BE1030468B1 - SYSTEM AND METHOD FOR RNA PRODUCTION - Google Patents

Abstract

The current invention relates to a system for producing RNA, said system comprising an in vitro transcription (IVT) unit and a processing unit for downstream processing of said compound, wherein the TIV unit comprises a plurality of chambers , wherein each of the plurality of chambers is configured to receive one or more TIV reagents and to perform a TIV reaction based on a DNA template, thereby obtaining a plurality of RNA molecules; and in which the processing unit comprises a purification device for carrying out at least one purification step downstream of said plurality of RNA molecules, said purification device comprising one or more sample holders, said sample holders are suitable for receiving a container enclosing a liquid medium comprising RNA molecules and magnetic particles, and in which a magnet is positioned at each sample, said magnet unit is configured to capture or introduce movement of said magnetic particles and into in which the sample holders are configured to perform mechanical movement, such that the magnetic particles are mixed with the liquid. The invention also discloses a method for the production of RNA, more preferably mRNA.

Description

1 BE2022/5293

SYSTEM AND METHOD FOR RNA PRODUCTION

FIELD OF THE INVENTION

The present invention relates to a system and method for RNA production, enabling efficient large-scale production of RNA for additional downstream application such as production of RNA vaccines.

CONTEXT

Therapeutic nucleic acids including RNA molecules represent an emerging class of drugs. RNA therapeutics include mRNA molecules encoding antigens for use as vaccines. Additionally, it is envisaged to use RNA molecules for — replacement therapies, e.g. providing missing proteins such as growth factors or enzymes to patients. Furthermore, the therapeutic use of RNA molecules for genome editing (e.g. guide RNAs

CRISPR/Cas9) is considered. As a result, RNA therapeutics used in immunotherapy, gene therapy and vaccination belong to the most promising and fastest developing therapeutic areas in modern medicine.

Commonly established manufacturing processes for RNA molecules involve many separate manufacturing steps. In particular, the respective manufacturing steps are carried out by several different devices. Additionally, RNA manufacturing requires a significant degree of manual handling in a GMP-regulated laboratory performed by well-trained technical staff. As a result, currently established manufacturing processes are time-consuming, expensive, and require extensive laboratory space and laboratory equipment.

SUMMARY OF THE INVENTION

The present disclosure and embodiments thereof serve to provide a solution to one or more of the above-mentioned disadvantages. To this end, the present disclosure provides an automated system and method for RNA production. The system and method make it possible to obtain production

2 BE2022/5293 rapid, efficient and automated large-scale RNA production that complies with quality requirements for use in therapeutic products.

Conventionally, in order to handle the production of a large quantity of therapeutic products such as RNA, a considerable number of large instruments is required. The compactness of the design and the amount of supporting resources, however, became an important issue. The COVID-19 pandemic and subsequent shortage of COVID-19 RNA vaccines have shown that there is a significant need for mobile, easy-to-transport, and easy-to-operate solutions for RNA production. and RNA-based therapeutic products.

By combining certain steps in an efficient and ingenious way, thus making optimal use of the available space, the overall size of the current system is kept low. As a result, the system can easily be transported, even to remote locations. Additionally, due to the highly automated methodology, the system is easy to operate and does not require highly trained or highly technical personnel. The present method and system are (almost) free of manual handling, thus also considerably reducing the risk of contamination and the risk of degradation of the final product. Due to the (semi)continuous process, large quantities of RNA can be produced in a short time.

DESCRIPTION OF FIGURES

Figure 1 shows a representation of a system according to preferred embodiments of the disclosure.

Figure 2 shows a representation of a TIV unit according to preferred embodiments of the disclosure.

Figure 3 shows representations of purification devices according to preferred embodiments of the disclosure.

Figure 4 presents the principle of capturing magnetic beads using magnetic unit. In Fig. 4A the balls are free and in Fig. 4B the balls are — captured.

Figure 5 shows a representation of a sample holder according to one embodiment of the disclosure.

3 BE2022/5293

Figure 6 shows a representation of the sample holder according to another embodiment of the disclosure.

Figure 7 shows a schematic representation of one embodiment of the downstream processing unit, showing two adjacent purification devices.

Figure 8 shows a schematic representation of one embodiment of a downstream processing unit, showing two nested devices.

Figure 9 schematically shows a robotic arm according to one embodiment of the invention.

Figure 10 shows a representation of a station according to preferred embodiments of the disclosure.

DETAILED DESCRIPTION OF THE INVENTION

US 2021/0261897 describes a bioreactor for in vitro transcription. However, the system is only suitable for performing a first step in the RNA production chain, and does not provide a fully integrated system for RNA production. Furthermore, the bioreactor described here does not allow (semi-)continuous production of RNA.

This disclosure is intended to address at least some of the problems and disadvantages mentioned above. Furthermore, the present disclosure describes a system and method for large-scale (semi-)continuous production of RNA.

Definitions

Unless otherwise defined, all terms used in the disclosure of the invention, including technical and scientific terms, have the meaning generally accepted by those skilled in the art to which this invention belongs. Definitions of terms are included for convenience to better appreciate the teaching of the present invention.

As used herein, the following terms have the following meanings:

4 BE2022/5293 “Un”, “une”, “le” and “la” as used herein mean both the singular and the plural, unless the context clearly indicates otherwise. For example, “a compartment” means one or more compartments. “Approximately” as used herein in reference to a measurable value such as a parameter, quantity, temporal duration and the like, is intended to encompass variations of +/-20% or less, preferably +/- 10% or less, more preferably +/-5% or less, even more preferably +/-1% or less, and even more preferably +/-0.1% or less of the value specified and in relation thereto, to the extent that such variations are appropriate in the disclosed invention. However, it is understood that the value to which the modifier "approximately" refers is itself also specifically disclosed. “Comprising”, “comprising”, and “includes” and “consisting of”, as used herein, are synonymous with “include”, “including”, “includes” or “contain”, “containing”, “ contains" and are inclusive or open terms which specify the presence of the following, for example a component, and do not exclude or preclude the presence of additional unmentioned components, features, elements, members, steps known in art or disclosed herein.

Furthermore, the terms first, second, third and the like in the description and in the claims are used to distinguish between similar elements and not necessarily to describe a sequential or chronological order, unless specified. It is understood that the terms so used are interchangeable in appropriate circumstances and that embodiments of the invention described herein may operate in sequences other than those described or illustrated herein.

Citation of numeric ranges by limits includes all numbers and fractions subsumed within that range, as well as the quoted limits.

The expression "weight percent" or "weight percent" herein and throughout the description, unless otherwise defined, refers to the relative weight of the respective component based on the overall weight of the formulation.

As used herein, the term "magnetic particle" and variations thereof are intended to mean a particle having a magnetic core, e.g., paramagnetic or superparamagnetic, coated with at least one material having a surface on which a compound of interest can bind reversibly. 5

While the terms "one or more" or "at least one", such as one or more or at least one member(s) of a group of members, are clear per se, by way of further exemplification, the term includes, among other things, a reference to any one of said members, or to any two or more of said members, such as, for example, any number > 3, > 4,2 5, > 6 or > 7 etc. of said members, and up to all of said members.

Unless otherwise defined, all terms used in the disclosure of the invention, including technical and scientific terms, have the meaning generally accepted by those skilled in the art to which this invention belongs. Definitions of terms used in the description are included for convenience to better appreciate the teaching of the present disclosure. Any terms or definitions used herein are provided solely to facilitate understanding of the invention.

The reference throughout this specification to "an embodiment" means that a particular (particular) feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. at least one embodiment of the present invention. Thus, the expression "in one embodiment" appearing in various places throughout this specification does not necessarily always refer to the same embodiment, but it can. Furthermore, the particular characteristic points, structures or characteristics may be combined in any suitable manner, as would be apparent to those skilled in the art from this disclosure, in one or more embodiments. Additionally, while some embodiments described herein include some but not other feature points included in other embodiments, the combinations of feature points of different embodiments are intended to be within the scope of the current disclosure. , and to form different embodiments, as those skilled in the art would understand. For example, in the following claims, any of the claimed embodiments may be used in any combination.

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Systems and system components

In a first aspect, the current disclosure addresses a system for the production of RNA. The term RNA or RNA molecules includes long-chain RNA, coding RNA, non-coding RNA, long non-coding RNA, single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), linear RNA ( RNAi), circular RNA (circRNA), messenger RNA (mRNA), self-amplifying mRNA (SAM), trans-amplifying mRNA, RNA oligonucleotides, antisense oligonucleotides, small interfering RNA (small RNAi ), small hairpin RNA (shRNA), antisense RNA (asRNA), CRISPR/Cas9 guide RNAs, ribosgulators, immunostimulatory RNA (siRNA), ribozymes, aptamers, ribosomal RNA (rRNA), a transfer RNA (tRNA), a viral RNA (vRNA), a

Retroviral RNA or replicon RNA, small nuclear RNA (small nRNA), small nucleolar RNA (small noRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA). In some embodiments, said RNA includes modified RNA molecules. In some embodiments, modification of the RNA molecule includes chemical modifications including backbone modifications as well as sugar modifications or base modifications. In this context, a modified RNA molecule as defined herein includes nucleotide analogues/modifications, e.g. skeletal modifications, sugar modifications, or base modifications. A backbone modification related to the present disclosure is a modification in which phosphates of the backbone of nucleotides contained in an RNA molecule are chemically modified. A sugar modification related to the present disclosure is a chemical modification of the sugar of the nucleotides of the RNA molecule. Furthermore, a base modification related to the present disclosure is a chemical modification of the base group of the nucleotides of the RNA molecule. In this context, the nucleotide analogs or modifications are selected from the nucleotide analogs, which are applicable to transcription and/or translation. In additional embodiments, the modified RNA comprises nucleoside modifications selected from 6-aza-cytidine, 2-thio-cytidine, a-thio-cytidine, pseudo-iso-cytidine, 5- aminoallyl-uridine, 5-iodo-uridine, N1-methylI-pseudouridine, 5,6-dihydrouridine, a-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5- hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, a-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8- oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-

7 BE2022/5293 methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, a-thio-adenosine, 8-azido-adenosine, 7 -deaza-adenosine.

In one embodiment, a system for producing RNA is disclosed, said system comprising an in vitro transcription (IVT) unit and a processing unit for downstream processing of said RNA molecules, wherein the processing unit

TIV includes a plurality of chambers, wherein each of the plurality of chambers is configured to receive one or more TIV reagents and to perform a TIV reaction based on a DNA template, thereby obtaining a plurality of molecules RNA; and in which the processing unit comprises a purification device for carrying out at least one purification step downstream of said plurality of RNA molecules, said purification device comprising one or more sample holders, said sample holders are suitable for receiving a container enclosing a liquid medium comprising RNA molecules and magnetic particles, and in which a magnet is positioned at each sample, said magnet unit is configured to capture or introduce movement of said magnetic particles and into in which the sample holders are configured to perform mechanical movement, such that the magnetic particles are mixed with the liquid. Advantageously, the system as disclosed herein allows the automation of the IVT of the RNA molecule and the downstream purification of said RNA molecules while performing the procedures continuously and with high precision without requiring human intervention. The production protocols can be implemented directly on the system as disclosed herein without any upscaling of the process since the device mimics small-scale manual operations performed in a laboratory.

The TIV unit should be understood as the part of said system which accommodates a TIV reaction starting from a DNA template, by means of a DNA-dependent RNA polymerase. In some embodiments, the VDNA-dependent RNA polymerases include at least one of a T7 RNA polymerase, a T3 RNA polymerase, an SP6 RNA polymerase, an RNA polymerase I, an polymerase II, an RNA polymerase III, an RNA polymerase IV, an RNA polymerase V, and a single subunit RNA polymerase. The DNA template for in vitro RNA transcription can be obtained by cloning a nucleic acid, in particular cDNA corresponding to the respective RNA to be transcribed in vitro, and introducing the same into a vector appropriate

8 BE2022/5293 for in vitro transcription of RNA, for example in circular plasmid DNA. cDNA can be obtained by reverse transcription of mRNA or chemical synthesis. Additionally, the DNA template for in vitro RNA synthesis can also be obtained by gene synthesis.

In some embodiments, the DNA template is a DNA molecule comprising a nucleic acid sequence encoding the RNA sequence. The template DNA is used as a template for in vitro RNA transcription to produce VRNA encoded by the template DNA. Therefore, template DNA includes all elements necessary for in vitro RNA transcription, in particular a promoter element for the binding of a DNA-dependent RNA polymerase such as e.g. RNA polymerases T3, T7 and SP6 5’ of the DNA sequence encoding the target RNA sequence. The poly(A) tail can either be encoded in the DNA template or added enzymatically to the RNA in a separate step after — in vitro transcription. In some embodiments, the template DNA includes primer binding sites 5' and/or 3' of the DNA sequence encoding the target RNA sequence to determine the identity of the target RNA sequence. DNA encoding the target RNA sequence e.g. by PCR or DNA sequencing. In some embodiments, the DNA template includes a DNA vector, such as plasmid DNA, that includes a nucleic acid sequence encoding the RNA sequence. In some embodiments, the DNA template includes a linear or circular DNA molecule.

In some embodiments, the chambers of the IVT unit are housed in a cartridge. In a further embodiment, the cartridge includes between 2 and 48 chambers.

In certain embodiments, the cartridge comprises at least 2 chambers, 3 chambers, 4 chambers, 5 chambers, 6 chambers, 7 chambers, 8 chambers, 9 chambers, 10 chambers, 11 chambers, 12 chambers, 13 chambers, 14 chambers, 15 rooms, 16 rooms, 17 rooms, 18 rooms, 19 rooms, 20 rooms, 21 rooms, 22 rooms, 23 rooms, 24 rooms, 25 rooms, 26 rooms, 27 rooms, 28 rooms, 29 rooms, 30 rooms, 31 rooms , 32 rooms, 33 rooms, 34 rooms, 35 rooms, 36 rooms, 37 rooms, 38 rooms, 39 rooms, 40 rooms, 41 rooms, 42 rooms, 43 rooms, 44 rooms, rooms, 46 rooms, 47 rooms, 48 rooms , 49 rooms or 50 rooms. In certain embodiments, the cartridge comprises at least

9 BE2022/5293 approximately 60 rooms, approximately 70 rooms, approximately 80 rooms, approximately 90 rooms, approximately 100 rooms, approximately 110 rooms, approximately 120 rooms, approximately 130 rooms, approximately 140 rooms, approximately 150 rooms, approximately 160 rooms, approximately 170 rooms, approximately 180 rooms, approximately 190 rooms or approximately 200 rooms.

In one embodiment, the TIV unit includes 1 cartridge, 2 cartridges, 3 cartridges, 4 cartridges, 5 cartridges, 6 cartridges, 7 cartridges, 8 cartridges, 9 cartridges or 10 cartridges. In another embodiment, said TIV unit comprises between 1 and 10 cartridges, between 1 and 20 cartridges, between 1 and 30 cartridges or between 1 and 40 cartridges, 1 and 50 cartridges, 1 and 60 cartridges, 1 and 70 cartridges , 1 and 80 cartridges, 1 and 90 cartridges, or 1 and 100 cartridges.

In one embodiment, the chamber of the IVT unit includes 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 a sensor and/or a probe.

In one embodiment, the cross section of the chamber is polygonal, such as trigonal, tetragonal, pentagonal or hexagonal. In a preferred embodiment, the cross section of the chamber is hexagonal. A plurality of hexagonal chambers, e.g. combined in a cartridge, can create a honeycomb-like structure. Without wishing to be bound by theory, the hexagonal shape is known in geometry to present the best fit to a plane having units of equal size without leaving any unused space. Additionally, the hexagonal arrangement also minimizes the perimeter for a given area due to its 120-degree angles, and thus the hexagonal shape of the chamber ensures less use of raw materials.

In one embodiment, the open end of the chamber is configured to receive a removable plug to at least partially close said open end. The cap prevents unwanted components from entering said chamber (e.g., RNases, microbial contamination or other degradative compounds or organisms) and protects the contents of said chamber from the external environment. In one embodiment, said plug also prevents the contents of said chamber from evaporating. In certain embodiments, the chamber includes the plug to limit the exchange with

10 BE2022/5293 the environment. In some embodiments, the chamber plug is removable. In some embodiments, the chamber plug is not removable. Alternatively, the bedroom is not covered.

In some embodiments, the plug can prevent excessive evaporation of water and loss of other critical volatile components. In some embodiments, the cap can prevent oxidation of the reagents or any other components. In some embodiments, the cap may provide protection from light (if necessary). In some embodiments, the cap prevents contamination of any other potential chemical compounds.

In some embodiments, the cap includes at least one opening for filling, draining, and sampling. In some embodiments, the at least one opening is positioned above the cap.

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

In some embodiments, the chamber may be removed from the cartridge.

In some embodiments, the chamber cannot be removed from the cartridge.

A system according to any preceding claim, wherein the chambers comprise a volume of approximately 0.1 ml to 500 ml.

In some embodiments, the chamber includes a volume of 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 ml, 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 mi, about 20 ml , about 25ml, about 30ml, about 35ml, about 40ml, about 45ml, about 50ml, about 55ml, about 60ml, about 65ml, about 70ml, about 75ml, about 80ml, about 85ml, approx. 90ml, approx. 95ml or approx. 100ml. In some embodiments, the chamber includes a volume of not more than about 100 ml, not more than about 95 ml, not more than about 90 ml, not more than about 85 ml, not more than about 80 ml ml, not more than about 75 ml, not more than about 70 ml, not more than about 65 ml, not more than about 60 ml, not more than about 55 ml, not more than about 50 ml, no more than about 45 ml, no more

11 BE2022/5293 of approximately 40 ml, not more than approximately 35 ml, not more than approximately 30 ml, not more than approximately 25 ml, not more than approximately 20 ml, not more than approximately 15 ml, not more than about 10 ml, not more than about 9 ml, not more than about 8 ml, not more than about 7 ml, not more than about 6 ml, not more than about 5 ml, not more of about 4 ml, not more than about 3 ml, not more than about 2 ml, not more than about 1 ml, not more than about 0.5 ml, not more than about 0.3 ml or not more than about 0.1 mi. In some embodiments, the chamber includes a volume between about 1 ml to about 100 ml, between about 10 ml to 90 ml, between about 15 ml to about 80 ml, between about 20 ml to about 70 ml, between about 25 ml to approximately 60 ml or between approximately 30 ml to approximately 50 ml.

In some embodiments, the chamber includes a volume of 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 600ml, about 650ml, about 700ml, about 750ml, about 800ml, about 850ml, about 900ml, about 950ml, about 1000ml, about 2000ml, about 3000ml, about 4 000ml, approx. 5000ml, approx. 6000ml, approx. 7000ml, approx. 8000ml, approx. 9000ml, approx. ml, about 35,000 ml, about 40,000 ml, about 45,000 ml or about 50,000 mi. In some embodiments, the chamber includes a volume of not more than about 50,000 ml, not more than about 45,000 ml, not more than about 40,000 ml, not more than about 35,000 ml, not more of approximately 30,000 ml, not more than approximately 25,000 ml, not more than approximately 20,000 ml, not more than approximately 15,000 mi, not more than approximately 10,000 ml, not more than approximately 9,000 ml, not more than about 8,000 ml, not more than about 7,000 ml, not more than about 6,000 ml, not more than about 5,000 ml, not more than about 4,000 ml, not more than 'about 3,000 ml, not more than about 2,000 ml, not more than about 1,000 ml, not more than about 950 ml, not more than about 900 ml, not more than about 850 ml, not more of approximately 800 ml, not more than approximately 750 ml, not more than approximately 700 ml, not more than approximately 650 ml, not more than approximately 600 ml, not more than approximately 550 ml, not more than about 500 ml, not more than about 450 ml, not more than about 400 ml, not more than about 350 ml, not more than about 300 ml, not more than about 250 ml, not more than about 200 ml, not more than about 150 ml. In some embodiments, the chamber comprises a volume between approximately 150 ml to approximately 50,000 ml, between approximately 200 ml to approximately 45,000 ml, between approximately 250 ml to approximately 40,000 ml, between approximately 300 ml to approximately 35,000 ml, between approximately 350 ml to approximately 30,000 ml, between approximately 400 ml to approximately 25,000 ml, between approximately

12 BE2022/5293 450 ml to approximately 20,000 ml, between approximately 500 ml to approximately 15,000 ml, between approximately 550 ml to approximately 10,000 ml, between approximately 600 ml to approximately 9,000 ml, between approximately 650 ml to 8,000 ml , between approximately 700 ml to approximately 7000 ml, between approximately 750 ml to approximately 6000 ml, between approximately 800 ml to approximately 5000 ml, between approximately 850 ml to approximately 4000 ml, between approximately 900 ml to approximately 3000 ml , between approximately 950 ml to approximately 2000 ml or between approximately 1000 ml to approximately 1500 ml.

In some embodiments, the TIV unit includes at least one handling apparatus configured to dispense and/or remove a reagent, a mixture of reagents, or a liquid into said chambers. Without being limiting, the liquid dispensed and/or eliminated may be deionized water, purified water, a buffer solution, a cleaning solution, a reagent, an enzyme, a DNA template, a transcription buffer , a solution comprising nucleotide triphosphates (NTPs), an RNase inhibitor, or an RNA polymerase, or a combination thereof, or a waste product. Non-limiting examples of buffers are magnesium acetate, HEPES, Tris, or Tris HCl. In a preferred embodiment, the liquid dispensed and/or disposed of is a TIV premix comprising water, RNA polymerase buffer, linearized DNA template, and dNTPs. In a further preferred embodiment, the TIV premix includes water, T7 polymerase buffer, linearized DNA template, and dNTPs.

The devices manipulating the IVT unit may be an injector or a robotic arm with one or more nozzles, needles and/or tips. Any injectors and robotic arms which are known in the art and are capable of dispensing and/or removing a component or liquid may be used with the system as disclosed herein. Non-limiting examples of handling devices are syringe pumps, vacuum pumps, peristaltic pumps, centrifugal pumps, or a combination thereof. In certain embodiments, the pipettes, micropipettes, needles or tips are removable and/or single-use. In other embodiments, the pipettes, micropipettes, needles or tips are not removable.

Alternatively, the distribution and/or elimination of components is carried out by the handling devices directly via the tubing with which they are equipped.

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In one embodiment, the IVT unit as disclosed herein includes a robotic arm. In another embodiment, the TIV unit includes one or more injectors. In yet another embodiment the TIV unit comprises one or more pumps. In yet another embodiment, the IVT unit includes a robotic arm and one or more injectors. In yet another embodiment, the IVT unit includes a robotic arm and one or more pumps. In a preferred embodiment, the IVT unit includes a robotic arm, one or more injectors, and one or more pumps.

The manipulation devices of the TIV unit are configured to perform movements. In some embodiments of the TIV unit, said movement is vertical, horizontal, centrifugal or 3D movement. In one embodiment, said handling devices are controlled by motorized units, preferably electric motors.

In some embodiments, the TIV unit includes a control system arranged and adapted to control the distribution and/or removal of a reagent, a mixture of reagents or a liquid by said handling apparatus . Based on the input information, such as sensor data and predefined algorithms, the control system regulates the performance of the handling devices.

In one embodiment of the TIV unit, the handling apparatuses are adapted to dispense a quantity of a TIV premix and/or one or more enzymes into said chambers.

In one embodiment, the system includes a storage unit for storing one or more reagents, said storage unit can be cooled to a temperature below 10°C. In one embodiment, one reagent, two reagents, three reagents, four reagents, five reagents, six reagents, seven reagents, eight reagents, nine reagents or ten reagents, preferably four reagents are stored in said storage unit. Reagents may be selected from, but are not limited to, water, T7 reaction buffer, linearized DNA template, and NTPs.

In some embodiments, the storage unit may be cooled below 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, 4°C, 3°C, or 1 °C, preferably cooled storage is at 4 °C.

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The storage unit is in fluid connection with a pump system, such as a peristaltic pump or a syringe pump. In additional embodiments, the pump includes a vacuum pump, a peristaltic pump, a centrifugal pump, or a combination thereof.

In one embodiment, said fluid connection is made via at least one tube. In some embodiments the tubing comprises a material, and is preferably made of a material selected from the group consisting of polyethylene (PE), nylon, urethane, copper, stainless steel, aluminum, polyvinyl chloride (PVC), polypropylene (PP), polyurethane (PU), vinyl, polyvinylidene fluoride (PVDF), fiberglass, glass, rubber, and combinations of these. In some embodiments, the tubing is chemical resistant.

In one embodiment, the pump system is configured to deliver a desired amount of one or more reagents to prepare a TIV premix to a TIV premix container.

In one embodiment, the system as disclosed herein includes heaters for heating the TIV premix container. In one embodiment, the TIV premix container is heated to a temperature between 35°C and 55°C, preferably between 35°C and 50°C, 35°C and 45°C, 35°C and 40°C. °C, or 35°C and 37°C. Alternatively, the TIV premix container is heated to a temperature between 37°C and 55°C, preferably between 40°C and 45°C and 55°C or 40 and 55°C. In a preferred embodiment, the premix container is heated to 37°C.

The system in some embodiments includes an intermediate reservoir for pooling the contents of a finalized TIV reaction from the plurality of chambers. In some embodiments, the intermediate storage tank is in fluid communication with the chambers. In other embodiments, the intermediate storage tank is not in fluid communication with the chamber. In some embodiments, the intermediate storage tank is configured to store RNA molecules produced by

TIV. In some embodiments, the intermediate storage tank is configured to detect contamination of RNA molecules. In certain embodiments, the intermediate storage tank contains a means for carrying out

15 BE2022/5293 some chemical, physical or mechanical treatment of RNA molecules and/or unwanted residues - for example, binding of the product, partial removal of residues, or adjustment of the pH value.

In one embodiment, the purification device of the processing unit disclosed herein comprises a sample plate, said sample plate comprises a base portion, and sample holders are provided on said base portion. The purification device makes it possible to obtain separation and/or purification of the RNA molecules which result from the TIV reaction, thus carrying out the downstream purification of RNA molecules in an automated manner.

In one embodiment, each sample holder of the purification device is configured to perform mechanical movement independently of the remaining sample holders. This makes it possible to carry out distinct steps of the separation and/or purification process in each sample holder. In other embodiments, the sample holders perform simultaneous movements.

In one embodiment, the mechanical movement of the sample holders is driven by a motor unit or an electromagnetic unit.

In a preferred embodiment, an electric motor is used. In another embodiment, a plurality of electromagnets are energized in sequence to generate movement of the sample holder. In another embodiment, the mechanical movement of the sample holders is driven by a shaker unit. The mechanical movement of the sample holders can be a rotation around an axis of said sample holder or a shaking or vibration movement. The mechanical movement of the sample holders can be turned on and off. The objective of mechanical movement includes, but is not limited to, homogenizing the mixture formed by the sample and the buffers for separation and/or purification, and/or facilitating contact between magnetic particles. and the sample.

In one embodiment, the sample holders can be positioned above the base plate or are positioned in pockets or recesses of said base plate.

In one embodiment, the magnet unit of each sample holder comprises a permanent magnet, a temporary magnet or an electromagnet, preferably one

16 BE2022/5293 permanent magnet. When an electromagnet is used, the magnetic field can be quickly changed by controlling the amount of electric current. In another embodiment each magnet unit comprises an array of magnets where each magnet can be a permanent magnet, a temporary magnet or an electromagnet.

In a preferred embodiment, magnet unit is positioned along a side portion of said sample holder and extends above said sample holder.

In another embodiment, the magnet unit is positioned around the sample holder.

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, said magnet units are positioned along different lateral portions of said sample holder. Alternatively, all the magnets are positioned along the same lateral portion of said sample holder.

In one embodiment, the magnet unit has the same length as the sample holder, or a shorter length. In a particularly preferred embodiment, the magnet unit is rod-shaped and at least 1/5th 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 carrier. In a preferred embodiment, the magnet unit has a height which is proportional to the height of the liquid level in the sample container. In a preferred embodiment, the height and thickness of said magnet unit are directly proportional to the surface area of the sample container, in order to ensure a sufficiently large magnetic field to capture the magnetic particles. In another embodiment, the magnet unit extends at least the entire length of the sample container, preferably extending below the sample container.

In yet another embodiment the magnet is in the shape of a bar, horseshoe, disc, sphere, cylinder or ring.

In one embodiment, the magnet unit is arranged in a housing, which is preferably open at the side facing the sample holder. Alternatively,

17 BE2022/5293 the housing is positioned around the sample holder and contains multiple magnets. In another embodiment, the magnet unit is ring-shaped and completely surrounds the sample holder.

The housing may be made from any suitable material known in the art, such as, but not limited to, polymers, thermoplastics, metals or metal alloys.

The magnet unit can be in a fixed or removable position. For example, the magnet unit can perform vertical or lateral movements. These movements can influence whether or not the magnetic particles will be attracted to the magnet.

If it is positioned too far away, the magnetic particles will remain in the liquid.

In one embodiment, an adapter is present in the sample holder, to adapt the size of said sample holder. The use of adapters allows the device to be used with sample containers of various shapes and sizes.

Adapters may be made of any material suitable in the art such as

PC (polycarbonate), PP (polypropylene), PAI (polyamide-imide) (e.g. Torlon), PI (polyimide) (e.g. Tecasint), PPS (polyphenylsulfide) (e.g. Tecatron), PPSU (polyphenylsulfone) (e.g. Tecason P), PSU (Polysulfone) (e.g.

Tecason S), PEI (polyetherimide) (e.g. Tecapei), glass (e.g. borosilicate glass), technical ceramics (e.g. FRIDURIT®),

Polyaryletherketone (e.g., Polyetheretherketone (PEEK)), thermoplastics (e.g., DuraForm® Pa or DuraForm® GF), a metal or an alloy of metals.

The sample plate of the purification device disclosed here is configured to rotate around an axis, preferably the central axis of said 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 movement. The rotation of the sample plate is driven by a motor unit, preferably an electric motor. Rotation of the sample plate allows delivery of the sample container positioned in the sample holder, to different stationary dispensers, where a component or liquid can be added to the sample container or removed from the sample container .

18 BE2022/5293

The base portion of said sample plate may be rectangular, polygonal, circular, ellipsoidal or annular. In one embodiment, the sample plate is circular and has a diameter between 20 and 50 cm, more preferably between 20 and 40 cm, such as 35 cm or 30 cm. In another embodiment, the sample plate is rectangular. The sample plate may be made from any suitable material known in the art such as polymers, metal, metal alloys, resins, or any non-magnetic or paramagnetic material. Polymers include, but are not limited to, polystyrene, PVC, Perspex or Lucite.

In one embodiment, the sample plate includes a plurality of sample holders, and said sample holders are positioned at regular intervals along the circumference of said base plate. In another embodiment, said plurality of sample holders are positioned at irregular intervals along the circumference of said base plate. Alternatively, the sample holders are positioned along the sides of said base plate.

In one embodiment, the plate may include a sample holder or a plurality of sample holders. The sample plate may comprise between 1 and 100, more preferably between 1 and 50 sample holders, more preferably between 1 and 20 sample holders, more preferably between 1 and 16 sample holders, more preferably between preferred 12 sample holder.

For example, a 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.

In one embodiment, the sample holder has a shape that is adapted to the shape of the sample container. In a preferred embodiment, the cross section of the sample holder is hexagonal. It would be obvious, however, to those skilled in the art that the cross section of the sample holder can have any shape, as described above, which can be adapted to the shape of the sample container.

In some embodiments, the sample holder can be removed from the sample plate. In some embodiments, the sample holder is a fixed part of the sample plate.

19 BE2022/5293

In one embodiment, the sample holders may include identification (ID) tags, to identify a sample container when present in said sample holder. The ID tags may include an RFID tag, a smart tag, or a reader for reading an RFID tag or a smart tag. Smart labels include, but are not limited to, QR codes and barcodes.

In some embodiments, the processing unit of the system includes at least one handling apparatus configured to dispense and/or remove a component or liquid from a sample container present in a sample holder of said purification device. Said particle component may be a magnetic particle and said liquid may be deionized water, purified water, buffered solution, wash buffer, elution buffer, reagent, or a combination thereof. this, or a residual product. It should be obvious that said liquid is not limited to these.

The handling devices used in conjunction with the purification device may include any device suitable in the art, such as an injector or a robotic arm, for example equipped with one or more nozzles, needles and/or tips. Alternatively, distribution by the handling devices is carried out directly via the tubing of the handling devices. Any injector, pump or robotic arm that is known in the art and is capable of dispensing and/or removing a component or liquid may be used with the system as disclosed herein. Non-limiting examples of handling devices are syringe pumps, vacuum pumps, peristaltic pumps, centrifugal pumps, or a combination thereof.

In one embodiment, the injectors and pumps are fixed dispensers placed near said sample plate and the sample container is brought to them by the rotation of the sample plate. In one embodiment, the robotic arm can move along three separate axes and is capable of accessing the sample container independent of rotation of the sample plate. In another embodiment, the robotic arm is positioned on the sample plate or in the center of said sample plate.

In one embodiment, at least one robotic arm is used in conjunction with the purification device. In another embodiment, one or more

20 BE2022/5293 injectors are used in conjunction with the purification device. In yet another embodiment one or more pumps are used in conjunction with the purification device. In yet another embodiment, a robotic arm and one or more injectors are used in conjunction with the purification device. In yet another embodiment, a robotic arm and one or more pumps are used in conjunction with the purification device.

In a preferred embodiment, a robotic arm, one or more injectors, and one or more pumps are used in conjunction with the purification device.

The handling devices used in conjunction with the purification device are connected to one or more reagent storages, waste tanks, and/or harvest tanks. Any reagent storage, waste tank and harvest tank known in the art may be used in conjunction with the system. Non-limiting examples include bags, vials, tubes, bottles, carboys or drums.

In one embodiment, the manipulation devices used in conjunction with the purification device are controlled by motorized units, preferably electric motors, and are configured to perform movements. In some embodiments, said movement is vertical, horizontal, centrifugal or 3D movement. In a preferred embodiment, the manipulators are operably coupled to at least one computer processor to control the manipulators.

The system may further comprise a control system arranged and adapted to control the distribution and/or elimination of a component or a liquid by said handling devices used in conjunction with the purification device. Based on the input information, such as sample ID and sensor data, and predefined algorithms, the control system regulates the performance of the handling devices.

In one embodiment, the system for producing RNA molecules as disclosed herein comprises a plurality of purification devices as described above. In another embodiment, the system includes two purification devices as described above. In one embodiment

21 BE2022/5293 additional said purification devices are positioned adjacent to each other. Alternatively, said purification devices are in a nested configuration. A multiple device system allows multiple concurrent purification steps to be carried out such that different steps of separation and/or purification can be carried out at the same time. In addition, a multiple device system allows for continuous operation of said system and processing of a large number of samples.

The system may include at least one harvest tank, for harvesting a compound of interest. In one embodiment, the system may include multiple harvest tanks. In another embodiment, the harvest reservoir is positioned in a port in the sample plate of the device. In yet another embodiment, the harvest tank is positioned in the system near the purification device. In yet another embodiment, all the purification devices are provided with at least one collection tank. In yet another embodiment, a purification device is equipped with a harvest tank while the other purification devices do not have a harvest tank.

The system includes one or more sample containers. Said sample containers are positioned in the sample holders of the purification device.

In one embodiment, the sample containers are disposable. In another embodiment, said containers can be reused multiple times.

In some embodiments, a material of the harvest tank and/or sample container of the purification device comprises a material that is resistant to, e.g., cleaning procedures (chemical resistant), temperature extremes ( e.g. denaturation of nucleic acids), extreme pH values (disinfection of the reactor with bases and acids, e.g. with NaOH), mechanical forces (e.g. friction caused by magnetic particles), and /or corrosion. In additional embodiments, the material of the harvest tank and/or sample container comprises a — correct light permeation material (transparent, translucent or opaque) for a corresponding objective. In some embodiments, the material of the harvest tank and/or sample container comprises a material of

22 BE2022/5293 correct gas permeation for a corresponding objective. In additional embodiments, the materials of the harvest tank and/or sample container must be thermally conductive at service temperatures between 37°C and 65°C (e.g., W/(mK) values of at least 10, preferably at least 15). In some embodiments, the internal surface of the harvest tank and/or sample container includes a surface material that does not release unwanted compounds that can contaminate the final product. In additional embodiments, the materials of the harvest tank and/or sample container and/or the internal surface thereof are PC (polycarbonate), PP (polypropylene), PAI (polyamide- imide) (e.g.

Torlon), PI (polyimide) (e.g. Tecasint), PPS (polyphenylsulphide) (e.g.

Tecatron), PPSU (polyphenylsulfone) (e.g. Tecason P), PSU (Polysulfone) (e.g. Tecason S), PEI (polyetherimide) (e.g. Tecapei), glass (e.g. glass borosilicate), technical ceramics (e.g. FRIDURIT®), Polyaryl Ether Ketone (e.g. Polyetheretherketone (PEEK)), thermoplastics (e.g. DuraForm® Pa or DuraForm® GF), all of which are resistant to chemicals, pH resistant and heat resistant. In additional embodiments, the materials of the harvest tank and/or sample container include a single-use material including, but not limited to, polyethylene terephthalate and other polyethylenes, polyvinyl acetate , polyvinyl chloride. In some embodiments, the materials of the harvest tank and/or sample container include a material exhibiting resistance to the sterilization process including steam treatment or exposure to ethylene oxide (EtO)/ gamma irradiation even before adding any reagent linked to the reaction. In some embodiments, the materials of the harvest tank and/or sample container provide light shielding (if necessary) for the media contained in the harvest tank and/or sample container. In some embodiments, the harvest reservoir and/or sample container are made of any non-magnetic or paramagnetic material known in the art.

In certain embodiments, the harvest tank and/or the sample container of the purification device are shaped like a piece of pie, shaped like a regular or irregular polygon, shaped like a concave polygon, shaped like a convex polygon, trigon shaped, quadrilateral polygon shaped, pentagon shaped, hexagon shaped, equilateral polygon shaped, equiangular polygon shaped, heptagon shaped, octagon shaped, shaped

23 BE2022/5293 nonagon, decagon shaped, hendecagon shaped, dodecagon shaped, tridecagon shaped, tetradecagon shaped, pentadecagon shaped, hexadecagon shaped, heptadecagon shaped, octadecagon shaped, enneadecagon shaped, icosagon shaped, n-gon shaped, or elliptical shaped, preferably circular shaped.

In some embodiments, the harvest reservoir and/or sample container of the purification device includes a volume of at least about 0.1 ml, about 0.3 ml, about 0.5 ml, about 1 ml , about 1.5ml, about 2ml, about 2.5ml, about 3ml, about 4ml, about 5ml, about 6ml, about 7ml, about 8ml, about 9ml, about 10ml, about 12ml, about 15ml, about 17ml, about 20ml, about 25ml, about 30ml, about 35ml, about 40ml, about 45ml, about 50ml, about 55ml, about 60ml, about 65ml , about 70 mi, about 75 mi, about 80 ml, about 85 ml, about 90 ml, about 95 ml or about 100 ml. In some embodiments, the harvest reservoir and/or sample container comprises a volume of not more than about 100 ml, not more than about 95 ml, not more than about 90 ml, not more than about about 85 ml, not more than about 80 ml, not more than about 75 ml, not more than about 70 ml, not more than about 65 ml, not more than about 60 ml, not more than about 55 ml, not more — about 50 ml, not more than about 45 ml, not more than about 40 ml, not more than about 35 ml, not more than about 30 ml, not more than about 25 ml , not more than about 20 ml, not more than about 15 ml, not more than about 10 ml, not more than about 9 mi, not more than about 8 ml, not more than about 7 ml, not more than about 6 ml, not more than about 5 ml, not more than about 4 ml, not more than about 3 ml, not more than about 2 ml, not more than about 1 ml, not more than 'about 0.5 ml, not more than about 0.3 ml or not more than about 0.1 ml. In some embodiments, the harvest reservoir and/or sample container includes a volume between about 1 ml to about 100 ml, between about 10 ml to 90 ml, between about 15 ml to about 80 ml, between about 20 ml ml to approximately 70 ml, between approximately 25 ml to approximately 60 ml or between approximately 30 ml to approximately 50 ml.

In some embodiments, the harvest reservoir and/or sample container of the purification device includes a volume of at least about 150 ml, about 200 ml, about 250 ml, about 300 ml, about 350 ml, about 400 mi, 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 mi, about 1000 ml, about 2000 ml, about 3000 ml,

24 BE2022/5293 approx. 4,000 ml, approx. 5,000 ml, approx. 6,000 ml, approx. 7,000 ml, approx. 8,000 ml, approx. 9,000 ml, approx. 10,000 ml, approx. 15,000 ml, approx. 20,000 ml, approx. 25,000ml, approx. 30,000ml, approx. 35,000ml, approx. 40,000ml, approx. 45,000ml or approx. 50,000ml. In some embodiments, the harvest tank and/or sample container comprises a volume of not more than about 50,000 ml, not more than about 45,000 ml, not more than about 40,000 ml, not more than about 35,000 mi, not more than about 30,000 ml, not more than about 25,000 ml, not more than about 20,000 ml, not more than about 15,000 ml, not more than about 10 000 ml, not more than about 9,000 mi, not more than about 8,000 ml, not more than about 7,000 ml, not more than about 6,000 ml, not more than about 5,000 ml, not more of approximately 4,000 ml, not more than approximately 3,000 ml, not more than approximately 2,000 ml, not more than approximately 1,000 ml, not more than approximately 950 ml, not more than approximately 900 ml, not more than about 850 ml, not more than about 800 ml, not more than about 750 ml, not more than about 700 ml, not more than about 650 ml, not more than about 600 ml, not more of approximately 550 ml, not more than approximately 500 ml, not more than approximately 450 ml, not more than approximately 400 ml, not more than approximately 350 ml, not more than approximately 300 ml, not more than about 250 ml, not more than about 200 ml, not more than about 150 ml. In some embodiments, the harvest reservoir and/or sample container include a volume between about 150 ml to about 50,000 ml, between about 200 ml to 45,000 ml, between about 250 ml to about 40,000 ml, between approximately 300 ml to approximately 35,000 ml, between approximately 350 ml to approximately 30,000 ml, between approximately 400 ml to approximately 25,000 ml, between approximately 450 ml to approximately 20,000 ml, between approximately 500 ml to approximately 15,000 ml, between approximately 550 ml to approximately 10,000 ml, between approximately 600 ml to approximately 9,000 ml, between ...— approximately 650 ml to approximately 8,000 ml, between approximately 700 ml to approximately 7,000 ml, between approximately 750 ml to approximately 6,000 ml , between approximately 800 ml to approximately 5000 ml, between approximately 850 ml to approximately 4000 ml, between approximately 900 ml to approximately 3000 ml, between approximately 950 ml to approximately 2000 ml or between approximately 1000 ml to approximately 1500 ml ml.

Preferably, the sample containers of the purification device are configured to hold a volume of 0.25 to 100 ml, more preferably 0.25 to 500 ml, more preferably 0.25 to 1000 ml , and more preferably from 0.25 to 1500 ml.

In some embodiments, the harvest tank and/or sample container includes a lid or cap, to prevent unwanted components from entering said harvest tank and/or container.

25 BE2022/5293 of sample (e.g. RNases, microbial contamination or other degrading compounds or organisms) and to protect the contents of the harvest tank and/or sample container from the external environment. In some embodiments, the harvest tank and/or the sample container include the cap to limit exchange with the environment. In some embodiments, the cap of the harvest tank and/or sample container is removable. In some embodiments, the cap of the harvest tank and/or sample container is not removable. Alternatively, the harvest tank and/or sample container does not have a lid.

In some embodiments, the plug can prevent excessive evaporation of water and loss of other critical volatile components. In some embodiments, the cap can prevent oxidation of the reagents or any other components. In some embodiments, the cap may provide protection from light (if necessary). In some embodiments, the cap prevents contamination of any other potential chemical compounds.

In some embodiments, the cap includes at least one opening for filling, draining, and sampling. In some embodiments, the at least one opening is positioned above the cap.

In one embodiment, the sample containers of the purification device comprise a liquid containing said compound of interest and magnetic particles.

In one embodiment, said liquid results from a TIV reaction. In another or additional embodiment, said liquid contains DNA, RNA, modified RNA, polypeptides, proteins and/or modified proteins. In yet another embodiment, said liquid contains at least one reagent. In yet another embodiment said liquid contains one or more reagents used for TIV. In yet another embodiment, said liquid comprises impurities. In a further embodiment said impurities are nucleotides, enzymes, proteins, DNA templates, dsRNA, or any other TIV by-product known in the art.

26 BE2022/5293

The magnetic particles may be any size suitable for binding to a nucleic acid, including commercially available sizes, such as a diameter ranging from about 0.3 µm to about 10 µm in diameter, e.g. e.g., approximately 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 µm in diameter, including all ranges and sub-ranges therebetween.

In one embodiment, said magnetic particles are silica-based magnetic particles, carboxyl-coated paramagnetic particles, silica-based paramagnetic particles, or combinations thereof. The silica-based magnetic particles may comprise, in some embodiments, a paramagnetic core coated with silica oxide, thereby providing a hydrated silica oxide adsorbent surface to which a nucleic acid can bind (e.g., a surface comprising silanol groups). The magnetic particles can, in additional embodiments, be surface modified to produce functionalized surfaces, such as weakly or strongly positively charged, weakly or strongly negatively charged, or hydrophobic surfaces, to name a few. . Alternatively, the magnetic particles may be polystyrene divinylbenzene particles, polymethacrylate particles, cross-linked agarose particles, or allyl dextran particles with N—N-bis acrylamide. It will be apparent to those skilled in the art that any material which is suitable for binding to nucleic acids, proteins or other biomolecules, can be used with the device or system as disclosed herein.

The magnetic particles of the system can be distributed to the sample containers by one of said handling devices.

The compound of interest is capable of binding to said magnetic particles by means of an affinity bond. In one embodiment, the compound of interest binds to the magnetic particle in the presence of a binding buffer, preferably a binding buffer containing a chaotropic agent. In the presence of the chaotropic agent, the compound of interest binds reversibly to the magnetic particle.

In another embodiment, the magnetic particle is coated with a ligand that interacts with the compound of interest.

27 BE2022/5293

The system as disclosed herein can be arranged in a station, preferably with a unit for generating laminar flow. In one embodiment, the storage unit for storing ingredients is positioned outside said station.

In one embodiment, the station is designed to allow the supply of filtered and sterile air to be circulated inside the units. The means of air filtration may include, for example, an HVAC system comprising filters

HEPA.

The station housing may be made of any material suitable in the art such as a metal alloy, metal or plastic. In one embodiment, a station is manufactured from a material including aluminum or stainless steel. In a specifically preferred embodiment, said station is made of a material including stainless steel.

Preferably, the system and station are designed and operated in such a way that they require only limited manipulation by the operator. This is intended to avoid contamination and disruption of process conditions.

If irregularities are observed, the operator can manipulate the process through one or more control devices present inside or outside the station. These controllers control the process (or parts thereof) taking place in the station. The station may be coupled to one or more controllers that are configured to perform multivariate analysis, automatically control process operation, and optionally communicate with remote components (using, for example, network protocols) to control operation in the unit(s).

Each station is preferably mobile and equipped with a means of transport. The means of transportation may include any means suitable in the art, controlled both manually and/or electronically, and include, but not limited to, wheels, tracks or rollers.

In one embodiment, the system as disclosed herein includes at least one computer processor operably coupled to the TIV unit and/or the processing unit. In one embodiment, the system comprises at least

28 BE2022/5293 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 computer processors. In another embodiment, the TIV unit and the processing unit are operably coupled to the same computer processor. In another embodiment, the TIV unit and the processing unit are operatively coupled to different computer processors. In yet another embodiment the plurality of chambers and manipulators of the TIV unit are operably coupled to different computer processors. In yet another embodiment the plurality of chambers and manipulation apparatuses of the TIV unit are operably coupled to the same computer processors. In yet another embodiment the purification device and the devices for manipulating said device are operationally coupled to different computer processors. In yet another embodiment the purification device and the devices for manipulating said device are operationally coupled to the same computer processors. In yet another embodiment all the manipulation devices are coupled to the same computer processor.

Methods

The disclosure also includes methods for producing RNA. In a particularly preferred embodiment, said method is executed by means of a system as described in one of the embodiments above.

In one embodiment, the TIV reaction is carried out in a plurality of chambers and includes an in vitro transcription step starting from a DNA template. The steps of the ICT may be: (i) providing one or more TIV reagents into a first chamber of the plurality of chambers; (ii) repeating step (i) until at least a portion of the plurality of chambers is provided with one or more TIV reagents; (ii) carrying out said TIV reaction in chambers equipped with one or more TIV reagents; and wherein the resulting TIV reaction is then subjected to at least one purification step.

29 BE2022/5293

Without wishing to be bound by theory, the reaction conditions that must be satisfied in a TIV reaction are the provision of linear DNA which serves as a template which is copied as RNA by a polymerase, dNTPs, RNA polymerase which incorporates dNTPs, a magnesium-containing buffer which catalyzes the reaction and a specific reaction temperature, typically 37°C. It will be apparent to those skilled in the art that any reaction condition which yields mRNA can be used with the method as described herein.

In one embodiment of the method disclosed herein, at least a portion of said TIV reagents are combined in a TIV premix. In a further embodiment said TIV premix comprises at least dNTPs, template DNA, and one or more RNA polymerase buffer components.

In one embodiment, the TIV reagents are stored in a storage unit, wherein the storage unit is in fluid connection with a pump system and wherein said pump system is configured to provide the desired amount of one or more TIV reagents to prepare a premix of

TIV to a TIV premix container. Said storage unit can be cooled to a temperature below 10°C, preferably 4°C, as described above.

The total volume of the premix is greater than or equal to the sum of the volumes of the individual TIV reactions in said chambers, in one embodiment.

In one embodiment, the TIV premix is heated to a temperature between 35°C to 55°C before a portion of said premix is supplied to one of said plurality of chambers. In one embodiment, said TIV premix is heated to a temperature between 35°C and 55°C, preferably between 35°C and 50°C, 35°C and 45°C, 35°C and 40°C , or 35°C and 37°C, before a portion of said premix is supplied to one chamber of said plurality of chambers. Alternatively said TIV premix is heated to a temperature between 37°C and 55°C, preferably between 40°C and 45°C and 55°C or 40 and 55°C before a portion of said premix is supplied to one chamber of said plurality of chambers.

In one embodiment, the TIV premix container is heated to a temperature between 35°C and 55°C, preferably between 35°C and 50°C, 35°C and °C, 35°C and 40°C. C, or 35°C and 37°C. Alternatively, the premix container

30 BE2022/5293 from TIV is heated to a temperature between 37°C and 55°C, preferably between 40°C and 45°C and 55°C or 40 and 55°C. In a preferred embodiment, the premix container is heated to 37°C.

In one embodiment of the method disclosed herein, an RNA polymerase is added to at least one chamber of said plurality of chambers. In other embodiments, a plurality of RNA polymerases are added to the chamber, preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10 RNA polymerases. In a further embodiment, said RNA polymerase is added before or after said chamber has received a portion of said TIV premix. Combining the TIV premix with the RNA polymerase triggers the start of the reaction. In some embodiments, it is desirable that the premix is heated in the absence of the RNA polymerase and thus the start of the reaction is delayed until the premix and the RNA polymerase are combined into the plurality of bedrooms. In other embodiments, said RNA polymerase is part of said TIV premix.

The RNA polymerase used is as described previously.

In some embodiments of the method, the TIV reagents include a capping reagent for co-transcriptional capping. Non-limiting examples of co-transcriptional capping reagents include CleanCap (TriLink) or ARCA (CellScript). Alternatively, capping takes place after transcription using any capping-like structure known in the art.

Preferably, the system as disclosed herein is used to purify an mRNA molecule from a TIV process, or for purification during pre- and/or post-capping. In one embodiment, when the mRNA molecule is co-transcriptionally capped, said mRNA is purified after capping. In another embodiment, when the mRNA is post-transcriptionally capped, the purification step is performed before capping. In yet another or additional embodiment, the purification is carried out after styling. In yet another embodiment, a purification step is performed before styling and a second purification step is performed after styling. The resulting product of IVT contains in addition to the desired mRNA product, a network of reaction by-products such as salts, nucleotides, enzymes, proteins, DNA or dsVRNA templates. These can interfere with the styling process, and reduce the efficiency of the transaction and the overall purity of the final product. Enzymatic capping immediately after IVT, without intermediate treatment of the product

31 BE2022/5293 reaction, produces reduced quantities or approaching 0% of capped mRNA molecules. The system disclosed here is designed to perform mRNA purification with high precision, in an automated manner under GMP-compliant conditions, and is adaptable to perform purification upstream and/or downstream of capping. The system provides continuous production of small or medium volumes of the compound of interest.

In some embodiments of the method, a finalized TIV reaction from one of the plurality of chambers is transferred to an intermediate reservoir. In a further embodiment, the TIV reactions of said plurality of chambers are grouped in said intermediate reservoir. In yet another embodiment, the transfer and pooling of the TIV reaction is performed using a robotic arm.

The TIV reaction is terminated by means of the addition of at least one DNA-ase in one embodiment of the method. In another embodiment, the TIV reaction is terminated by the addition of EDTA. In yet another embodiment, DNA-ase and/or EDTA are both added to terminate the TIV reaction. DNA-ase enzymatically digests the DNA template and thus terminates the TIV reaction while EDTA is a chelating agent which depletes the reaction of Mg?* ions, which also causes the TIV reaction to terminate.

In one embodiment, DNA-ase and/or EDTA is added to the plurality of chambers. In another embodiment, said DNA-ase and/or said EDTA is added to the intermediate reservoir. In an additional embodiment, DNA-ase and/or EDTA is added using a pump, injector or robotic arm.

In one embodiment, at least one purification step after TIV uses magnetic particles capable of binding to said RNA molecules. In a further embodiment, the TIV reaction comprising RNA molecules or a portion of said TIV reaction is supplied to one or more sample containers positioned in a sample holder. In a further embodiment said magnetic particles are added to said sample container. Alternatively, the sample container comprises magnetic particles and the TIV reaction comprising RNA molecules or a portion of said TIV reaction is provided to said container.

32 BE2022/5293

In some embodiments of the method, the RNA binds to the magnetic particles and then the magnetic particles are captured or induced to move toward a magnet unit present near the sample holder, thereby causing separation of the molecules. of RNA bound to said magnetic particles and any remaining TIV liquid. In some embodiments, a binding buffer is used to mediate reversible binding between the compound of interest and the magnetic particles. In a further embodiment said binding buffer may comprise a chaotropic agent, an alcohol, a PEG, a salt, or a mixture thereof. Said chaotropic agent can be chosen from guanidine salts, such as a guanidium hydrochloride (GuHCl) and a thiocyanate (GuSCN); lithium salts, such as lithium acetate and lithium perchlorate; or sodium salts such as NaCl and combinations thereof. In one embodiment, said binding buffers are free of chaotropic agents. — The alcohol may be chosen from isopropanol, ethanol, methanol, butanol, and combinations thereof. In one embodiment, said alcohol is present at a concentration of 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20, 15% to 20% v/v, including all ranges and subranges between them.

In one embodiment, said binding buffer may comprise a PEG, either as an alternative to alcohol or in combination with said alcohol. The concentration of PEG in the binding buffer may range from 10% to 40%, 20% to 40%, 20% to 35%, 20% to 30%, or 25% to 35%, including all ranges and subranges between them. In one embodiment, 30% PEG is used. In one embodiment, said PEG used in the binding buffer is chosen 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.

The at least one salt may be present in the binding buffer in a concentration ranging from 0.1 M to 5 M, for example, 0.1 to 4 M, 0.1 M to 3 M , from 0.1 M to 2 M, from 0.1 M to 1 M, from 0.5 to 1 M, from 0.5 to 2 M, from 1 M to 2 M and from 2 M to 3 M and from 3M to 5M, including all ranges and subranges therebetween.

According to various embodiments, the at least one salt may be sodium chloride (NaCl).

33 BE2022/5293

In one embodiment, the binding buffer includes Tris-HCl, NaCl, EDTA, and ethanol.

In embodiments, said binding buffer may have a pH in a 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 in between. In embodiments, said binding buffer comprises at least one first alcohol and/or PEG, at least one salt and at least one optional chelating agent such as EDTA.

The magnetic particle may be present in the binding buffer in a concentration ranging from, for example, about 0.1 ug/µl to about 60 µg/µl, such as 0.5 µg to about 60 µg/µl. /pl, from about 0.75 ug/ul to about 55 ug/ul, from about 1 ug/ul to about 50 ug/ul, from about 2 ug/ul to about 45 pg/ul, from about 3 ug/yl to about 40 ug/yl, from about 4 ug/ul to about 35 ug/yl, from about 5 ug/yul to about 30 ug/yl, from about 6 ug/ul to about 25 ug /l, from about 7 ug/yl to about 20 ug/ul, from about 8 ug/ul to about 15 ug/ul, or from about 9 pg/yl to about 10 ug/yl, including all ranges and subranges between them. As a non-limiting embodiment, the at least one magnetic particle may be selected from Qbeads and may be present in the binding buffer B1 in a concentration ranging from about 0.5 ug/ul to about 5 ug/ul. In alternative embodiments, at least one magnetic particle may be chosen from the Grace beads and may be present in the binding pad.

Bi in a concentration ranging from about 2 ug/ul to about 60 ug/ul.

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

In an additional embodiment, the remaining TIV fluid is removed after separation of the RNA molecules from the remaining TIV fluid.

34 BE2022/5293

In yet another embodiment, after removal of the remaining liquid, additional liquid is added to said RNA bound to the magnetic beads and the separation step is repeated.

The sample holder is subjected in some embodiments of the method to mechanical movement, thereby allowing mixing of said TIV reaction or additional liquid with said magnetic particles.

In some embodiments of the method, the sample holder with a sample container rotates between or after a capture step to at least one subsequent position. The sample holders with containers are positioned on a base portion of a sample plate and said sample plate is rotatable. Rotation of the sample plate allows delivery of the sample container to different dispensers such as injectors, robotic arms or pumps, where a component or liquid can be added to the sample container or removed from the sample container .

In other embodiments of the method, the sample plate rotates between or after a capture method step, thereby moving the sample holder with a sample container to a subsequent position.

In some embodiments of the method, the magnetic particles are added to the sample container by means of an injector or robotic arm having one or more nozzles, needles and/or tips, as previously described.

In additional embodiments, the addition and/or removal of a liquid takes place by means of one or more injectors, or robotic arms having one or more nozzles, needles and/or tips, such as described previously.

In one embodiment, the mechanical movement of the sample holder that allows the RNA to mix with the magnetic particles is shaking or agitation. Shaking or agitation of the sample holder causes the movement of the magnetic particles which are suspended in the liquid medium and come into contact with the compound of interest.

The magnet unit includes a permanent magnet in one embodiment of the method disclosed herein. The mechanical movement prevents magnetic particles from being captured or introduced into said magnet unit.

35 BE2022/5293

In a particularly preferred embodiment, the magnetic particles used in the method disclosed here are magnetic particles based on silica, as described above. In one embodiment of the method, a binding buffer as described above is used to mediate the binding of RNA to said magnetic particles.

Upon binding of the nucleic acid to the magnetic particles and after separation of the modified magnetic particles using a magnet, the magnetic particles may in some embodiments be combined, rinsed or washed with one or more wash buffers. A washing buffer may comprise, for example, at least one alcohol and optionally at least one salt.

The modified magnetic particles may be rinsed once or multiple times with the wash buffer, and any additional washes may employ the same or different compositions, concentrations and/or volumetric amounts. According to various embodiments, at least one alcohol can be chosen from isopropanol, methanol, ethanol, butanol, and combinations thereof. According to various embodiments, at least one salt can be chosen from ammonium sulfate ((NH4)2S04), ammonium acetate (NH4Ac), lithium acetate (LiAc), potassium acetate (KAc), sodium acetate (NaAc), sodium chloride (NaCl), and combinations thereof.

In embodiments of the methods as taught herein, said wash buffer comprises at least one second alcohol in a concentration ranging from 50% to 100% volume/volume (v/v), 55% at 95%, 60% to 85%, or 60% to 80% v/v, including all ranges and subranges in between. In embodiments, 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 as the first alcohol or different from the first alcohol in the binding buffer. In some embodiments, the second alcohol may be ethanol. In non-limiting embodiments, the washing buffer optionally comprises at least one salt. The optional salt, if any, in the binding buffer, is in a concentration ranging from 0.1 M to 5 M, e.g., 0.3 M to 4 M, 0.1 M to 3M, 0.1M to 2M, 0.1M to 1M, and 1M to 2M, including all ranges and subranges therebetween. In various embodiments, the salt in the wash buffer may be sodium chloride (NaCl). In certain embodiments, the modified magnetic particles can be washed one or more times with at least one of said washing buffers. For example, the said

36 BE2022/5293 modified magnetic particles can be washed once, twice, or more with the wash buffer with intervals separating the modified magnetic particles using a magnet between washes.

In some embodiments, the modified magnetic particles are rinsed once or several times with said wash buffer, and additional wash steps may be employed with, for example, the same or different compositions, concentrations and/or volumetric quantities.

After the addition and removal of wash buffer, the magnetic particles modified with the compound of interest reversibly bound to the surface are substantially free of contaminants such as salts, proteins, enzymes, etc.

According to various embodiments, the modified magnetic particles thus produced can then be incubated with one or more elution buffers to release the bound compound of interest and separate it from the magnetic particles.

According to various embodiments, the elution buffer is a low conductivity solution in which the conductivity of the buffer is in a range of 0.001 to 40 mS/cm, more preferably 0.01 to 40 mS/cm, from 0.1 to 40 mS/cm, from 0.5 to 40 mS/cm, more preferably from 0.5 to 30 mS/cm, from 0.5 to 20 mS/cm, from 0.5 to 10 MS/cm, including all ranges and subranges between them.

In another or additional embodiment, said elution buffer comprises a salt concentration between 0.01 and 50 mM, more preferably between 0.1 and 40 mM, more preferably between 0, 1 and 30 mM, more preferably between 0.1 and 20 MM. Possible salts include sodium citrate, sodium chloride, sodium phosphate, potassium chloride, potassium phosphate, and combinations thereof.

The pH of the elution buffer may range from, for example, 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 between them.

For example, in some cases the elution buffer may include water; in others water and VEDTA, or only Tris, or Tris and VEDTA, or

37 BE2022/5293 sodium citrate, or phosphate buffer, or phosphate-buffered saline (PBS). The concentration of sodium citrate, if used as an elution buffer, may be in the range of 0.5 mM to 10 MM, e.g. 0.6 MM to 5 mM, 1 mM to 2 MM, including all ranges and subranges therebetween. The pH of sodium citrate, if used as an elution buffer, can be in the range of 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 may comprise water or Tris-

10 MM HCl, 1 mM VEDTA, pH 7.4, or 10 MM Tris-HCl, pH 7.4, or citrate

Na 1 MM, pH 6.4.

In embodiments, said elution buffer is free of toxic chaotropic agents such as guanidine salts (guanidinium thiocyanate or guanidinium thiocyanate), iodide, perchlorate and trichloroacetate, preferably guanidine salts .

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

In one embodiment, when said elution buffer is added, the sample holder is subjected to mechanical movement, thus allowing the mixing of said magnetic particles with the elution buffer. Once the movement stops, the magnetic particles move towards said magnet unit, thus causing a separation of the elution buffer comprising the compound of interest and the magnetic particles. In a further embodiment, the elution buffer comprising the RNA is removed from said sample container and stored in a harvest reservoir.

The purification step includes or precedes a post-transcriptional capping step in some embodiments of the method disclosed herein. In one embodiment, the purification step is part of a pre-capping step and is followed by a post-transcriptional capping step.

In some embodiments, after styling, a second purification step is performed. In an additional embodiment, said second

38 BE2022/5293 purification step uses magnetic particles capable of binding to said capped RNA molecules. The second purification step is carried out as described previously for the first purification step.

Said second purification step can be carried out using the same purification device. In other embodiments, the second purification step is carried out in a second purification device. The two purification devices can be placed adjacent to each other or in a nested configuration.

In one embodiment, the system as disclosed herein is used in conjunction with end filtration. End filtration of mRNA can be performed after the TIV reaction and before loading onto the purification device. In another embodiment, end filtration is performed after the mRNA is purified with the purification device disclosed herein.

The method as disclosed herein is executed by means of a device or system according to embodiments described above. In one embodiment, the method is semi- or fully automated. The method is fast and efficient while reproducing repetitive manual procedures on a small scale. Said method complies with GMP requirements, is not prone to human error, and provides a high purity product.

The present disclosure will now be further illustrated with reference to the following examples. The present disclosure is in no way limited to the examples given or the embodiments presented in the figures.

FIGURES

The present disclosure is in no way limited to the embodiments described in the examples and/or shown in the figures. Rather, the methods according to the present disclosure can be performed in many different ways without departing from the scope of the disclosure.

Fig. 1. shows an embodiment of a system for producing RNA (11) comprising a TIV unit (34) and a downstream processing unit (33) and handling devices: robotic arms (7, 7”) , injectors (20, 21, 22, 27), and

39 BE2022/5293 pumps (19). Robotic arms (7, 7”) use needles and/or tips (18). In one embodiment, the compound of interest obtained using the method disclosed herein is further processed downstream in a container (26).

The TIV unit (34) is shown in Fig. 2. In one embodiment, a desired amount of reagents to prepare a TIV premix is supplied to a TIV premix container (24). The TIV unit (34) includes a heating unit (25) for heating the TIV premix container (24) to 37°C.

The premix contains in some embodiments dNTPs, template DNA, and one or more RNA polymerase buffer components. In one embodiment, heating is carried out before adding the RNA polymerase.

In another embodiment, the RNA polymerase is also added to the TIV premix container (24) and heated.

In a preferred embodiment, the TIV premix at 37°C is transferred to a plurality of chambers (23) where the TIV reaction is performed in the presence of RNA polymerase, resulting in a plurality of RNA molecules.

The IVT unit (34) includes manipulation devices, such as a robotic arm (7), injectors (20, 21, 22, 27) and/or pumps (19), configured to dispense and/or removing a reagent, reagent mixture or liquid into the premix container (24) and the plurality of chambers (23) arranged in cartridges (35). In the example of Fig. 2, the TIV unit (34) has 4 cartridges (35).

The RNA molecules are further purified in the downstream processing unit (33) which contains a purification device (1).

A purification device (1) for separating and/or purifying an RNA molecule obtained by TIV according to one embodiment of the current specification is illustrated in FIG. 3 in which the purification device is in the form of a carousel. According to the particular embodiment illustrated in Fig. 3, the device (1) comprises a sample plate (2) equipped with sample holders (3) arranged on the base portion (4) of the sample plate (2). In the embodiment of FIG. 3 the sample plate (2) includes eight sample holders (3). However, it will appear obvious to those skilled in the art that this number is flexible.

Accordingly, other embodiments are envisaged in which a different number of sample holders (3) are provided. Sample containers

40 BE2022/5293 (8) comprising the compound of interest in a liquid medium are positioned inside the sample holders (3).

A magnet unit (5) is positioned at each sample holder (3).

The magnet unit (5) as shown in the embodiment of Fig. 3 is arranged in a housing (6) which is open at the side facing the sample holder (3). In some embodiments, magnet unit (5) comprises a permanent magnet, in other embodiments comprises a temporary magnet or an electromagnet.

The sample holders (3) can perform mechanical movements which in some embodiments are rotations around their axis and in other embodiments are shaking movements. The sample holders (3) can move independently of each other or can synchronize their movements. In the example shown in Fig. 3 The movement is driven by a motor unit or a shaker unit. The sample plate (2) is configured to rotate about an axis clockwise and counterclockwise.

In the embodiment shown in Fig. 3A, a robotic arm (7) is positioned in the center of the sample plate (2). In the embodiment of FIG. 3B, the robotic arm (7) is positioned outside the sample plate (2) or the device (1). The robotic arm (7), equipped with a nozzle tool (13), a pipetting tool (17), needles and/or tips, is configured to dispense and/or remove components and/or or liquids from sample containers (8).

In one embodiment, the RNA to be purified is produced by a TIV reaction. A sample containing said RNA suspended in a reaction medium is distributed into a sample container (8) by a robotic arm (7). — Magnetic particles (9) are also distributed into the sample container (8) by the robotic arm (7).

The capture of magnetic particles (9) is shown in Fig. 4. The magnet unit (5) is configured to attract and capture said particles (9) residing in the sample container (8). The movement of the sample holder (3) determines the captured or free status of the silica beads (9). When the sample holder (3) is moving, the magnetic particles (9) are free to move in the liquid

41 BE2022/5293 and interact with RNA molecules (Fig. 4A). The RNA molecules bind reversibly to said silica beads (9). When the sample holder (3) stops its movement, the silica beads (9) become captured (Fig. 4B). In one embodiment where an electromagnet is used, the captured or free status of the magnetic particles is controlled by switching the electric current on and off.

While the magnetic particles (9) are captured inside the sample container (8), the liquid contents of said container can be removed or replaced without losing the magnetic particles (9) or the RNA bound thereto. . The distribution and removal of liquids in the sample container is carried out by handling devices such as robotic arms (7), injectors (20, 21, 22, 27) or pumps (19). The robotic arm (7) can move to any random location and can access any sample container (8). The injectors (20, 21, 22, 27) or pumps (19) are located at fixed positions and the sample containers (8) are brought thereto by the rotation of the sample plate (2) . The robotic arm (8) and other handling devices are connected to one or more reagent storages (29), waste tanks, and harvest tanks (28).

In one embodiment, the purification device (1) is designed to perform one or more RNA purification steps: binding of the RNA to magnetic particles in the presence of a chaotropic agent, washing of the bound RNA to silica beads, eluting the RNA, and collecting the purified RNA in a harvest tank.

This makes it possible to carry out multiple automated and precise washing steps, without losing the RNA. The device is configured to allow repetitive separation and/or purification of RNA and can be operated continuously.

In a preferred embodiment, the magnetic particles are silica-based magnetic beads.

In Fig. 5 an embodiment of the sample holder (3) with a sample container (8) having a capacity of 50 ml, is shown. Said sample container (8) is installed directly in the sample holder (3). It will be apparent that other sample containers may also be used in the context of the current disclosure.

42 BE2022/5293

Fig. 6 shows an embodiment of a sample holder (3) with a sample container (8") having a capacity of 2 ml. Said sample container (8') is installed in an adapter (10) which adapts the sample holder (3) to the size of the sample container (8).In other embodiments, sample containers (8') of other sizes and volumes are used.

A particular embodiment of the downstream processing unit (33) is shown in Fig. 7, wherein said downstream processing unit (33) comprises two purification devices (1 and 1”) and two robotic arms (7 and 7’) are provided.

In this embodiment, said purification devices are positioned adjacent to each other. This configuration makes it possible to obtain greater production capacity while keeping the size of the purification devices to a minimum. Alternatively, this configuration can be used for different purification processes. For example, a first purification device (1) can be used for pre-styling purification, while a second purification device (1%) can be used in a post-styling process. The capping reaction can take place in the final position of the first purification device (1), or in an intermediate reservoir positioned downstream of the first purification device (1) and upstream of the second purification device (1”).

An alternative embodiment is provided in Fig. 8, in which two purification devices (1 and 1’) are in a nested configuration and a single robotic arm (7) is provided.

A multi-purification device system as shown in Fig. 7 and Fig. 8, makes it possible to carry out multiple concomitant steps in such a way that different steps of separation and/or purification can be carried out at the same time.

The robotic arm (7) for handling liquid media and sample containers from/to a container according to one embodiment of the invention is illustrated in Fig. 9. Robotic arms (7) are used for the manipulation steps that take place both in the IVT unit (34) and in the downstream processing unit (33). One or more robotic arms (7, 7') can be used with the system (11).

43 BE2022/5293

The robotic arm shown in Fig. 9 includes a base (12), a pipetting tool (17), and a nozzle tool (13) configured to manipulate one or more liquid media. The nozzle tool (13) is positioned at a distal end of the robotic arm (7).

In the following, the robotic arm (7) is described as being mounted on a horizontal surface. Other modes of installation are of course possible, and adaptation of the following to such other modes of installation, falls within the scope of the skills of those skilled in the art, and is considered to be part of the scope of the invention. For example, the robotic arm (7) can be mounted on a vertical surface, resulting in a 90° rotation for all subsequent orientations.

The robotic arm (7) is an articulated robotic arm (7) comprising joints (14) and wherein the robotic arm 1 is manufactured by sequentially connecting these joints (14) by multiple links (15). The robotic arm (7) as shown in Fig. 7 includes six joints (14), allowing movement in six degrees of freedom. More specifically, in a first joint (14a), a base and a proximal end portion of a first link (15a) are connected so as to be rotatable about an axis extending in the vertical direction. The first joint (14a) is a torsion joint.

In a second joint (14b), a distal end portion of the first link (15a) and a proximal end portion of a second link (15b) are connected so as to be able to rotate around an axis extending in the horizontal direction. The second joint (14b) is a revolution joint.

In a third joint (14c), a distal end portion of the second connection (15b) and a proximal end portion of a third connection (15c) are connected so as to be able to rotate around an axis s extending in the horizontal direction, in this case, parallel to the axis for the second connection (15b).

It should be noted that deviations from the horizontality of this third axis are possible, but that the third axis will always have at least one horizontal component, its full horizontality being the most efficient version.

The third joint (14c) is in this case a revolution joint.

44 BE2022/5293

In a fourth joint (14d), a distal end portion of the third link (15c) and a proximal end portion of a fourth link (15d) are connected so as to be able to rotate around an axis in the longitudinal direction of the fourth link (15d). The fourth joint (14d) is a torsion joint.

In a fifth joint (14e), a distal end portion of the fourth link (15d) and a proximal end portion of a connector (16) are connected so as to be able to rotate around an axis orthogonal to the fourth axis. The fifth joint (14th) is a revolution joint but it should be noted that this joint can be easily adapted to a torsion joint or a rotation joint.

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

Each of the joints (14) is provided with a drive motor, for example an actuator for the relative rotation of the two parts connected by the joint (14). The drive motor is, for example, a servo motor which is servo-controlled via a servo amplifier by a control signal transmitted from the control device. In addition, each of the joints is provided with a rotation angle sensor to detect the rotation angle of the drive motor and a current sensor to detect the current of the drive motor.

In one embodiment, the system (11) is arranged in a station (32), preferably with a unit for generating laminar flow, as shown in Fig. 10. The station (32) includes a reactive storage unit (29) which in some embodiments is cooled down to 4°C, a unit for storing the compound of interest after processing (30), and a unit filter (31). The filter unit (31) allows the supply of filtered and sterile air to be circulated inside the station (32). The air filtration means may include, in some embodiments, an HVAC system having HEPA filters. — Numbers in Figures 1, 1': purification device

45 BE2022/5293 2: sample plate 3: sample holder 4: base portion of sample plate 5: magnet unit 6: housing

7, 7': robotic arm 8, 8': sample container 9: magnetic particles: adapter

10 11: system 12: base of the robotic arm 13: nozzle tool 14a-f: joints 15a-e: connections

16: connector 17: pipetting tool 18: needles or tips 19: syringe pumps 20, 21, 22, 27: injectors

23: plurality of chambers 24: TIV premix container: heating unit 26: container for downstream processing 28: harvest tank

25 29: reagent storage unit: compound storage unit 31: filter unit 32: station 33: downstream processing unit

30 34: TIV unit 35: cartridge

Claims (96)

46 BE2022/5293 CLAIMS 1. System for producing RNA, said system comprising an in vitro transcription (TIV) unit and a processing unit for downstream processing of said compound, wherein the TIV unit comprises a plurality of chambers, wherein each of the a plurality of chambers is configured to receive one or more TIV reagents and to perform a TIV reaction based on a DNA template, thereby obtaining a plurality of RNA molecules; and in which the processing unit comprises a purification device for carrying out at least one purification step downstream of said plurality of RNA molecules, said purification device comprising one or more sample holders, said sample holders are suitable for receiving a container enclosing a liquid medium comprising RNA molecules and magnetic particles, and in which a magnet is positioned at each sample, said magnet unit is configured to capture or introduce movement of said magnetic particles and into in which the sample holders are configured to perform mechanical movement, such that the magnetic particles are mixed with the liquid. 2. System according to claim 1, wherein the chambers of the TIV unit are housed in a cartridge. 3. System according to claim 2, in which the cartridge comprises between 2 and 48 chambers. 4. System according to any one of the preceding claims, wherein the chambers comprise a volume of approximately 0.1 ml to 500 ml. 5. System according to any one of the preceding claims, wherein the TIV unit comprises at least one handling apparatus configured to distribute and/or remove a reagent, a mixture of reagents or a liquid into said chambers. 6. System according to claim 5, in which said handling devices are chosen from an injector, or a robotic arm equipped with one or more nozzles, needles and/or tips. 7. System according to any one of claims 5 or 6, wherein said manipulation devices are configured to carry out a movement. 8. System according to any one of claims 5 to 7, comprising a control system arranged and adapted to control the distribution and/or elimination of a reagent, a mixture of reagents or a liquid by said handling devices. 47 BE2022/5293 9. System according to any one of claims 5 to 8, wherein said handling devices are adapted to distribute a quantity of a premix of TIV and/or one or more enzymes into said chambers. 10. System according to claim 1, wherein the system comprises a storage unit for storing one or more reagents, said storage unit can be cooled to a temperature below 10°C. 11. System according to claim 10, wherein the storage unit is in fluid connection with a pump system, such as a peristaltic pump or a syringe pump. 12.The system of any one of claims 10 or 11 wherein the pump system is configured to deliver a desired quantity of one or more reagents for preparing a TIV premix to a TIV premix container. 13. System according to any preceding claim wherein the system comprises heating means for heating the TIV premix container. 14. System according to any one of the preceding claims wherein the system comprises an intermediate reservoir for pooling the contents of a finalized TIV reaction from the plurality of chambers. 15. System according to any one of the preceding claims, wherein said purification device of said processing unit comprises a sample plate, said sample plate comprises a base portion and wherein said sample holder is provided on said portion basic. 16. System according to any one of the preceding claims, wherein each sample holder of said purification device is configured to carry out the mechanical movement independently of the remaining sample holders. 17. System according to any one of the preceding claims, wherein the mechanical movement of the sample holders is driven by a motor unit or an electromagnetic unit. 18. System according to any one of the preceding claims, in which the mechanical movement of the sample holders is driven by a shaker unit. 19. System according to any one of the preceding claims, in which the mechanical movement can be activated and deactivated. 48 BE2022/5293 20. System according to any one of the preceding claims, wherein said mechanical movement is a rotation around an axis of said sample holder or a shaking movement. 21. System according to any one of the preceding claims, wherein the sample holders are positioned above the base plate or are positioned in pockets or recesses of said base plate. 22. System according to any one of the preceding claims, wherein said sample plate is configured to rotate around an axis, preferably the central axis of said base portion or is configured to perform linear movement. 23. The system of claim 22, wherein said sample plate is configured to rotate clockwise and counterclockwise. 24. 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. 25. System according to claim 24, wherein said magnet unit is positioned along a lateral portion of said sample holder and extends above said sample holder. 26. System according to any one of claims 24 or 25 in which the magnet unit is rod-shaped and in which the magnet unit extends at least over the entire length of the sample container, preferably extending below the sample container. 27. System according to any one of claims 24 to 26, wherein the magnet unit is arranged in a housing. 28. System according to claim 27, wherein the housing is open at the side facing the sample holder. 29. System according to any one of claims 24 to 28, wherein the magnet unit is in a fixed or movable position. 30. System according to any one of the preceding claims, wherein an adapter is present in said sample holder, to adapt the size of said sample holder. 31. System according to any one of the preceding claims wherein the base portion of said sample plate is rectangular, polygonal, circular, ellipsoidal or annular. — 32. System according to any one of the preceding claims, wherein the sample plate comprises a plurality of sample holders, said holders 49 BE2022/5293 sample are positioned at regular or irregular intervals along the circumference of said base plate. 33. System according to claim 32, wherein said sample plate comprises between 1 and 20 sample holders, more preferably between 1 and 16 sample holders, more preferably 12 sample holders. 34. System according to any one of the preceding claims, wherein the sample holders include identification labels, to identify a sample container when present in said sample holder. 35. The system of claim 34, wherein the identification tags may include an RFID tag, a smart tag, or a reader for reading an RFID tag or a smart tag. 36. System according to any one of the preceding claims, wherein the processing unit comprises at least one handling apparatus configured to dispense and/or remove a component or liquid from a sample container present in a holder. sample of said purification device. 37. System according to claim 36, wherein said handling devices are chosen from an injector, or a robotic arm equipped with one or more nozzles, needles and/or tips. 38. System according to any one of the preceding claims, wherein said handling devices are connected to one or more reagent storages, waste tanks and/or harvest tanks. 39, System according to any one of the preceding claims, wherein said manipulation devices are configured to carry out a movement. 40. System according to any one of the preceding claims, comprising a control system arranged and adapted to control the distribution and/or elimination of a component or a liquid by said handling devices. 41. System according to any one of the preceding claims, comprising a plurality of devices according to claims 15 to 35. 42. System according to claim 41, wherein said system comprises at least two purification devices. 43. System according to any one of claims 41 to 42, wherein said devices are positioned adjacent to each other. 44. System according to any one of claims 41 to 43, wherein said devices are in a nested configuration. 45. System according to any one of the preceding claims, comprising at least one harvest tank, for harvesting the final product. 50 BE2022/5293 46. System according to any one of the preceding claims, said system comprising one or more sample containers. 47.System according to claim 46, wherein said sample containers are disposable. 48. The system of claim 46, wherein said sample containers are configured to hold a volume of 0.25 to 100 ml, more preferably 0.25 to 500 ml, more preferably 0.25 to 1250ml. 49. System according to claim 46, wherein said sample containers comprise a liquid containing said compound of interest and magnetic particles. 50. System according to claim 49, wherein said magnetic particles are distributed into said sample containers by one of said handling devices. 51. System according to claim 50, wherein said magnetic particles are silica-based magnetic particles. 52. System according to any one of claims 50 to 51, wherein said RNA is capable of binding to said magnetic particles. 53. System according to any one of the preceding claims, wherein said system is arranged in a station, preferably with a unit for generating laminar flow. 54. System according to claim 53, wherein the storage unit for storing ingredients is positioned outside said station. 55. System according to any one of the preceding claims, comprising at least one computer processor operatively coupled to the TIV unit and/or the processing unit. 56. A method for producing RNA, said method comprising an in vitro transcription step starting from a DNA template, wherein the TIV reaction is carried out in a plurality of chambers, said in vitro transcription step comprising: (1) providing one or more TIV reagents into a first chamber of the plurality of chambers; (ii) repeating step (i) until at least a portion of the plurality of chambers is provided with one or more TIV reagents; (iii) carrying out said TIV reaction in chambers equipped with one or more TIV reagents; and wherein the resulting TIV reaction is then subjected to at least one purification step. 51 BE2022/5293 57. The method of claim 56, wherein at least a portion of said TIV reagents are combined in a TIV premix. 58. The method of claim 57, wherein said TIV premix comprises at least dNTPs, template DNA, and one or more RNA polymerase buffer components. 59. Method according to claim 57, wherein the TIV reagents are stored in a storage unit, wherein the storage unit is in fluid connection with a pump system and wherein said pump system is configured to provide a desired amount of one or more TIV reagents to prepare a TIV premix to a TIV premix container. 60. The method of claim 59, wherein said TIV premix is heated to a temperature between 35°C to 55°C before a portion of said premix is supplied to one of said plurality of chambers. 61. Method according to any one of claims 57 to 60, wherein the total volume of the premix is greater than or equal to the sum of the volumes of the individual TIV reactions in said chambers. 62. The method of claim 61, wherein an RNA polymerase is added to at least one chamber of said plurality of chambers. 63. The method of claim 62, wherein said RNA polymerase is added before or after said chamber has received a portion of said TIV premix. 64. Method according to any one of claims 57 to 63, wherein said RNA polymerase is part of said TIV premix. 65. Method according to any one of claims 57 to 64, wherein said TIV reagents comprise a capping reagent for co-transcriptional capping. 66. Method according to claim 65, wherein the capping takes place after transcription. 67. Method according to any one of claims 56 to 66, wherein a finalized TIV reaction originating from one chamber of said plurality of chambers is transferred to an intermediate reservoir. 68. Method according to claim 67, wherein the TIV reactions of said plurality of chambers are grouped in said intermediate reservoir. 69. Method according to any one of claims 56 to 68, wherein the TIV reaction is terminated by means of the addition of at least one DNA-ase and/or VEDTA. 52 BE2022/5293 70. Method according to any one of claims 56 to 69, in which the at least one purification step after the TIV uses magnetic particles capable of binding to said RNA molecules. 71.The method of claim 70, wherein said TIV reaction comprising RNA molecules or a portion of said TIV reaction is supplied to one or more sample containers, wherein said sample container is positioned in a sample holder and wherein said magnetic particles are added to said sample container. 72.The method of claim 71, wherein said TIV reaction comprising RNA molecules or a portion of said TIV reaction is supplied to one or more sample containers, wherein said sample container is positioned in a sample holder and wherein said sample container comprises magnetic particles. 73. Method according to any one of claims 70 to 72, allowing said RNA to bind to said magnetic particles and then capturing or introducing movement of the magnetic particles towards a magnet unit present in the vicinity of said sample holder, thereby causing a separation of the RNA molecules bound to said magnetic particles and any remaining TIV liquid. —74.A method according to claim 73, wherein the remaining TIV fluid is removed. 75. The method of claim 74, wherein after removal of the remaining liquid, additional liquid is added to said RNA bound to the magnetic beads and the separation step is repeated. 76. Method according to any one of claims 71 or 72, wherein said sample holder is subjected to mechanical movement, thereby allowing mixing of said TIV reaction or additional liquid with said magnetic particles. 77. Method according to claim 76, wherein the sample holders with containers are positioned on a base portion of a sample plate, and wherein said sample plate is rotatable. 78. The method of claim 77, wherein the sample plate rotates between or after a capture step, thereby moving the sample holder with a container to a subsequent position. 79. Method according to any preceding claim, wherein the magnetic particles are added to the sample container by means 53 BE2022/5293 of an injector, or a robotic arm equipped with one or more nozzles, needles and/or tips. 80. Method according to claim 79, wherein the addition and/or removal of a liquid takes place by means of one or more injectors, pumps or robotic arms provided with one or more nozzles, needles and/or tips. 81. Method according to claim 76, wherein the mechanical movement is shaking or agitation. 82. The method of claim 73 wherein the magnet unit comprises a permanent magnet. 83.A method according to claim 76, wherein the mechanical movement prevents the capture or introduction of movement of the magnetic particles toward said magnet unit. 84. Method according to any one of the preceding claims, wherein said magnetic particles are silica-based magnetic particles. 85. A method according to any one of the preceding claims, wherein in a final step RNA is eluted from said magnetic particles by means of adding an elution buffer to said sample container. 86. Method according to claim 85, in which when adding said elution buffer, the sample holder is subjected to mechanical movement, thus allowing the mixing of said magnetic particles with the elution buffer and in which once the movement stops, the magnetic particles move towards said magnet unit, thus causing a separation of the elution buffer comprising the RNA molecules and the magnetic particles. 87. Method according to any one of claims 85 or 86, wherein the elution buffer comprising the RNA molecules is removed from said sample container. 88. Method according to claim 87 wherein said RNA molecules are transferred to a harvest reservoir or to an intermediate reservoir. 89. Method according to any one of claims 85 to 87 in which said elution buffer has a conductivity between 0.001 and 40 mS/cm or in which the total salt concentration is from 0 to 50 mM. 90. Method according to any one of claims 70 to 89, in which the purification step includes or precedes a post-transcriptional capping step. 91. Method according to any one of claims 70 to 89, in which the purification step is part of a pre-capping step and is followed by a post-transcriptional capping step. 54 BE2022/5293 92. Method according to any one of claims 90 or 91, in which after styling, a second purification step is carried out. 93. Method according to claim 92, wherein said second purification step uses magnetic particles capable of binding to said capped RNA molecules. 94. Method according to any one of claims 92 to 93, wherein said second purification step is as described in any one of claims 70 to 91. 95. Method according to any one of claims 56 to 94, wherein said method is carried out by means of a system according to any one of the preceding claims. 96. Use of a system according to any one of the preceding claims for the production of RNA, preferably mRNA, preferably capped mRNA.