (KAc), sodium acetate (NaAc), sodium chloride (NaCl) and combinations thereof.

[0164] In an embodiment of the method taught herein, the wash buffer comprises at least one second alcohol in a concentration range of 50% to 100% volume / volume (v / v), 55% to 95%, 60% to 85%, or 60% to 80% (v / v), including all ranges and sub-ranges therebetween. In an embodiment, the second alcohol may be selected from isopropanol, methanol, ethanol, butanol, and combinations thereof. The second alcohol in the wash buffer may be the same or different from the first alcohol in the binding buffer. In some embodiments, the second alcohol may be ethanol. In a non-limiting embodiment, the wash buffer may optionally have at least one salt. If present, the concentration range of the optional salt in the binding buffer may be 0.1M to 5M, such as 0.3M to 4M, 0.1M to 3M, 0.1M to 2M, 0.1M to 1M, and 1M to 2M, including all ranges and sub-ranges therebetween. According to various embodiments, the salt in the wash buffer is sodium chloride (NaCl). In some embodiments, the modified magnetic particles may be washed once or multiple times with at least one of the wash buffers. For example, the modified magnetic particles can be washed once, twice, or more times with a wash buffer, and the modified magnetic particles can be separated using a magnet between washes.

[0165] In some embodiments, the modified magnetic particles are washed one or more times with the wash buffer, and additional wash steps can be performed using, for example, the same or different compositions, concentrations, and / or volumes.

[0166] After adding and removing the wash buffer, the modified magnetic particles with the target compound reversibly bound to the surface are substantially free of contaminants such as salts, proteins, enzymes. According to various embodiments, the modified magnetic particles thus produced can then be incubated with one or more elution buffers to release the bound target compound and separate it from the magnetic particles.

[0167] According to different embodiments, the elution buffer is a low conductivity solution, wherein the conductivity of the buffer ranges from 0.001 mS / cm to 40 mS / cm, more preferably from 0.01 mS / cm to 40 mS / cm, 0.1 mS to 40 mS / cm, 0.5 mS / cm to 40 mS / cm, more preferably from 0.5 mS / cm to 30 mS / cm, 0.5 mS / cm to 20 mS / cm, 0.5 mS / cm to 10 mS / cm, including all ranges and sub-ranges therebetween.

[0168] In another or further embodiment, the elution buffer contains a salt concentration of 0.01 mM to 50 mM, more preferably 0.1 mM to 40 mM, more preferably 0.1 mM to 30 mM, more preferably 0.1 mM to 20 mM. Possible salts include sodium citrate, sodium chloride, sodium phosphate, potassium chloride, potassium phosphate and combinations thereof.

[0169] The pH of the elution buffer can range, for example, from 5 to about 10, such as 5.5 to about 9, 6 to 8, or 6.4 to about 7.5, including all ranges and subranges therebetween.

[0170] For example, in some cases, the elution buffer comprises water; in other cases, the elution buffer comprises water and EDTA, or only Tris, or Tris and EDTA, or sodium citrate, or phosphate buffer, or phosphate buffered saline (PBS). If used as elution buffer, the concentration range of sodium citrate can be 0.5mM to 10mM, such as 0.6mM to 5mM, 1mM to 2mM, including all ranges and subranges therebetween. If used as elution buffer, the pH range of sodium citrate is pH 5.4 to 7.5, pH 6 to pH 7, pH 6 to pH 6.5, including all ranges and subranges therebetween. According to non-limiting embodiments, the elution buffer can comprise water or 10mM Tris-HCl, 1mM EDTA, pH7.4; or 10mM Tris-HCl, pH7.4; or 1mM sodium citrate, pH6.4.

[0171] In an embodiment, the elution buffer is free of toxic chaotropic agents such as guanidine salts (guanidinium thiocyanate or guanidine thiocyanate), iodide, perchlorate and trichloroacetate, preferably guanidine salts.

[0172] In embodiments, at least one modified particle is incubated with the elution buffer for a period of time of from 30 seconds to 30 minutes, from 1 minute to 20 minutes, from 1 minute to 10 minutes, including all ranges and subranges therebetween.

[0173] In one embodiment, after adding the elution buffer, the sample holder is mechanically moved so that the magnetic particles are mixed with the elution buffer. Once the movement stops, the magnetic particles move toward the magnet unit, resulting in the elution buffer containing the target compound being separated from the magnetic particles. In a further embodiment, the elution buffer containing the RNA is removed from the sample container and stored in a harvesting vessel.

[0174] In some embodiments of the methods disclosed herein, the purification step includes a capping step or precedes a post-transcriptional capping step. In one embodiment, the purification step is part of a pre-capping step followed by a post-transcriptional capping step.

[0175] In some embodiments, after capping, a second purification step is performed. In further embodiments, the second purification step utilizes magnetic particles that can bind to the capped RNA molecules. The second purification step is performed in the same manner as the first purification step described above.

[0176] The second purification step can be performed using the same purification device. In other embodiments, the second purification step is performed in a second purification device. The two purification devices can be placed adjacent to each other or in a nested configuration.

[0177] In one embodiment, the system disclosed herein is used in combination with dead-end filtration. Dead-end filtration of mRNA can be performed after the IVT reaction and before loading into the purification device. In another embodiment, dead-end filtration is performed after purification of mRNA using the purification device disclosed herein.

[0178] Alternatively, any filtration method may be used with the systems disclosed herein, such as, but not limited to, diafiltration or tangential flow filtration (TFF).

[0179] The method disclosed herein is carried out by the device or system of the aforementioned embodiment. In one embodiment, the method is semi-automatic or fully automatic. The method is fast and efficient, and reproduces manual operations repeated on a small scale. The method meets GMP conditions, is not prone to human error, and provides a high-purity product.

[0180] The present disclosure will now be further illustrated with reference to the following examples.The present disclosure is not limited to the embodiments given in the examples or shown in the figures.

[0181] Attached photos

[0182] The present disclosure is not limited to the implementations described in the embodiments and / or shown in the drawings. On the contrary, the method of the present disclosure can be implemented in many different ways without departing from the scope of the present disclosure.

[0183] Figure 1An embodiment of a system (11) for producing RNA is shown, comprising an IVT unit (34) and a downstream processing unit (33) and a delivery device: a robotic arm (7, 7'), a syringe (20, 21, 22, 27) and a pump (19). The robotic arm (7, 7') utilizes a needle and / or a tip (18). In one embodiment, the target compound obtained using the methods disclosed herein will be further processed downstream in a container (26).

[0184] Figure 2 An IVT unit (34) is depicted in FIG. In one embodiment, the required amount of reagents for preparing the IVT premix is ​​delivered to the IVT premix container (24). The IVT unit (34) comprises a heating unit (25) for heating the IVT premix container (24) at 37° C. In some embodiments, the premix comprises dNTPs, template DNA, and one or more RNA polymerase buffer components. In one embodiment, heating is performed prior to adding the RNA polymerase. In another embodiment, the RNA polymerase is also added to the IVT premix container (24) and heated.

[0185] In a preferred embodiment, the IVT premix at 37° C. is transferred to a plurality of chambers ( 23 ), wherein an IVT reaction is performed in the presence of RNA polymerase in the chambers ( 23 ), thereby obtaining a plurality of RNA molecules.

[0186] The IVT unit (34) comprises a transport device, such as a robotic arm (7), a syringe (20, 21, 22, 27) and / or a pump (19), which is configured to dispense and / or remove reagents, reagent mixtures or liquids in a premix container (24) and a plurality of chambers (23) arranged in a cartridge (35). Figure 2 In the example of , the IVT unit ( 34 ) has four cartridges ( 35 ).

[0187] The RNA molecules are further purified in a downstream processing unit (33) containing the purification device (1).

[0188] FIG3 shows a purification device (1) for separating and / or purifying RNA molecules obtained by IVT according to an embodiment of the present specification, wherein the purification device is in the form of a carousel. Figure 1As shown in Figure 3, the device (1) can be configured to rotate about an axis. Alternatively, the purification device can be fixed and therefore cannot rotate (not shown). According to the specific embodiment shown in Figure 3, the device (1) includes a sample plate (2) equipped with a sample holder (3), and the sample holder (3) is arranged on the base portion (4) of the sample plate (2). In the embodiment of Figure 3, the sample plate (2) includes 8 sample holders (3). However, it is obvious to those skilled in the art that this number is flexible. Therefore, other embodiments in which different numbers of sample holders (3) are provided are also considered. A sample container (8) containing a target compound in a liquid medium is located in the sample holder (3). It is obvious to those skilled in the art that the sample plate can have a variety of forms. In some embodiments, it may not be Figure 1 and the circular shape shown in Figure 3. In another embodiment, the purification device (1) does not comprise a sample plate (2), but comprises one or more sample holders (3) present in the system (not shown in the figure).

[0189] Each sample holder (3) is equipped with a magnet unit (5). As shown in the embodiment of Figure 3, the magnet unit (5) is installed in a housing (6) which is open on the side facing the sample holder (3). In some embodiments, the magnet unit (5) comprises a permanent magnet, and in other embodiments comprises a temporary magnet or an electromagnet.

[0190] The sample holder (3) is capable of mechanical movement, in some embodiments a rotation about an axis, and in other embodiments a shaking movement. The sample holders (3) can move independently of each other or in sync. In the example shown in FIG3 , the movement is driven by a motor unit or an oscillator unit. The sample plate (2) is configured to rotate clockwise and counterclockwise about an axis.

[0191] exist Figure 3A In the embodiment shown, the robot arm (7) is located in the center of the sample plate (2). Figure 3B In an embodiment, the robot arm (7) is located outside the sample plate (2) or the device (1). The robot arm (7) is equipped with a nozzle tool (13), a pipetting tool (17), a needle and / or a tip, and is configured to dispense and / or remove components and / or liquids from the sample container (8).

[0192] In one embodiment, the RNA to be purified is produced by an IVT reaction. A sample containing the RNA suspended in a reaction medium is dispensed into a sample container (8) by a robotic arm (7). Magnetic particles (9) are also dispensed into the sample container (8) by the robotic arm (7).

[0193] The capture of magnetic particles (9) is shown in FIG4 . The magnet unit (5) is configured to attract and capture the particles (9) residing in the sample container (8). The movement of the sample holder (3) drives the capture or free state of the silica beads (9). When the sample holder (3) is in motion, the magnetic particles (9) move freely in the liquid and interact with the RNA molecules ( Figure 4A ). The RNA molecules are reversibly bound to the silica beads (9). When the sample holder (3) stops moving, the silica beads (9) are captured ( Figure 4B ). In embodiments using electromagnets, the captured or free state of the magnetic particles is controlled by activating and deactivating an electric current.

[0194] When the magnetic particles (9) are captured in the sample container (8), the liquid contents in the container can be removed or replaced without losing the magnetic particles (9) or the RNA bound thereto. The distribution and removal of the liquid in the sample container is performed by a transport device, such as a robotic arm (7), a syringe (20, 21, 22, 27) or a pump (19). The robotic arm (7) can move at any random position and can enter any sample container (8). The syringe (20, 21, 22, 27) or pump (19) is located at a fixed position and the sample container (8) is transported to these positions by the rotation of the sample plate (2). The robotic arm (8) and other transport devices are connected to one or more reagent reservoirs (29), waste containers and harvest vessels (28).

[0195] In one embodiment, the purification device (1) is designed to perform one or more RNA purification steps: binding RNA to magnetic particles in the presence of a chaotropic agent, washing the silica beads bound to the RNA, eluting the RNA, and collecting the purified RNA in a harvesting vessel. This allows multiple automatic, precise cleaning steps to be performed without losing RNA. The device is configured to allow repeated separation and / or purification of RNA, and can be performed continuously. In a preferred embodiment, the magnetic particles are silica-based magnetic beads.

[0196] Figure 5 An embodiment of a sample holder (3) having a sample container (8) with a capacity of 50 ml is shown. In some embodiments, the capacity may vary depending on the process. The sample container (8) is mounted directly in the sample holder (3). It is apparent that other sample containers may also be used in the context of the present disclosure.

[0197] Figure 6An embodiment of a sample holder (3) having a sample container (8') with a capacity of 2 ml is shown. The sample container (8') is mounted in an adapter (10) for adapting the sample holder (3) to the size of the sample container (8'). In other embodiments, sample containers (8') of other sizes and capacities are used.

[0198] Figure 7 A specific embodiment of a downstream processing unit (33) is shown, wherein the downstream processing unit (33) comprises two purification devices (1 and 1') and two robotic arms (7 and 7') are provided. In this embodiment, the purification devices are placed adjacent to each other. This configuration can provide higher productivity while keeping the footprint of the purification device to a minimum. Alternatively, this configuration can also be used for different purification processes. For example, the first purification device (1) can be used for pre-capping purification, while the second purification device (1') can be used for post-capping process. The capping reaction can occur at the final position of the first purification device (1), or in an intermediate vessel located downstream of the first purification device (1) and upstream of the second purification device (1').

[0199] Figure 8 Another embodiment is provided, wherein two purification devices (1 and 1') are in a nested configuration and are equipped with a single robotic arm (7).

[0200] Figure 7 and Figure 8 The multiple purification device system shown allows multiple steps to be performed simultaneously, thereby enabling different stages of separation and / or purification to be performed simultaneously.

[0201] Fig. 9 A robotic arm (7) for removing / transporting liquid culture medium and sample solution from / to a receiver according to embodiments described herein is shown. The robotic arm (7) is used for operation steps in the IVT unit (34) and the downstream processing unit (33). One or more robotic arms (7, 7') can be used with the system (11).

[0202] Fig. 9 The robot arm shown comprises a base (12), a pipetting tool (17) and a nozzle tool (13) for processing one or more liquid culture media. The nozzle tool (13) is located at the distal end of the robot arm (7).

[0203] In the following, the robot arm (7) is described as being mounted on a horizontal surface. Of course, other mounting arrangements may be used, and adjustments to such other mounting arrangements described below are within the capabilities of those skilled in the art and are considered to form part of the scope of the embodiments described herein. For example, the robot arm (7) may be mounted on a vertical surface, resulting in a 90° rotation in all subsequent directions.

[0204] The robot arm (7) is an articulated robot arm (7) including joints (14), wherein the robot arm 1 is made by sequentially connecting these joints (14) with a plurality of connecting rods (15). Figure 7 The robot arm (7) shown comprises six joints (14) to allow movement in six degrees of freedom. More specifically, in the first joint (14a), the base and the proximal part of the first link (15a) are connected so as to be able to rotate around an axis extending in a vertical direction. The first joint (14a) is a torsion joint.

[0205] In the second joint (14b), the distal end portion of the first link (15a) is connected to the proximal end portion of the second link (15b) so as to be rotatable around an axis extending in the horizontal direction. The second joint (14b) is a rotary joint.

[0206] In the third joint (14c), the distal portion of the second link (15b) and the proximal portion of the third link (15c) are connected so as to be able to rotate around an axis extending in the horizontal direction, which in this case is parallel to the axis of the second link (15b). It should be noted that the horizontality of the third axis can deviate, but the third axis will always have at least one horizontal component, and completely horizontal is the most effective form. The third joint (14c) in this case is a revolute joint.

[0207] In the fourth joint (14d), the distal end of the third link (15c) is connected to the proximal end of the fourth link (15d), so as to be rotatable around the axis in the length direction of the fourth link (15d). The fourth joint (14d) is a torsion joint.

[0208] In the fifth joint (14e), the distal end portion of the fourth link (15d) is connected to the proximal end portion of the connector (16) so as to be rotatable about an axis orthogonal to the fourth axis. The fifth joint (14e) is a rotation joint, but it should be noted that it is easy to adjust the joint to a torsion joint or a rotation joint.

[0209] In the sixth joint (14f), the distal end portion of the connector (16) and the proximal end of the nozzle tool (13) are connected so as to be rotatable in a plane orthogonal to the length direction of the connector (16).

[0210] Each joint (14) is equipped with a drive motor as an example of an actuator for causing two components connected by the joint (14) to rotate relative to each other. The drive motor is, for example, a servo motor, which is servo-controlled via a servo amplifier by a control signal sent from a controller. In addition, each joint is equipped with a rotation angle sensor for detecting the rotation angle of the drive motor and a current sensor for detecting the current of the drive motor.

[0211] In one embodiment, if Fig.10 As shown, the system (11) is arranged in a cabinet (32), preferably with a laminar flow generating unit. The cabinet (32) contains a reactive storage unit (29). In some embodiments, the unit is cooled to 4°C), and a unit (30) for storing the treated target compound (cooled if necessary), and a filtration unit (31). The filtration unit (31) provides filtered sterile air that circulates in the cabinet (32). In some embodiments, the air filtration device includes an HVAC system with a HEPA filter. In one embodiment, the cabinet or a portion of the cabinet can have wheels for easy transportation.

[0212] The system can be designed so that the cabinet (32) containing the system fits into a transport container (e.g. Fig.11 As shown) or a small clean room. Therefore, it can be suitable for building modular mobile laboratories with multiple systems to produce large amounts of RNA.

[0213] Figure No.

[0214] 1,1': Purification unit

[0215] 2: Sample plate

[0216] 3: Sample holder

[0217] 4: Base of the sample plate

[0218] 5: Magnet unit

[0219] 6: Shell

[0220] 7,7': Robotic arm

[0221] 8,8': Sample container

[0222] 9: Magnetic particles

[0223] 10: Adapter

[0224] 11: System

[0225] 12: Robotic arm base

[0226] 13: Nozzle tool

[0227] 14a-f: Joints

[0228] 15a-e: Connecting rod

[0229] 16: Connector

[0230] 17: Pipetting tools

[0231] 18: Needle or pointed end

[0232] 19: Syringe Pump

[0233] 20,21,22,27: Syringes

[0234] 23: Multiple Rooms

[0235] 24: IVT premix container

[0236] 25: Heating unit

[0237] 26: Containers for downstream processing

[0238] 28: Harvest Vessel

[0239] 29: Reagent storage unit

[0240] 30: Compound storage unit

[0241] 31: Filter unit

[0242] 32: Cabinet

[0243] 33: Downstream processing unit

[0244] 34: IVT Unit

[0245] 35: Tube

Claims

1. A system for producing RNA, the system comprising: An in vitro transcription (IVT) unit for producing a plurality of RNA molecules, wherein the IVT unit comprises: a plurality of chambers, wherein the chambers are configured to receive one or more IVT reagents and perform an IVT reaction based on a DNA template to produce the plurality of RNA molecules; and a processing unit for processing the plurality of RNA molecules after producing the plurality of RNA molecules, Wherein, the processing unit includes: one or more containers, wherein the containers are configured to contain a liquid medium containing the plurality of RNA molecules and the plurality of magnetic particles; and One or more magnet units, wherein the magnet units are configured to capture the plurality of magnetic particles in the container or guide the movement of the plurality of magnetic particles. 2 . The system of claim 1 , further comprising one or more sample holders, wherein the sample holders are configured to receive the containers.

3. The system according to claim 1 or 2, wherein: The container is configured to perform mechanical movement to mix the plurality of magnetic particles within the liquid medium.

4. The system according to any one of claims 1 to 3, wherein: The processing unit comprises a purification device for purifying the plurality of RNA molecules.

5. A system according to any one of claims 1 to 5, wherein: The system includes an intermediate vessel configured to collect and store final IVT reaction products from the plurality of chambers.

6. A system according to any one of the preceding claims, wherein: A portion of the final IVT reaction product is transferred to a container of the purification apparatus via a carrier device.

7. A system according to any one of the preceding claims, wherein: The IVT unit is housed in a cartridge.

8. The system according to claim 7, wherein: The cartridge contains from 2 to 48 chambers.

9. A system according to any one of the preceding claims, wherein: The volume of the chamber is about 0.1 mL to 500 mL.

10. A system according to any one of the preceding claims, wherein: The system comprises at least one delivery device configured for dispensing and / or removing reagents, reagent mixtures or liquids.

11. The system according to claim 10, wherein: The delivery device comprises a syringe or a robotic arm equipped with one or more nozzles, needles and / or gun tips.

12. The system according to claim 10 or 11, wherein: The vehicle is configured to move.

13. The system of claims 10 to 12, further comprising a control system configured to control the dispensing and / or removal of the reagent, reagent mixture or liquid by the carrier.

14. The system according to claim 10 to 13, wherein: The delivery device is configured to dispense a quantity of the IVT premix and / or one or more enzymes within the chamber.

15. A system according to any one of the preceding claims, wherein: The system comprises a storage unit for storing one or more reagents of the IVT reaction, wherein the storage unit is capable of being cooled to a temperature below 10°C.

16. The system of claim 15, wherein: The storage unit is fluidly connected to a pump system, wherein the pump system comprises a peristaltic pump or a syringe pump.

17. The system according to claim 15 or 16, wherein: The pump system is configured to dispense a desired amount of one or more IVT premix preparation reagents to an IVT premix container located in the IVT unit.

18. A system according to any preceding claim, wherein: The system includes a heating device for heating the IVT premix container.

19. A system according to any one of the preceding claims, wherein: The sample holder of the purification device is configured to independently perform mechanical movement.

20. The system of claim 19, wherein: The mechanical movement of the sample holder is driven by a motor unit or an electromagnetic unit.

21. The system according to claim 19 or 20, wherein: The mechanical movement of the sample holder is driven by an oscillator unit.

22. A system according to claims 19 to 21, wherein: The mechanical movement can be started and stopped.

23. A system according to claims 19 to 22, wherein: The mechanical movement is a rotational or oscillatory movement around the axis of the sample holder.

24. A system according to any one of the preceding claims, wherein: The purification device of the processing unit contains a sample plate, the sample plate comprises a base portion, and wherein the sample holder is placed on the base portion.

25. The system of claim 24, wherein: The sample holder is located on top of the sample plate or in a pocket or recess of the sample plate.

26. A system according to claim 24 or 25, wherein: The sample plate is configured to rotate about an axis, preferably about a central axis of the base portion, or to perform a linear motion.

27. The system of claim 26, wherein: The sample plate is configured to rotate clockwise and counterclockwise.

28. The system of claim 24 or 25, wherein: The sample plate is fixed or non-removable.

29. A system according to any one of the preceding claims, wherein: The magnet unit comprises a permanent magnet, a temporary magnet or an electromagnet, preferably a permanent magnet.

30. A system according to any one of the preceding claims, wherein: The magnet unit is disposed along one side of the sample holder and extends above the sample holder.

31. A system according to any one of the preceding claims, wherein: The magnet unit is rod-shaped and extends at least over the entire length of the sample container, preferably below the sample container.

32. A system according to any one of the preceding claims, wherein: The magnet unit is arranged in a housing.

33. The system of claim 32, wherein: The housing is open at a side facing the sample holder.

34. A system according to any one of claims 1 to 33, wherein: The position of the magnet unit is fixed or movable.

35. The system of claims 1 to 34, wherein: An adapter is present in the sample holder to adjust the size of the sample holder.

36. The system of claims 24 to 26, wherein: The base portion of the sample plate is rectangular, polygonal, circular, elliptical or ring-shaped.

37. The system of claims 24 to 26, wherein: The sample plate comprises the plurality of sample holders, wherein the plurality of sample holders are placed at regular or irregular intervals along the circumference of the sample plate.

38. The system of claim 37, wherein: The sample plate comprises 1 to 20 sample holders, more preferably 1 to 16 sample holders, more preferably 12 sample holders.

39. A system according to any one of the preceding claims, wherein: The sample holder includes an identification tag for identifying the sample container when the sample container is located in the sample holder.

40. The system of claim 39, wherein: The identification tag includes an RFID tag, a smart tag, or a reader for reading an RFID tag or a smart tag.

41. A system according to any one of the preceding claims, wherein: The processing unit comprises at least one transport device configured for dispensing and / or removing components or liquids from sample containers present in a sample holder of the purification apparatus.

42. The system of claim 41, wherein: The delivery device is selected from a syringe, or a robotic arm equipped with one or more nozzles, needles and / or gun tips.

43. A system according to claim 41 or 42, wherein: The transport device is connected to one or more reagent reservoirs, waste vessels and / or harvest vessels.

44. A system according to claims 41 to 43, wherein: The carrier vehicle is configured to move.

45. A system according to any one of claims 4 to 44, wherein: The system contains at least two purification units.

46. ​​The system of claim 45, wherein: The devices are placed adjacent to each other.

47. A system according to claim 45 or 46, wherein: The devices are in a nested configuration.

48. The system according to any of the preceding claims, comprising at least one harvesting vessel for harvesting the end product.

49. A system according to any of the preceding claims, comprising one or more sample containers.

50. The system of claim 49, wherein: The sample container is disposable.

51. A system according to claim 49 or 50, wherein: The sample container is configured to have a volume of 0.25 mL to 100 mL, more preferably 0.25 mL to 500 mL, more preferably 0.25 mL to 1250 mL.

52. The system of claims 49 to 51, wherein: The sample container contains a liquid containing the target compound and magnetic particles.

53. The system of claim 52, wherein: The magnetic particles are dispensed into the sample container by one of the transport devices.

54. A system according to claim 52 or 53, wherein: The magnetic particles are silica-based magnetic particles.

55. The system of claims 52 to 54, wherein: The plurality of RNA molecules are capable of binding to the magnetic particles.

56. A system according to any one of the preceding claims, wherein: The system is arranged in a cabinet, preferably with a laminar flow generating unit.

57. The system of claim 56, wherein: A storage unit for storing ingredients is located outside the cabinet.

58. A system according to any of the preceding claims, comprising at least one computer processor operably connected to the IVT unit and / or processing unit.

59. A method for producing RNA, the method comprising an in vitro transcription step starting from a DNA template, wherein the IVT reaction is performed in multiple chambers, the in vitro transcription step comprising: (i) providing one or more IVT reagents in a first chamber of the plurality of chambers; (ii) repeating step (i) until at least a portion of the plurality of chambers have one or more IVT agents; (iii) performing the IVT reaction in a chamber having one or more IVT reagents; and Wherein the resulting IVT reaction is subsequently subjected to at least one purification step.

60. The method of claim 59, wherein: At least one purification step following the IVT uses magnetic particles capable of binding to the RNA molecules.

61. The method of claim 59 or 60, wherein: The final IVT reaction from one of the plurality of chambers is transferred to an intermediate vessel.

62. The method of claim 61, wherein: The IVT reactions of the multiple chambers were pooled in the intermediate vessel.

63. The method according to claims 61 to 62, wherein: A portion of the final IVT reaction from the intermediate vessel is subjected to at least one purification step.

64. The method according to claims 60 to 63, wherein: At least a portion of the IVT reagents are combined into an IVT premix.

65. The method of claim 64, wherein: The IVT premix contains at least dNTPs, template DNA, and one or more RNA polymerase buffer components.

66. The method of claim 64 or 65, wherein: The IVT reagents are stored in a storage unit, wherein the storage unit is fluidly connected to a pump system, and wherein the pump system is configured to provide a desired amount of one or more IVT reagents for preparing an IVT premix to an IVT premix container.

67. The method of claims 64 to 66, wherein: Prior to providing a portion of the IVT premix to one of the plurality of chambers, the IVT premix is ​​heated to a temperature of 35°C to 55°C.

68. The method according to any one of claims 64 to 67, wherein: The total volume of the premix is ​​equal to or greater than the sum of the volumes of the individual IVT reactions in the plurality of chambers.

69. The method according to any one of claims 60 to 68, wherein: RNA polymerase is added to at least one chamber of the plurality of chambers.

70. The method of claim 69, wherein: The RNA polymerase is added before or after the chamber receives a portion of the IVT premix.

71. The method of any one of claims 64 to 68, wherein: The RNA polymerase is part of the IVT premix.

72. The method of any one of claims 60 to 71, wherein: The IVT reagents include a capping reagent for co-transcriptional capping.

73. The method of claim 72, wherein: The capping occurs post-transcriptionally.

74. The method of any one of claims 60 to 73, wherein: The IVT reaction is terminated by adding at least DNAse and / or EDTA.

75. The method according to any one of the preceding claims 60 to 74, wherein: The IVT reaction or a portion of the IVT reaction containing RNA molecules is provided to one or more sample containers, wherein the magnetic particles are added to the sample containers.

76. The method according to any one of the preceding claims 60 to 74, wherein: The IVT reaction or a portion of the IVT reaction comprising RNA molecules is provided to one or more sample containers, wherein the sample containers comprise magnetic particles.

77. According to the method described in any one of the preceding claims 61 to 73, the RNA is bound to the magnetic particles, and then the magnetic particles are captured or guided to move toward a magnet unit located near the sample holder, thereby separating the RNA molecules bound to the magnetic particles from any remaining IVT liquid.

78. The method of claim 77, wherein: Remove remaining IVT fluid.

79. The method of claim 78, wherein: After removing the remaining liquid, additional liquid is added to the RNA bound to the magnetic beads and the separation step is repeated.

80. The method of any one of claims 62 to 77, wherein: The sample holder is subjected to mechanical movement to mix the IVT reaction or additional liquid with the magnetic particles.

81. The method of claim 80, wherein: The sample holder with the container is located on the base portion of the sample plate.

82. The method of claim 81, wherein: The sample plate is rotatable and can be rotated between or after capture steps to move the sample holder with the containers to a subsequent position.

83. The method according to any one of the preceding claims 61 to 82, wherein: The magnetic particles are added to the sample container via a syringe or a robotic arm equipped with one or more nozzles, needles and / or gun tips.

84. The method of claims 60 to 83, wherein: The addition and / or removal of liquids is performed by one or more syringes, pumps or a robotic arm equipped with one or more nozzles, needles and / or gun tips.

85. The method of claim 80, wherein: The mechanical motion is oscillation or agitation.

86. The method of claim 80, wherein: The mechanical movement prevents the magnetic particles from being captured or directed toward the magnet unit.

87. The method according to any one of the preceding claims 60 to 86, wherein: The magnetic particles are silica-based magnetic particles.

88. The method according to any one of the preceding claims 60 to 87, wherein: In the final step, RNA is eluted from the magnetic particles by adding elution buffer to the sample container.

89. The method of claim 88, wherein: After adding the elution buffer, the sample holder is subjected to mechanical movement, thereby mixing the magnetic particles with the elution buffer, and wherein once the movement stops, the magnetic particles move toward the magnet unit, thereby causing the elution buffer containing the RNA molecules to separate from the magnetic particles.

90. The method according to any one of claims 88 or 89, wherein The elution buffer containing the RNA molecules is removed from the sample container.

91. The method of claim 90, wherein: The RNA molecules are transferred to a harvest vessel or an intermediate vessel.

92. The method of any one of claims 88 to 90, wherein: The conductivity of the elution buffer is 0.001 mS / cm to 40 mS / cm, or the total salt concentration thereof is 0 mM to 50 mM.

93. The method of any one of claims 60 to 93, wherein: The purification step includes or precedes a post-transcriptional capping step.

94. The method of any one of claims 60 to 93, wherein: The purification step is part of a pre-capping step, which is followed by a post-transcriptional capping step.

95. The method according to any one of claims 93 or 94, wherein: After capping, a second purification step was performed.

96. The method of claim 96, wherein: The second purification step utilizes magnetic particles capable of binding the capped RNA molecules.

97. The method according to any one of claims 95 to 96, wherein: The second purification step is as described in any one of claims 61 to 96.

98. The method of any one of claims 60 to 98, wherein: The method is performed by a system as claimed in any one of the preceding claims.

99. Use of the system of any of the preceding claims for producing RNA, preferably mRNA, preferably capped mRNA.

Images