Bioreactor for in vitro transcription of RNA

Abstract

The present invention relates to a bioreactor for RNA in vitro transcription, a method for RNA in vitro transcription, a module for transcription of DNA into RNA and an automated device for RNA manufacturing. Furthermore, the use of a bioreactor for RNA in vitro transcription as described herein is part of the present invention. The present invention relates to an RNA in vitro transcription reactor designed to be operable in an automated manner under GMP compliant conditions. In particular, the RNA in vitro transcription reactor allows for repeated use of a DNA template for various RNA in vitro transcription reactions. Furthermore, the present invention relates to a device for RNA manufacturing comprising (a) a module for template DNA synthesis, (b) a module for transcription of DNA into RNA, the module comprising the RNA in vitro transcription reactor; and optionally (c) a module for RNA formulation.

Description

TECHNICAL FIELD

[0001] The present invention relates to a bioreactor for RNA in vitro transcription, a method for RNA in vitro transcription, a module for transcribing DNA into RNA and an automated device for RNA manufacturing. Furthermore, the use of a bioreactor for RNA in vitro transcription as described herein is part of the present invention. The present invention relates to an RNA in vitro transcription reactor designed to be operable in an automated manner under GMP compliant conditions. In particular, the RNA in vitro transcription reactor allows for the reuse of a DNA template for various RNA in vitro transcription reactions. Furthermore, the present invention relates to a device for RNA manufacturing comprising: (a) a module for template DNA synthesis; (b) a module for transcribing DNA into RNA, the module comprising the RNA in vitro transcription reactor; and optionally (c) a module for RNA formulation. BACKGROUND

[0002] Therapeutic nucleic acids, including RNA molecules, represent an emerging class of drugs. RNA-based therapies include mRNA molecules encoding antigens as vaccines (Fotin-Mleczek et al., 2012. J. Gene Med. 14(6):428-439). In addition, it is also envisaged to use RNA molecules for replacement therapies, e.g. to provide patients with missing proteins, such as growth factors or enzymes (Kariko et al., 2012. Mol. Ther. 20(5):948-953; Kormann et al., 2012. Nat. Biotechnol. 29(2): 154-157). Furthermore, non-coding, immunostimulatory RNA molecules (e.g. WO 2009 / 095226 A2) and other non-coding RNAs, such as microRNAs, and long non-coding RNAs suitable for genome editing (e.g. CRISPR / Cas9 guide RNAs) or therapeutic use of RNAs are considered. Thus, RNA-based therapies belong to the most promising and fastest growing therapeutic areas in modern medicine with respect to their application in immunotherapy, gene therapy and vaccination.

[0003] Currently established manufacturing processes for RNA molecules approved by regulatory agencies implement many individual manufacturing steps. In particular, the individual manufacturing steps are performed by a plurality of different devices. Furthermore, various individual quality controls are performed at the DNA level and at the RNA level as described in detail in WO 2016 / 180430 A1.

[0004] A key step in RNA production is the generation of suitable DNA templates, which is a major cost factor on an industrial scale. Currently, DNA templates can only be used for a single RNA in vitro transcription reaction, which subsequently needs to be destroyed by DNAse digestion and finally removed by RNA purification to ensure the effectiveness and safety of RNA-based therapies.

[0005] The manufacturing of RNA requires a lot of manual handling by trained technicians in GMP supervised laboratories. As a result, the currently established manufacturing process is time consuming, cost intensive and requires a lot of laboratory space and laboratory equipment. SUMMARY

[0006] As mentioned above, there are problems associated with common manufacturing equipment and processes, i.e. RNA in vitro transcription currently requires a lot of manual handling by trained technicians. Therefore, it is desirable to provide an improved bioreactor for RNA in vitro transcription and an automated device for RNA production to save time, space, equipment and manpower.

[0007] An advantage of the improved bioreactor can be that it can allow the reuse of DNA templates in multiple RNA production processes, which reduces costs because less starting material (i.e. DNA templates) has to be used and DNAse treatment can be omitted or substantially minimized. Furthermore, the improved bioreactor can allow robust production of RNA with a higher purity profile (no residual DNAse, no residual DNA fragments in the RNA end product). An advantage of the automated device for RNA production is that the whole manufacturing process can be more robust and reliable (due to minimizing human errors) and can accelerate the production of RNA.

[0008] Furthermore, an acceleration of RNA manufacturing would be highly advantageous and of great importance for public health, especially in the case of a pandemic. In this case, it would be further advantageous to produce RNA therapeutics in the region of an outbreak, however, this would require a portable RNA production device.

[0009] The above-described problems are solved by the subject matter of the independent claims, wherein further embodiments are incorporated in the dependent claims. It should be noted that the features of the application described below apply equally to the bioreactor for RNA in vitro transcription, the method for RNA in vitro transcription, the module for transcribing DNA into RNA, the automated device for RNA manufacturing and the uses described herein.

[0010] In a first aspect, the present application relates to a bioreactor for RNA in vitro transcription, comprising:

[0011] - a reaction vessel, and

[0012] - a magnet unit located at the reaction vessel.

[0013] The reaction vessel is adapted to contain at least one of magnetic particles, DNA template, DNA immobilization buffer, DNA magnetic particles and RNA in vitro transcription (IVT) master mix. Thus, the DNA magnetic particles are DNA templates immobilized on freely floating magnetic particles. The magnet unit is arranged to capture or induce a movement of the magnetic particles and the DNA magnetic particles are kept in the reaction vessel. By this movement, a mixing or agitation of the magnetic particles and / or the DNA magnetic particles can be induced. Thus, depending on the number of additional components kept in the reaction vessel, a mixing or agitation of the magnetic particles and / or the DNA magnetic particles and at least one of the DNA template, the DNA immobilization buffer and the IVT master mix can be induced by the magnetic unit. For example, in case of a DNA template and freely floating magnetic particles as components kept in the reaction vessel, a mixing or agitation of the magnetic particles induced by the magnet unit can result in mixed DNA magnetic particles, wherein the DNA magnetic particles are the DNA template immobilized on magnetic particles. In case of a mixing or agitation of the DNA magnetic particles and the IVT master mix due to the movement of the DNA magnetic particles induced by the magnetic unit, a more homogeneous mixture of DNA magnetic particles and IVT master mix established thereby supports the in vitro transcription of the template DNA into RNA.

[0014] The bioreactor according to the present application can also be suitable for use under regulated conditions (GMP) suitable for pharmaceutical applications, e.g. pharmaceutical nucleic acid production. The bioreactor can allow for a continuous production or a repeated batch production of a liquid nucleic acid composition, preferably a ribonucleic acid (RNA) composition. In the context of the present application, the term RNA is used to denote any type of ribonucleic acid. Thus, the term “RNA” can refer to a selected from the group consisting of long-chain RNA, coding RNA, non-coding RNA, single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), linear RNA (linRNA), circular RNA (circRNA), messenger RNA (mRNA), RNA oligonucleotides, small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA (asRNA), CRISPR / Cas9 guide RNA, riboswitch, immune-stimulatory RNA (isRNA), aptamer, ribozyme, ribosomal RNA (rRNA), transfer RNA (tRNA), viral RNA (vRNA), retroviral RNA or replicon RNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), circular RNA (circRNA) and Piwi-interacting RNA (piRNA).

[0015] In one embodiment, the inner surface of the reaction vessel has an ellipsoid or oval inner geometry. The inventors found that an ellipsoid shape or oval inner geometry allows for better mixing results. In addition, such a shape allows for better liquid drop or run-off and can allow for better cleanability. The latter can prevent the formation of liquid droplets which otherwise can dry unfavorably on the inner surface of the bioreactor. This can be particularly relevant for protein residues of the fluid held in the reaction vessel, which can harden or solidify at temperatures of 37°C or higher, for example.

[0016] In one embodiment, the inner surface of the reaction vessel has an egg-shaped inner geometry. Such an egg shape can provide the same or improved advantages as described above with respect to the ellipsoid shape. The egg shape can also provide optimal pressure distribution, optimal behavior of magnetic beads during mixing or turning, distribution for holding magnetic beads at the inner surface of the reaction vessel, during the cleaning process. For example, the egg shape can be obtained from two halves of a spheroid with the same base radius, wherein one of the spheroids is a half sphere with a height equal to the base radius, and the other spheroid has a height greater than the base radius. Alternatively, the inner surface of the reaction vessel can have a spheroid shape, in particular a sphere shape, or the inner surface can have a pill shape. The inner surface of the reaction vessel can also have a shape which is a combination of an egg shape and an ellipsoid shape or a combination of an egg shape and a cylindrical shape. By such a combination, a part of the inner surface of the reaction vessel has, for example, the shape of an egg, while the remaining part of the inner surface has, for example, a cylindrical shape.

[0017] In one embodiment, the inner surface of the reaction vessel can have a spherical inner geometry. Such a sphere can provide the same advantages as the egg-shaped reaction vessel described above or improve the advantages of the egg-shaped reaction vessel.

[0018] In one embodiment, the inner surface of the reaction vessel has a shape without edges (e.g., a cuboid with rounded edges). Such a shape likewise supports optimal run-off of liquid droplets, thereby preventing hardening of protein residues of the fluid held in the reaction vessel. This shape (without edges) allows for an efficient cleaning process.

[0019] In one embodiment, the reaction vessel can have an inner surface without wide gaps or cracks. In this case, a gap or crack of more than 2 pm, preferably a gap or crack of more than 1 pm, more preferably a gap or crack of more than 0.8 pm is still considered a "wide" gap or crack. Such a shape (without wide gaps) allows for an efficient cleaning procedure, as larger gaps can provide ecological niches for microbial contamination and biofilm or residues.

[0020] In one embodiment, the movement of the magnetic particles and / or DNA magnetic particles is set in such a way that deposition of the particles kept in the reaction vessel is avoided. Additionally or alternatively, the movement of the magnetic particles and / or DNA magnetic particles is set in such a way that the particles contained on the reaction vessel are kept free-floating, so that deposition at the bottom of the reaction vessel can be prevented. Furthermore, the mixing or vortexing process is improved by keeping the particles free-floating in the vessel and / or preventing or reducing the coagulation of the beads. Advantageously, keeping the magnetic particles and / or DNA magnetic particles free-floating and / or avoiding deposition of the magnetic particles and / or DNA magnetic particles improves the biochemical reactions in the bioreactor, i.e. DNA immobilization and RNA in vitro transcription.

[0021] In one embodiment, the magnet unit of the bioreactor is given by an array of electromagnets. The array of electromagnets can be positioned on or close to the outer surface of the reaction vessel. The individual electromagnets in the array can be turned on or off individually. In this way, the mixing or vortexing of the magnetic particles and / or DNA magnetic particles kept in the reaction vessel can be improved and better controlled. The array of electromagnets is preferably not movable and the bioreactor itself is not movable (not shaken, etc.) and the mixing or vortexing is introduced by the cooperation of the magnetic particles and / or DNA magnetic particles with the magnet unit.

[0022] Alternatively, in another embodiment, the magnet unit can be a permanent magnet or an electromagnet, which is movable in longitudinal direction along the longitudinal axis of the reaction vessel. In addition to or instead of this longitudinal movement, the permanent magnet or electromagnet can be moved in transversal direction towards and away from the reaction vessel. Similar to the case of the array of electromagnets, the longitudinally and / or transversally movable permanent magnet or electromagnet can allow for a better control of the mixing / vortexing and better mixing results.

[0023] Alternatively, in yet another embodiment, the magnet unit can be given by an electromagnet and preferably by at least one induction coil. In this case, the magnet unit is movable in longitudinal direction along the longitudinal axis of the reaction vessel. In addition, the magnet unit is rotatable around the vertical axis of the reaction vessel.

[0024] Suitably, the magnet unit can be arranged in the form of at least one Helmholtz coil.

[0025] The position of the magnet unit in the vicinity of the reaction vessel refers to the distance between the magnet unit and the reaction vessel, which still allows for the establishment of a suitable magnetic field within the reaction vessel when the magnet unit is turned on. Thus, the strength and form of the magnetic field has to be such that eddies / mixing of the magnetic particles on the inner surface of the reaction vessel can be induced and / or the magnetic particles can be trapped.

[0026] In an embodiment, the magnet unit is arranged to rotate around a longitudinal axis of the reaction vessel, wherein a direction of rotation of the magnet unit is switchable during mixing. The magnet unit can induce motion of the magnetic particles in a radial direction of the reaction vessel by inducing the magnetic particles in a radial direction relative to the longitudinal axis of the reaction vessel. By rotating the magnet unit around the reaction vessel to induce rotation, a magnetic force can be generated statically or dynamically, thereby mixing the magnetic particles. The direction of rotation of the magnet unit can be clockwise or counterclockwise relative to the longitudinal axis of the reaction vessel, and / or alternately changed. Thus, the magnetic particles can be kept free-floating in a non-contacting manner, and therefore mixing of components can be improved. Once rotation of the magnet unit is stopped, the magnet particles (e.g. DNA magnetic particles) are captured at an inner surface of the reaction vessel and no longer rotate. Thus, the magnet unit is arranged to (i) rotate around a longitudinal axis of the reaction vessel to induce motion of the magnetic particles as described above, and to (ii) capture the magnetic particles when rotation is stopped.

[0027] In an embodiment, the magnet unit comprises a magnetic ring, wherein the magnetic ring is designed to surround the reaction vessel. To facilitate assembly and rotation of the magnet unit around the reaction vessel, the magnet unit can be formed as a ring. In other words, the reaction vessel can be positioned in the center of a ring-shaped magnet unit, such that the magnet unit encircles the reaction vessel.

[0028] In an embodiment, the magnetic ring comprises at least a first rod and a second rod extending from an inner circumference of the magnetic ring to a center of the magnetic ring, such that free ends of the first and second rods face each other. In an embodiment, the free end of the first rod comprises a magnet with an N-pole, and the free end of the second rod comprises a magnet with an S-pole.

[0029] A disc-shaped or ring-shaped magnet unit can comprise magnets arranged in a circumferential direction of the magnetic ring. The magnets can be arranged directly at the magnetic ring and in contact with the magnetic ring, or offset from the magnetic ring in a manner closer to the reaction vessel located in the center of the magnetic ring, to reduce a gap between the magnets and the reaction vessel. To keep the magnets separate from the ring, a magnet holder can be used which is attached to the inner surface and extends to the center of the ring. The magnet holder can be designed to hold a rod such that one end of the holding rod is attached to the inner circumference of the magnetic ring, and the other end of the holding rod holds the magnet. The magnetic ring and the holding rod can be manufactured separately and attached to each other, or manufactured as one part, for example by molding.

[0030] To effectively initiate the movement of the magnetic particles, the magnetic ring can comprise at least two rods spaced apart from each other along the circumference of the magnetic ring such that the free ends of the rods face each other. Furthermore, permanent magnets having a N-pole and an S-pole can be alternately attached to each free end of the rods. Thus, when rotating the magnetic ring, the magnetic particles can be initiated to be rotatable around the reaction vessel, which results in an improved mixing of the components in the reaction vessel.

[0031] To effectively capture the magnetic particles, the rotation of the magnetic ring can be stopped after the mixing of the components in the reaction vessel.

[0032] In another embodiment, the magnetic ring comprises a plurality of rods, wherein the plurality of rods extends from an inner circumference of the magnetic ring to a center of the magnetic ring and are arranged in a star shape evenly spaced apart from each other. Preferably, magnets having a N-pole and magnets having an S-pole are alternately arranged at each free end of the rods.

[0033] In a preferred embodiment, the magnetic ring can comprise an even number of rods such that the plurality of rods and respectively the plurality of magnets attached to each free end of the rods are arranged in pairs to provide a non-uniform or periodic magnetic field. Furthermore, rods evenly spaced apart along the circumference of the magnetic ring allow to initiate a symmetrical magnetic field of the magnet particles within the reaction vessel.

[0034] In one embodiment, the magnetic ring and the rods are arranged to form a laminated stack for shielding external peripheral components from the magnetic field. The magnetic ring and the rods can be made of magnetizable laminated electrical sheets. The laminated electrical sheets can comprise electrical steel and can be used for electrical insulation. The laminated stack can shield the magnetic field generated by the permanent magnets attached to the free ends of the rods and does not affect other devices except the reaction vessel. The shielding of the magnetic field is particularly advantageous and allows to integrate the bioreactor in a device comprising other devices / components that can be affected by the magnetic field.

[0035] In one embodiment, the magnetic ring comprises a plurality of guide plates extending from an inner circumference of the magnetic ring to a center of the magnetic ring. Preferably, each guide plate comprises an electrical coil arranged for generating a magnetic field. The magnetic ring can comprise at least one, preferably a plurality of electromagnets generating a magnetic field by the electrical coils. The guide plates can be arranged in a star shape along the circumference of the magnet ring and extend to the center of the magnet ring, at which the reaction vessel can be positioned. The electrical coils rapidly vary the magnetic field by controlling the amount of electric current.

[0036] In one embodiment, the magnetic ring is arranged in a housing with a cooling device. The cooling device can be integrated in the housing of the magnetic ring along the circumference of the magnetic ring to take away heat caused by high currents through the electromagnetic coil. The cooling device can be a cooling channel in which a cooling medium, such as water, is circulated. The cooling device can preferably be integrated in the magnetic ring comprising the electromagnetic coil. The cooling device can not be integrated in the magnetic ring comprising the permanent magnet (and not comprising the electromagnetic coil).

[0037] In one embodiment, the magnet unit further comprises a first drive device arranged to rotate the magnetic ring around a longitudinal axis of the reaction vessel and a second drive device arranged to move the magnetic ring in a perpendicular direction along the longitudinal axis of the reaction vessel. The magnetic ring can be held by a frame that moves in the longitudinal direction of the reaction vessel. Thus, when the magnet ring is rotated and moved perpendicularly, a magnetic field can be provided and changed in the longitudinal direction and in the radial direction of the reaction vessel, which can result in an even better homogeneous mixing of the components in the reaction vessel.

[0038] The drive device for rotating the magnetic ring and the drive device for moving the magnetic ring in a perpendicular direction can be arranged separately. The first drive device for rotating the magnetic ring can be arranged directly to the magnetic ring and above the reaction vessel, while the second drive device for moving the magnetic ring perpendicularly can be connected to the magnetic ring via a frame that fixedly holds the magnet ring and allows the magnet ring to move perpendicularly.

[0039] In one embodiment, the reaction vessel is paramagnetic, such that magnetic particles and DNA magnetic particles can be held on the inner wall of the reaction vessel by the cooperation of the paramagnetic vessel and the magnet unit located at the reaction vessel. Thus, the entire reaction vessel can be paramagnetic, for example, by comprising paramagnetic or magnetically permeable material, or the inner surface of the reaction vessel can be paramagnetic. The term "magnetizable" throughout the present invention means that the reaction vessel or its inner surface can be temporarily magnetized, such that magnetic particles can be attracted and held on the reaction vessel wall. However, the magnetization of the reaction vessel or its inner surface can be reversed, such that the magnetic particles and DNA magnetic particles held on the reaction vessel wall can be released. Thus, it is important that the material of the bioreactor and / or the inner surface of the bioreactor is not permanently magnetized by switching on the magnet unit (i.e. non-ferromagnetic).

[0040] Thus, in a preferred embodiment, the reaction vessel is paramagnetic. In other embodiments, the reaction vessel is arranged to allow penetration of a magnetic field instead of being magnetizable.

[0041] In one embodiment, the magnet unit is configured to operate periodically to mix the magnetic particles and / or the DNA magnetic particles. Periodic activation of the magnet unit leads to improved mixing of the components compared to sequential activation. This periodic activation of the magnet unit, which results in improved mixing of the components, must be regulated in a manner that keeps the magnetic particles or the DNA magnetic particles free-floating and allows mixing to occur in an optimized manner for the biochemical reaction (all components involved in the biochemical reaction, e.g., in in vitro RNA transcription, are mixed and in contact with each other, thus leading to RNA synthesis). Equally important is the regulation of the mixing initiated by the periodically active magnet and the DNA magnetic particles / magnet particles, so that unwanted shear forces are minimized and heat formation is reduced (heat formation can be initiated by converting magnetic energy into heat or by frictional heat).

[0042] In one embodiment, the magnetic unit is configured to be activated to capture the DNA magnetic particles between two or more subsequent in vitro RNA transcriptions on the same DNA template (set in the form of DNA magnetic particles). This capture may be related to the magnetization of the reaction vessel, causing the DNA magnetic particles to remain on the inner surface of the reaction vessel, and / or may be related to magnetizable but chemically inert beads or spheres within the reaction vessel. Advantageously, such capture allows for the reuse of the DNA magnetic particles in two or more in vitro RNA transcription reactions, thereby reducing production time by decreasing the scale of template provision and the cost of the RNA product (the DNA template can be used several times).

[0043] In one embodiment, the magnetic unit is configured to be activated to remove magnetic particles and DNA magnetic particles. This removal of magnetic particles and DNA magnetic particles can be used for cleaning the reaction vessel. The removal of DNA magnetic particles can be performed after the final in vitro RNA transcription reaction (e.g., by pausing the rotation of the magnetic ring). This removal of DNA magnetic particles has the advantage that DNA can be removed without digestion by enzymes such as DNase, which reduces DNA and enzyme contamination in the final RNA product (no DNA digestion product, no DNase), and lowers the cost of the RNA product (no need to control DNase contamination in the final product, no DNase required).

[0044] In one embodiment, no mechanical motion introduction means for the magnetic particles and the DNA magnetic particles are included. According to this embodiment, no additional mechanical stirrer or agitator capable of causing mixing or agitation of the components contained in the reaction vessel is present, such that mixing is caused solely by the magnet unit. This is particularly advantageous in the context of the present application, as mechanical motion introduction means located within the reaction vessel can lead to the formation of undesired precipitates (e.g. on the mechanical agitation means). Furthermore, the absence of mechanical motion introduction means also improves the cleaning of the bioreactor (reduced surface, no edges within the reaction vessel).

[0045] In an alternative embodiment, a mechanical motion introduction means for the magnetic particles and the DNA magnetic particles in the form of a vibrator (e.g. an orbital vibrator) is included, wherein the vibrator is preferably located outside the reaction vessel.

[0046] In one embodiment, mixing or agitation of the components held in the reaction vessel can be introduced by a combination of (i) the cooperation of the magnetic particles and the magnet unit, (ii) the mechanical motion introduction means, and / or (iii) the introduction of process gas or process fluid into the reaction vessel.

[0047] In one embodiment, the reaction vessel comprises at least one flow disrupter arranged at least partially along the inner surface of the reaction vessel in the longitudinal direction of the reaction vessel. The flow disrupter can interfere with the uniform flow of components in the reaction vessel, thereby improving mixing. Furthermore, the flow disrupter can prevent the deposition of magnetic particles when the magnet ring stops rotating and / or changes the direction of rotation. Thus, the flow disrupter can be continuously designed without any recesses in which magnetic particles can accumulate, in particular in the horizontal direction perpendicular to the longitudinal direction of the reaction vessel.

[0048] The flow disrupter can protrude from the inner surface of the reaction vessel in the radial direction of the reaction vessel and extend along the longitudinal direction of the reaction vessel. The flow disrupter can continuously extend from the top to the bottom of the reaction vessel or comprise a plurality of elements arranged separately from each other along the longitudinal direction of the reaction vessel. Thus, the flow disrupter can comprise a plurality of protrusions, which are preferably spaced apart from each other.

[0049] In one embodiment, the reaction vessel comprises two flow disrupters spaced apart from each other along the circumference of the reaction vessel. The reaction vessel can comprise at least one, exactly two or more flow disrupters. The flow disrupters are preferably evenly distributed along the inner surface of the reaction vessel in the radial direction of the reaction vessel to improve mixing and prevent the deposition of magnetic particles.

[0050] In one embodiment, the flow disruptors are rib-shaped, and the rib-shaped flow disruptors can preferably comprise a T-shaped or L-shaped cross-section. The flow disruptors protruding from the inner surface of the reaction vessel in a direction towards the center of the reaction vessel can be formed along an arc of the curved inner surface of the reaction vessel and comprise a plurality of radii of curvature along the ellipsoidal inner geometry of the reaction vessel. The radial cross-section of the flow disruptors relative to the longitudinal axis of the reaction vessel can also vary. For example, the radial cross-section can be formed as a T-shape, L-shape, or convex shape. The protruding length of the radial cross-section of the flow disruptors can also vary along the inner surface from the top to the bottom of the reaction vessel. In one embodiment, the flow disruptors are corrugated. The rib-shaped flow disruptors can also be wavy along the inner surface of the reaction vessel, which can prevent the deposition of the magnetic particles. The wavy surface of the corrugated flow disruptors can be aligned perpendicular to the inner surface of the reaction vessel.

[0051] In one embodiment, a temperature element is located between the inner surface and the outer surface of the reaction vessel for regulating the temperature of the reaction vessel. In other words, the reaction vessel can comprise a thick wall made of a solid material, thereby allowing the temperature element to be integrated between the inner surface and the outer surface. Thus, a rapid temperature regulation with respect to heating and cooling of the reaction vessel can be facilitated.

[0052] In one embodiment, the temperature element comprises a heat exchange channel spirally surrounding the reaction vessel at least partially in the radial direction of the reaction vessel. The heat exchange channel can be integrated between the inner surface and the outer surface and adapted to regulate the temperature in the reaction vessel. In order to provide an efficient and uniform heating or cooling, the heat exchange channel can completely surround the reaction vessel, and a heat exchange medium can flow within the heat exchange channel.

[0053] In one embodiment, the heat exchange channel comprises a first end and a second end, wherein the first end is arranged at the top of the reaction vessel and the second end is positioned at the bottom of the reaction vessel. The spirally arranged heat exchange channel can have at least two ports for an inlet and / or an outlet of the heat exchange medium, wherein, when one port is arranged at the top of the reaction vessel and the other port is arranged at the bottom of the reaction vessel, where a gravitational force can be applied, an efficient distribution of the heat exchange medium can be facilitated.

[0054] In one embodiment, the heat exchange channel and / or the reaction vessel are manufactured by an additive manufacturing process. Thus, a complex geometry of the reaction vessel, including the heat exchange channel spirally surrounding the reaction vessel between the inner surface and the outer surface of the reaction vessel, can be easily realized.

[0055] In one embodiment, the reaction vessel further comprises a temperature element comprising a heating wire at least partially spirally surrounding the reaction vessel in a radial direction relative to a longitudinal axis of the reaction vessel. As an alternative to the heat exchange channel, the heating wire can be arranged on the reaction vessel to regulate the temperature of the components in the reaction vessel. The heating wire can also spirally surround the reaction vessel to provide uniform heating.

[0056] In one embodiment, the heating wire is at least partially integrated on or at least partially coated on an outer surface of the reaction vessel. In order to minimize heat loss and provide efficient heating by the heating wire, the heating wire can be fixed to the outer surface of the reaction vessel. Alternatively or additionally, the outer surface of the reaction vessel can be coated with a thermal insulation material, and the heating wire can be at least partially withdrawn in the thermal insulation material.

[0057] In one embodiment, the reaction vessel is dimensioned such that it can absorb at least 20 ml of fluid, or at least 50 ml of fluid, or at least 100 ml of fluid, or at least 500 ml of fluid. Preferably, it can absorb 20 ml to 100 ml or 20 ml to 50 ml of fluid. It can also be configured to absorb 50 ml to 100 ml of fluid. Notably, when used in the method of the second aspect, the reaction vessel is only filled to about 60% to about 80% to allow for sufficient shaking of the liquid. In one particular embodiment, the reaction vessel is dimensioned such that it can absorb about 100 ml of fluid, wherein only 60 ml to 80 ml of fluid is filled into the reaction vessel, corresponding to a filling of only about 60% to 80% of the reaction vessel. In another particular embodiment, the reaction vessel can absorb about 20 ml to 50 ml of fluid, wherein in this case the reaction vessel should be filled to about 60%.

[0058] According to one embodiment, the IVT premix can comprise ribonucleoside triphosphates and a DNA-dependent RNA polymerase. According to one embodiment, the DNA immobilization buffer can comprise DNA and a salt-containing buffer. The DNA template can be a linear double-stranded DNA template, which is preferably a PCR-amplified DNA template. In one embodiment, the magnetic particles can be given by magnetic beads, preferably streptavidin magnetic beads or chemically functionalized magnetic beads, most preferably paramagnetic streptavidin or chemically functionalized magnetic beads.

[0059] In one embodiment, the inner surface of the reaction vessel has a surface roughness value (Ra value) of Ra <= 0.8, preferably Ra <= 0.6. The inner surface can be, for example, electropolished or otherwise treated, for example chemically or mechanically, so that the above-mentioned Ra value is achieved. Such an Ra value is particularly advantageous because such a material can improve the cleanability of the reactor, since it can prevent or reduce the deposition and hardening of, for example, protein residues or biofilms at the inner surface of the reaction vessel.

[0060] In one embodiment, the bioreactor comprises a flow inlet port which allows the introduction of a fill media into the reaction vessel. Accordingly, the flow inlet port is arranged below the maximum fluid amplitude or fluid level. In the context of the present invention, the maximum fluid amplitude is understood to be the amplitude reached by the fluid contained in the reaction vessel and which reaches the maximum shaking or rotational movement on the inner surface of the reaction vessel. In the case of rotational movement, the centrifugal force acting on the fluid molecules causes the fluid to be pushed upwards towards the inner surface of the reaction vessel. The boundary between the wetted and dry areas on the inner surface defines the line which gives the maximum fluid amplitude. In other words, the maximum fluid amplitude can be associated with a line or area which is wetted during the shaking or rotational movement of the fluid contained in the reaction vessel. The fill media to be introduced through the flow inlet port of the reaction vessel can be provided, for example, by magnetic particles, DNA templates, immobilization buffers and / or IVT pre-mix. Other fill media can be cleaning, washing and process fluids, etc. Positioning the flow inlet port below the maximum fluid amplitude prevents the deposition and hardening of substances, such as proteins, DNA or particles or salts, etc., on the inner surface of the reaction vessel, for example in the case of a temperature of approximately 37°C.

[0061] In one embodiment, the reaction vessel comprises a media port at the bottom of the reaction vessel for supplying media into the reaction vessel and / or removing media from the reaction vessel and which port is connectable to a valve device. In other words, the bioreactor comprises a combined flow inlet and outlet port (flow inlet / outlet port), preferably at the lowest point of the reaction vessel. The valve device can allow the introduction of fill media into the reaction vessel and the discharge of media from the reaction vessel. Advantageously, the valve device can be arranged to retain, for example, magnetic particles and DNA magnetic particles within the reaction vessel when the valve device is closed or to allow, for example, the passage of fluids containing RNA products when the valve device is open.

[0062] In an embodiment, the valve arrangement comprises a magnetic trap. The magnetic trap is positioned at the culture medium port and is arranged to capture magnetic particles and DNA magnetic particles. In this way, magnetic particles and DNA magnetic particles can be captured when the reaction vessel is cleaned. Additionally or alternatively, magnetic particles and DNA magnetic particles that are not intended to leave the reaction vessel can be captured and thereby separated from, for example, the produced RNA. The magnetic trap can be located outside the reaction vessel and can at least partially surround the culture medium tube. The culture medium tube can be connected to the culture medium port of the reaction vessel and adjoin the culture medium port of the reaction vessel downstream. The culture medium port can be located at the lowest point of the reaction vessel. In this way, fluid can easily flow out of the reaction vessel under the drive of gravity.

[0063] In an embodiment, the magnetic trap comprises magnetisable or magnetic spheres or magnetisable or magnetic rings and / or a semi-permeable filter that allows the retention of magnetic particles and / or DNA magnetic particles. The magnetic trap can comprise an electromagnet or a permanent magnet. The magnetic trap can be controllable to prevent magnetic particles and / or DNA magnetic particles from escaping from the reaction vessel. Such control can be advantageously used when separating the produced RNA from the magnetic particles and DNA magnetic particles.

[0064] In an embodiment, the bioreactor comprises a multi-position valve. The multi-position valve is positioned downstream of the magnetic trap and is arranged for directing a cleaning gas or cleaning fluid through the port. The arrangement is for removing magnetic particles and DNA magnetic particles or other deposits collected at the port from the multi-position valve.

[0065] In an embodiment, the above multi-position valve is arranged to direct a process gas or process fluid into the reaction vessel. The process gas or process fluid introduced into the reaction vessel can cause mixing, stirring or vortexing of the magnetic particles or DNA magnetic particles.

[0066] In an embodiment, the bioreactor comprises a valve arrangement at the flow outlet port, the flow inlet port and / or the flow inlet / flow outlet port and is arranged to retain, for example, magnetic particles and DNA magnetic particles within the reaction vessel when the valve arrangement is closed or to allow, for example, fluid containing RNA product to pass through when the valve arrangement is open. Advantageously, the valve arrangement can be arranged to allow closing and opening of the flow outlet port and / or the flow inlet port or the combined flow inlet / flow outlet port. Suitably, such valve arrangement can be a ball valve, a butterfly valve, a control valve, a diaphragm valve, a gate valve, a needle valve or a pinch valve or a combination thereof.

[0067] In one embodiment, the bioreactor comprises at least a first leg and a second leg supporting the bioreactor vertically (along the longitudinal direction of the reaction vessel). The first leg comprises a first conduit and the second leg comprises a second conduit. The first conduit is arranged in fluid communication with the valve device and the second conduit is arranged in fluid communication with one end of the heat exchange channel of the temperature element. The first and second legs can be positioned at the bottom of the reaction vessel and arranged to stabilize the reaction vessel vertically. Furthermore, the first and second legs can comprise conduits within the respective legs. A first end of the first conduit located in the first leg can be connected to the valve device to supply and / or drain reaction components and a second end of the first conduit can be connected to a peripheral device that supplies and / or drains reaction media. Furthermore, a first end of the second conduit located in the second leg can be connected to a second end of the heat exchange channel that is spirally wound around the reaction vessel and a second end of the second conduit can be connected to a peripheral device that supplies and / or drains heat exchange media. Thus, the reaction vessel can be designed compactly.

[0068] In one embodiment, the bioreactor comprises an outlet port. The outlet port is connected to at least one of the exhaust conduit and the waste channel. For example, the outlet port can be connected to both the exhaust conduit and the waste channel via a multi-position valve. The outlet port can allow for receiving and draining one or more exhaust gases that occur within the reaction vessel. In the case of waste liquid or cleaning fluid, the outlet port can be used to drain the fluid out of the reaction vessel. The outlet port, exhaust conduit, and / or waste channel can hold at least one device for measuring and / or regulating pressure.

[0069] In one embodiment, the bioreactor further comprises a Hall sensor. The Hall sensor is arranged downstream of the magnetic trap and is used to detect a magnetic field. The Hall sensor can observe or control that the product (e.g. fluid) entering the capillary downstream of the magnetic trap is free of magnetic particles and / or DNA magnetic particles. In this way, the Hall sensor helps to control the correct operation of the magnetic trap. As a result of measuring a magnetic field emerging from, for example, magnetic particles or DNA magnetic particles by the Hall sensor, a fault signal can be given.

[0070] In the context of the present invention, "downstream" and "upstream" refer to the direction of movement of a fluid or gas within the process encompassed by the present invention. For example, when the Hall sensor is arranged downstream of the magnetic trap, this means that the magnetic trap and the Hall sensor are arranged, for example, around a capillary in which a fluid or gas is conducted, and the fluid or gas conducted within the capillary will first pass the magnetic trap and then the Hall sensor.

[0071] In one embodiment, the reaction vessel comprises a Titan. Titans possess low remanence, or remnant magnetism, which indicates the magnetization remaining in a ferromagnetic material after the external magnetic field is removed. Therefore, a reaction vessel made of Titan can provide direct interaction between the magnetic force generated by the magnetic rings and the magnetic particles contained within the reaction vessel.

[0072] Suitable, the reaction vessel is made of a material resistant to, for example, cleaning procedures (chemical resistance), extreme temperatures (e.g., for cleaning procedures of 75°C and 85°C), extreme pH values ​​(cleaning the reactor with alkalis and acids (e.g., with NaOH), mechanical forces (e.g., friction caused by magnetic particles), and / or corrosion. Furthermore, the material of the reaction vessel should have thermal conductivity at an operating temperature of approximately 20°C (e.g., a W / (mK) value of at least 10, preferably at least 15).

[0073] Suitablely, and particularly important in the context of this invention, the inner surface of the reaction vessel has a surface material that does not release unwanted compounds that could contaminate the final product. Suitable materials for the reaction vessel and / or the inner surface of the reaction vessel are austenitic stainless steel (e.g., 1.4404 (AISI 316L), 1.4435 (AISI 316L)), iron-free... Alloys or Titan (Ti1) possess paramagnetic properties, chemical resistance, pH resistance, temperature resistance, and thermal conductivity.

[0074] Other suitable materials for the reaction vessel and / or the inner surface of the reaction vessel are glass (e.g., borosilicate glass), industrial ceramics (e.g., ), polyaryletherketones (e.g., polyetheretherketone (PEEK)), thermoplastics (e.g.) Pa or All of these materials are non-magnetic, chemically resistant, pH- and temperature-resistant. The advantage of glass (e.g., borosilicate glass) is that it can be used as a reaction vessel that can be visually inspected.

[0075] In one embodiment, the bioreactor further includes a (semi-permeable) filter element at the culture medium port or culture medium tube. The filter helps retain magnetic particles and DNA magnetic particles within the reaction vessel. For effective filtration, the filter element may have a pore size of less than 1 μm. The semi-permeable filter may include a filter membrane having a molecular weight cutoff (MWCO) suitable for retaining magnetic particles and / or DNA magnetic particles. To prevent clogging of the filter port, the filter may preferably be a disposable filter.

[0076] In one embodiment, the temperature element is configured to regulate the temperature within the reaction vessel to a DNA immobilization or RNA transcription temperature of 20°C to 37°C. In addition, the temperature element can also be configured to regulate the temperature within the reaction vessel to a cleaning temperature of 75°C to 85°C. The appropriate temperature (e.g. 20°C to 37°C or 75°C to 85°C) can be controlled by at least one means for measuring and / or regulating the temperature (e.g. a temperature sensor) which can be suitably positioned at the inner surface of the reaction vessel and / or in the vicinity of the reaction vessel and / or in the vicinity of the temperature element. Temperature elements and means for measuring and / or regulating the temperature are of particular importance as, for example, magnetic energy or friction can generate unwanted heat (e.g. an unwanted temperature increase in the reaction) which can hamper the biochemical reaction.

[0077] In one embodiment, the bioreactor comprises an inlet flow cell and / or an outlet flow cell and / or an outlet flow cell. The inlet flow cell can be arranged upstream of the inlet port, the outlet flow cell can be arranged downstream of the outlet port, and the outlet flow cell can be arranged downstream of the outlet port. The inlet flow cell and / or the outlet flow cell and / or the outlet flow cell can be calibratable and can be arranged for monitoring the flow rate of a fluid or gas flowing into or out of the reaction vessel. In the context of the present application, the reaction vessel can contribute to the partial control of a process, for example RNA transcription or cleaning of the bioreactor.

[0078] In one embodiment, the reaction vessel is configured to additionally contain at least one of the following ingredients provided by a buffer suitable for RNA in vitro transcription, ribonucleoside triphosphates, cap analogs, modified ribonucleoside triphosphates, ribonuclease inhibitors, pyrophosphatase, MgCl2, antioxidants, polyamines, and solutions for cleaning and / or sterilization.

[0079] In one embodiment, the reaction vessel can further be configured to hold at least one means for measuring and / or regulating the pH or concentration of the contained components as well as the magnesium concentration, the phosphate concentration, the temperature, the pressure, the flow rate, the RNA concentration and / or the ribonucleotide triphosphate concentration. The means can be given by a corresponding sensor or a corresponding probe. Such one or more means can contribute to the monitoring of the process covered by the present application. The measuring means can be a measuring device or a sensor, and the regulating means can be a dosing device. For example, the means can be a sensor for measuring the pH of the components contained in the reaction vessel or a sensor for measuring the magnesium or salt concentration. In addition, by way of example, the means can be a device for measuring the temperature, the pressure or the flow rate. In the latter case, the means can be, for example, a flow cell inside the reaction vessel or at the outlet.

[0080] In one embodiment, the bioreactor is designed to operate in batch, repeated batch, continuous mode or in semi-continuous or continuous mode. Repeated batch RNA in vitro transcription (IVT) is preferred as it allows multiple reactions on the same DNA template with the advantages already outlined herein.

[0081] In embodiments where the bioreactor comprises a mechanical motion introduction device, the bioreactor can comprise a rotation device for rotating the reaction vessel. Such rotation can help to prevent deposition of magnetic particles and DNA magnetic particles at the flow outlet port.

[0082] In a second aspect, the present application relates to a method of RNA in vitro transcription. The method comprises the following steps:

[0083] - providing DNA magnetic particles and an IVT premix in a reaction vessel of a bioreactor, wherein the bioreactor is designed according to at least one of the above embodiments of the first aspect (S3a),

[0084] - mixing the freely floating DNA magnetic particles with the IVT premix by cooperation of the DNA magnetic particles and a magnet unit of the bioreactor and / or by a vibrator. To this end, the magnet unit can be arranged to induce motion of the components of the DNA magnetic particles and the IVT premix by a suitable electromagnetic field. As a result of the mixing, RNA in vitro transcribed is obtained (S3b).

[0085] The method of the second aspect can further comprise the following steps:

[0086] - providing magnetic particles, a DNA template, a DNA immobilization buffer in a reaction vessel of a bioreactor, wherein the bioreactor is designed according to at least one of the above embodiments of the first aspect (S1),

[0087] - mixing the magnetic particles, the DNA template and the DNA immobilization buffer. Mixing is performed by cooperation of the magnetic particles and a magnet unit and / or by a vibrator (S2). To this end, the magnet unit can be arranged to induce motion of the magnetic particles and the DNA magnetic particles by a suitable electromagnetic field. As a result of the mixing, DNA magnetic particles are obtained, which are DNA templates immobilized on freely floating magnetic particles. The DNA templates immobilized on freely floating magnetic particles can be mixed with an IVT premix to obtain RNA (as described above; S3). Thus, steps S1 and S2 are performed prior to step S3.

[0088] The method of the second aspect can further comprise the following steps:

[0089] - capturing the DNA magnetic particles by the magnet unit and collecting / harvesting the obtained RNA in vitro transcribed, e.g. through a flow outlet port (S4a),

[0090] - providing fresh IVT premix in the reaction vessel of the bioreactor of the first aspect (S4b),

[0091] - releasing the captured DNA magnetic particles to provide free-floating DNA magnetic particles (S4c),

[0092] - mixing the free-floating DNA magnetic particles with the IVT premix by cooperation of the DNA magnetic particles and the magnet unit and / or by a vibrator to obtain RNA (S4d).

[0093] Preferably, steps S4a to S4d are performed after step S3. The method steps (S4) are particularly suitable for embodiments in which more than one RNA in vitro transcription reaction is performed. Preferably, the method steps are performed at least 2 times, for example 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or up to 30 times.

[0094] In one embodiment, the method according to the application can further comprise a step of adjusting the pH and / or the salt concentration.

[0095] In one embodiment, the method according to the application further comprises a step of tempering the reaction vessel to a temperature between 20 °C and 37 °C. Such a temperature can be suitable for RNA in vitro transcription. The tempering can be performed before filling the reaction vessel.

[0096] In a further method step for immobilizing the DNA template on the magnetic particles, the reaction vessel can be tempered to a temperature between 20 °C and 25 °C, preferably 22 °C. Further, in a further method step, the reaction vessel can be tempered to a temperature between 75 °C and 85 °C during / for the time of the cleaning process of the reaction vessel.

[0097] In one embodiment, the method further comprises a step of cleaning the reaction vessel by a cleaning gas and / or a cleaning fluid. Before or during the cleaning, the reaction vessel can be heated, for example, to the above-mentioned temperature between 75 °C and 85 °C. After obtaining the in vitro transcribed RNA, the DNA magnetic particles can be removed by cooperation of the DNA magnetic particles and the magnet unit. This method step allows for removing the DNA template without, for example, performing a DNAse digestion.

[0098] In order to allow mixing or capturing / releasing of the magnetic particles or DNA magnetic particles, it is important that the magnetic particles are paramagnetic to avoid irreversible attachment to the walls of the reactor vessel during mixing, for example, introduced by the magnet unit. Examples of suitable magnetic particles are Dynabeads® Dynabeads® magnetic beads (ThermoFisher Scientific).

[0099] The method can further comprise different quality control steps which can allow to assess e.g. RNA identity, RNA integrity, RNA purity, etc. Said quality control can be performed in-line or on-line.

[0100] The method for RNA in vitro transcription outlined herein can be performed on one DNA template to produce an RNA composition comprising one RNA species. In other embodiments, the method for RNA in vitro transcription can be performed on at least two different DNA templates to produce a composition comprising at least two RNA species. For example, the method described in WO2017 / 1090134A1 can be adapted accordingly and performed in the reaction vessel of the bioreactor of the first aspect.

[0101] The method can further comprise a step of enzymatic RNA capping which can be performed in the reaction vessel of the bioreactor of the first aspect (e.g. using immobilized capping enzymes on magnetic particles; immobilized capping enzymes can be obtained using the method disclosed in WO2016 / 193226). The magnet unit can be set to induce movement of the magnetic particles and the capping enzymes on the RNA by an appropriate electromagnetic field. As a result of the mixing, capped RNA is obtained.

[0102] The method can further comprise a step of enzymatic RNA polyadenylation which can be performed in the reaction vessel of the bioreactor of the first aspect (e.g. using PolyA polymerase immobilized on magnetic beads; immobilized PolyA polymerase can be obtained using the method disclosed in WO2016 / 174271). The magnet unit can be set to induce movement of the magnetic particles and the PolyA polymerase on the RNA by an appropriate electromagnetic field. As a result of the mixing, polyadenylated RNA is obtained.

[0103] In a third aspect, the present invention relates to the use of the bioreactor described above in the method described above.

[0104] Furthermore, the bioreactor of the first aspect can be used for RNA in vitro transcription reactions wherein the DNA is free or immobilized on non-magnetic particles (e.g. agarose beads, sepharose beads, non-magnetic polystyrene beads and other suitable synthetic resins) and mixing is introduced by the cooperation of magnetic particles not carrying the DNA template and the magnetic unit of the bioreactor of the first aspect. In such embodiments, the RNA in vitro transcription reaction can only be performed once. Furthermore, the bioreactor can be used for any enzymatic method involving nucleic acids (e.g. polymerase chain reaction (PCR), isothermal DNA amplification, RNA reverse transcription to cDNA) wherein mixing is introduced by the cooperation of magnetic particles and the magnetic unit of the bioreactor of the first aspect.

[0105] In a fourth aspect, the present application relates to a module for the transcription of a DNA template into RNA. The module comprises a bioreactor according to at least one of the above embodiments of the first aspect, and at least one of a unit for the preparation of an IVT premix, a unit for the preparation of immobilization buffer, a device for the purification of the obtained RNA product, a device for RNA handling and / or a device for RNA sterile filtration.

[0106] In a preferred embodiment, the device for the purification of the obtained RNA product comprises an HPLC unit, preferably a unit for performing RP-HPLC. Particularly preferred in this context is RP-HPLC using the method disclosed in WO 2008 / 077592, preferably using a porous, non-alkylated poly(styrene-divinylbenzene) reverse phase, wherein the reverse phase is formed by beads or occurs as a polymeric block (e.g. monolith). Alternatively or in addition, the device for the purification of the obtained RNA product can comprise an oligo dT purification unit for affinity purification of the obtained polyadenylated RNA via an oligo dT functionalized matrix or beads or column (e.g. as described in WO 2014152031 Al).

[0107] In a preferred embodiment, the device for RNA handling comprises a tangential flow filtration unit. Particularly preferred in this regard is tangential flow filtration as described in WO 2016 / 193206, wherein TFF is used for diafiltration and / or concentration and / or purification of RNA.

[0108] In one embodiment, the module further comprises a media supply unit. The media supply unit is arranged to provide components of the IVT premix to the unit for the preparation of the IVT premix.

[0109] In one embodiment, the DNA template is a terminally modified or terminally functionalized PCR-generated DNA template. Preferably, the DNA template is a biotinylated PCR-generated DNA template, unmodified or terminally modified linearized plasmid DNA or unmodified or terminally modified linearized dogbone DNA.

[0110] In a fifth aspect, the present application relates to an automated device for RNA manufacturing comprising a bioreactor according to the above first aspect or a module according to the fourth aspect, wherein the device further comprises at least one of a module for DNA synthesis and a module for RNA formulation.

[0111] In one embodiment, the module for DNA synthesis is arranged to produce a sufficient amount of DNA suitable for use in the bioreactor of the first aspect or the module for transcribing a DNA template into RNA of the second aspect. In a preferred embodiment, the module for DNA synthesis can comprise a thermal cycler element for PCR-based DNA amplification and an element for purifying the obtained PCR product. Suitably, the DNA synthesis module can produce biotinylated DNA templates, preferably PCR-based biotinylated DNA templates.

[0112] In one embodiment, the module for RNA formulation is arranged to produce a lipid nanoparticle (LNP)-encapsulated RNA. Accordingly, the module for RNA formulation comprises an LNP formulation module, wherein the LNP formulation module can comprise, for example, a pump element (e.g. a syringe pump), a tangential flow element and a filtration element (e.g. comprising a sterile filter).

[0113] In one embodiment, the module for RNA formulation is arranged to produce RNA complexed with a polycationic peptide or protein (e.g. protamine or a polymeric carrier such as a polyethylene glycol / peptide polymer according to WO2012 / 013326). Accordingly, the module for RNA formulation comprises at least one of a protamine formulation / complexing module and / or a polyethylene glycol / peptide polymer formulation / complexing module. In this context, the RNA formulation module can suitably use methods and devices according to WO2016165825A1 and / or WO2018041921A1.

[0114] In one embodiment, the automated device is arranged in a closed container and preferably in a single container, wherein the container comprises a unit for producing a laminar air flow. Such an arrangement within a single container is particularly helpful to save space in addition to equipment and personnel. Furthermore, the automated device can be arranged to be portable, for example dimensioned in a way to allow transportation to regions of a pandemic outbreak.

[0115] In one embodiment, the automated device further comprises at least one of a DNA immobilization module, e.g. for immobilizing plasmid DNA (e.g. as described in PCT / EP2017 / 084264 or PCT / EP2018 / 086684), a DNA linearization module, e.g. for linearization of plasmid DNA or dogbone DNA, a RNA capping module, e.g. for adding a cap0 or cap1 structure to in vitro transcribed RNA, a RNA polyadenylation module, e.g. for adding a polyA tail to in vitro transcribed RNA, a RNA mixing module, e.g. for mixing at least two different RNA species, a RNA spray-drying module, e.g. according to WO2016 / 184575 or WO2016184576, e.g. for producing spray-dried or freeze-spray-dried RNA, a RNA lyophilization module, e.g. according to WO2016 / 165831 or WO2011 / 069586, for producing lyophilized RNA, and / or a module for final product storage.

[0116] In one embodiment, the automated device further comprises at least one of a NGS (next generation sequencing) module, e.g. for sequence analysis, a mass spectrometry (MS) module, a quality control module (e.g. comprising an HPLC unit for analysis of HPLC), a qPCR or ddPCR module, a capillary electrophoresis module, a media supply rack or media supply module, a documentation module and / or a module for computer-aided control of all process steps and an interface for a high-level control and documentation system.

[0117] In summary, a device and a method for economic, controllable, reproducible, continuous (repeated batch) and GMP-compatible RNA production is provided. The described method and device allow the repeated use of DNA templates in multiple RNA (mass) production processes. Thus, the device as described above allows an accelerated production of RNA manufacturing, for example. Furthermore, in case of production of RNA therapeutics in regions of a pandemic outbreak, the automation as described above and due to the appropriate size of the portable RNA production device is advantageous.

[0118] It is to be understood that the bioreactor for RNA in vitro transcription, the method for RNA in vitro transcription, the use of the bioreactor according to the method, the module for transcribing DNA into RNA, and the automated device for RNA manufacturing according to the independent claims have similar and / or identical preferred embodiments, in particular as defined in the dependent claims. It is also to be understood that preferred embodiments of the present application can also be any combination of dependent claims with the respective independent claim.

[0119] These and other aspects of the present application will become apparent upon reference to the following embodiments described below. BRIEF DESCRIPTION OF DRAWINGS

[0120] The drawings shown below are merely illustrative and will describe the present application in a further way. These drawings should not be interpreted as limiting the present application thereto.

[0121] Figure 1 A schematic view of a bioreactor according to an embodiment of the present application is shown.

[0122] Figure 2 A schematic view of a reaction vessel according to an embodiment of the present application is shown.

[0123] Figure 3A and Figure 3B A schematic view of a reaction vessel according to an embodiment of the present application is shown.

[0124] Figure 4 A schematic view of a magnet unit according to an embodiment of the present application is shown.

[0125] Figure 5 A schematic view of a magnetic ring according to another embodiment of the present application is shown.

[0126] Figure 6 A schematic view of a magnet unit according to another embodiment of the present application is shown.

[0127] Figures 7A to 7C A schematic view of a bioreactor according to another embodiment of the present application is shown.

[0128] Figures 8A to 8C A schematic view of a bioreactor according to another embodiment of the present application is shown.

[0129] Figures 9A to 9H A schematic view of a bioreactor according to another embodiment of the present application is shown.

[0130] Figure 10 A schematic view of a bioreactor according to another embodiment of the present application is shown.

[0131] Figure 11 A schematic view of a bioreactor according to another embodiment of the present application is shown.

[0132] Figure 12A and Figure 12B A schematic view of a bioreactor with a movable magnet unit according to an embodiment of the present application is shown.

[0133] Figure 13 A schematic view of a bioreactor with a rotatable magnet unit according to an embodiment of the present application is shown.

[0134] Figure 14A and Figure 14BA schematic of a bioreactor with orbital shaker is shown.

[0135] Figure 15 An exemplary assembly of modules for transcription of DNA to RNA is shown.

[0136] Figure 16 An embodiment of a method for transcription of DNA to RNA is shown, according to one embodiment.

[0137] Figure 17 An exemplary apparatus for automation of RNA production is shown, according to one embodiment.

[0138] Figure 18 An exemplary process overview for RNA production is shown, according to one embodiment.

[0139] Figure 19 Results of repeated batch in vitro transcription of RNA using the same immobilized DNA template in 3 IVT reactions are shown. Experiments were performed as described in Example 1.

[0140] Figure 20A And Figure 20B Potency of produced RNA (RAVG mRNA) expressed in HepG2 cells is shown. Experiments were performed as described in Example 1.

[0141] Definitions

[0142] For the sake of clarity and readability, the following definitions are provided. Any technical features mentioned in relation to these definitions can be read over each embodiment of the application. Further definitions and explanations can be specifically provided in the context of these embodiments.

[0143] Dog bone, dog bone DNA :

[0144] The term “Doggybone TM ” (dbDNA) as used herein denotes the smallest, closed linear DNA vector developed enzymatically by Touchlight Genetics Limited. Linear DNA is produced rapidly, plasmid-free and synthesized by an enzymatic process that produces a vector cassette containing only the encoded sequence of interest, a promoter, e.g. a polyA tail and a telomere end.

[0145] Mixing :

[0146] In the context of the present application, “mixing” generally refers to a process that involves manipulating a heterogeneous physical system to make it more homogeneous. Mixing is performed to allow mass transfer to occur between one or more streams, components, or phases. Mixing is essentially the evolution in time of a spatially dependent concentration to a more uniform state.

[0147] In the context of the present application, a magnet unit is used which allows to cooperate with magnetic particles and / or DNA magnetic particles in order to improve the mixing of components contained in a reaction vessel as defined herein, preferably without exerting any mechanical stress (e.g. shear stress) on said components. In particular, according to the present application, conventional mixing means are preferably avoided which are known to induce mechanical stress on the components to be mixed. For example, the mixing of fluids is preferably performed without shaking and / or stirring the reaction vessel. Alternatively, the magnet unit is arranged to generate an appropriate magnetic field which results in forces acting on the magnetic particles and / or DNA magnetic particles such that the DNA magnetic particles start to move within the reaction vessel, thereby resulting in the mixing of the components contained in the reaction vessel.

[0148] The initiated movement of the magnetic particles and / or DNA magnetic particles can introduce a turbulent flow in the components contained in the reaction vessel which is not induced by shaking or vibration, which allows to improve the mixing of the components in the reaction vessel to produce a homogeneous composition.

[0149] RNA in vitro transcription :

[0150] The term "RNA in vitro transcription" relates to a process in which RNA is synthesized in a cell-free system. RNA can be obtained by DNA-dependent RNA in vitro transcription of an appropriate DNA template, which according to the present application can be a linearized plasmid DNA template or a PCR-amplified DNA template. The promoter used to control RNA in vitro transcription can be any promoter of any DNA-dependent RNA polymerase. Particular examples of DNA-dependent RNA polymerases are T7, T3, SP6 or Syn5 RNA polymerases.

[0151] The DNA template (e.g. plasmid DNA, dogbone DNA) can be linearized with a suitable restriction enzyme and immobilized on magnetic beads (e.g. as described in PCT / EP2017 / 084264 or PCT / EP2018 / 086684) and then RNA in vitro transcription is performed. Alternatively, the DNA template can be provided as PCR-amplified DNA immobilized on magnetic particles (using biotinylated primers for PCR-based DNA template amplification and subsequent immobilization on streptavidin magnetic beads).

[0152] Reagents for RNA in vitro transcription typically include: a DNA template (linearized DNA or linear PCR product) with a promoter sequence having a high binding affinity for its respective RNA polymerase, e.g. bacteriophage encoded RNA polymerases (T7, T3, SP6 or Syn5); ribonucleotide triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil); optionally, a cap analog (e.g. m7G(5’)ppp(5’)G (m7G) or any cap structure derivable from the structure disclosed in claims 1-5 of WO2017 / 053297 or definable from the structure defined in claim 1 or 21 of WO2018075827); optionally, further modified nucleotides as defined herein; a DNA-dependent RNA polymerase (e.g. T7, T3, SP6 or Syn5 RNA polymerase) capable of binding to the promoter sequence within the DNA template; optionally, a ribonuclease (RNase) inhibitor to inactivate any potential contaminating RNase; optionally, a pyrophosphatase to degrade pyrophosphate (an inhibitor of RNA synthesis); MgCl2, which provides Mg 2+ ions as a co-factor for the polymerase; a buffer (TRIS or HEPES) to maintain a suitable pH value, which can also comprise an antioxidant (e.g. DTT) and / or a polyamine, such as spermidine at an optimal concentration, e.g. a buffer system comprising citrate and / or betaine, as disclosed in WO2017 / 109161.

[0153] The nucleotide mix for RNA in vitro transcription can additionally contain modified nucleotides as defined herein. In this case, the preferred modified nucleotides include pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine and 5-methoxyuridine. The nucleotide mix for the RNA in vitro transcription reaction (i.e. the fraction of each nucleotide in the mix) can be optimized for a given RNA sequence, preferably as described in WO2015188933.

[0154] RNA in vitro transcription pre-mix, IVT pre-mix:

[0155] The RNA in vitro transcription (IVT) pre-mix can comprise the components necessary to perform an RNA in vitro transcription reaction as defined above. Thus, the IVT pre-mix can comprise at least one component selected from the group consisting of a nucleotide mix, a cap analog, a DNA-dependent RNA polymerase, an RNAse inhibitor, a pyrophosphatase, MgCl2, a buffer, an antioxidant, betaine, citrate.

[0156] Semi-permeable filter :

[0157] A filter which allows certain particles to pass through the pores of the filter material when the particles are smaller than the pore size, thereby preventing the passage of particles larger than the pore size of the filter material.

[0158] If a group is defined below to include at least a certain number of embodiments, this is also meant to include a group which consists only of these embodiments.

[0159] As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0160] It is to be understood that the terms "including", "comprising", "consisting" and "consisting essentially of" are not restricted to listed members but include equivalents which are not expressly listed. DETAILED DESCRIPTION

[0161] The present application relates to a bioreactor for RNA in vitro transcription which is set up to be operated in an automated manner under GMP compliant conditions. A schematic view of a bioreactor for RNA in vitro transcription according to an embodiment of the present application is provided in particular in Figure 1 and 7.

[0162] The bioreactor 1 comprises a reaction vessel 2 for containing magnetic particles, DNA template, DNA immobilization buffer, DNA magnetic particles and IVT premix. The inner surface 21 of the reaction vessel 2 has an oval inner geometry. Alternatively, the inner surface 21 of the reaction vessel 2 according to the present application can be ellipsoidal or ovoid. In any case, it is preferred that the inner surface 21 of the reaction vessel 2 has a shape without edges. This can be particularly important for the mixing properties of the bioreactor 1. Furthermore, the ellipsoidal, ovoid or egg shape, in particular without edges, is advantageous for cleanability (GMP compliance is important) and reduces the risk of forming unwanted precipitates in the bioreactor. Moreover, the oval shape has the advantage over e.g. a flat circular shape that the fluid (e.g. the RNA product) can easily flow out of the bioreactor 1 through the media port 6 into the media tube 66 (see also Figure 11 ).

[0163] Furthermore, the above-mentioned inner geometry helps to prevent adhesion and drying of e.g. protein residuals at the inner surface, typically a shape without edges, more specifically an ellipsoid, oval or egg shape supports a good draining of the fluid. In addition, the ellipsoid, oval or egg shape has the advantage over e.g. a "conical" shape that the risk of assembly of DNA magnetic particles at the bottom of the reactor is minimized, which can reduce the yield of the RNA in vitro transcription (e.g. RNA polymerase cannot access those DNA templates) or clog the media port 6. In order to further prevent clogging of the media port 6, liquid can be flushed through the media port 6 into the bioreactor 1 at regular intervals during the transcription reaction. These flushes can additionally improve the mixing properties of the biochemical reactions (e.g. IVT reaction, DNA immobilization reaction) in the bioreactor.

[0164] The bioreactor 1 is set up to allow repeated RNA in vitro transcription reactions on DNA templates immobilized on freely floating magnetic particles ("DNA magnetic particles"). For example, the DNA templates can be provided as PCR amplified DNA immobilized on magnetic beads (using biotinylated primers for PCR based DNA template amplification and subsequent immobilization on streptavidin magnetic beads) or linearized plasmid DNA immobilized on magnetic beads (as described in PCT / EP2017 / 084264 or PCT / EP2018 / 086684).

[0165] The bioreactor 1 further comprises a magnet unit 3 located at the reaction vessel 2. The magnet unit 3 enables contactless mixing of reactions containing magnetic particles or DNA magnetic particles, which means that no mixing devices have to be implemented in the mixing process, which is an advantageous feature in case of sufficient cleanability of the bioreactor 1 (e.g. in pharmaceutical production of RNA). Furthermore, mixing of the RNA in vitro reaction can be performed without rotating / shaking the bioreactor 1. This is particularly advantageous because rotation or vibration would be strongly impaired due to the different flow inlet and outlet ports that have to be mounted on the bioreactor 1.

[0166] Furthermore, the magnet unit 3 can be used to capture the DNA magnetic particles before starting another RNA in vitro transcription cycle, thereby allowing repeated batch RNA in vitro transcription (IVT) on the same DNA template, which significantly reduces the overall RNA production costs. Furthermore, the magnet unit 3 can be used to remove the DNA magnetic particles for final cleaning or sterilization of the bioreactor 1. Thus, the DNA can be removed without the need for an enzymatic DNAse treatment, which (i) reduces costs because no such enzyme is needed, (ii) reduces the risk of contaminating the final RNA product with additional components (i.e. DNAse) and (iii) reduces the risk of contaminating the final RNA product with DNA fragments or partially digested DNA fragments.

[0167] In Figure 1 particular, the magnet unit 3 is formed as a ring (see also Figure 4 ) and the reaction vessel 2 is accommodated in the center 33 of the magnet unit 3 so that the magnet unit 3 can be rotated around the reaction vessel 2. The magnet unit 3 is attached to a spindle axis 36 via an arm 37, wherein the spindle axis 36 can move the magnet unit 3 in a vertical direction. By moving the magnet unit 3 up and down, a magnetic field can be generated along the longitudinal direction of the reaction vessel 2. Thus, a uniform mixing of the components in the reaction vessel 2 can be achieved by inducing magnetic particles in the radial and longitudinal direction. A rotary drive 38 for the magnet unit 3 is arranged on the arm 37 directly above the reaction vessel 2 and a drive 39 for the spindle axis 36 is arranged directly above the spindle axis 36.

[0168] Figure 2 A perspective view of the reaction vessel 2 is shown, Figure 3A a bottom view of the reaction vessel 2 is shown, Figure 3B a top view of the reaction vessel 2 is shown. The reaction vessel 2 can be made of a material such as Teflon, which is chemically resistant, resistant to extreme temperatures, extreme pH values, mechanical forces and / or corrosion.

[0169] In all embodiments of the bioreactor 1 according to the application, the inner surface 21 of the reaction vessel 2 has a shape without edges, preferably an ellipsoidal, oval or egg shape. It is further preferred that the inner surface 21 of the reaction vessel 2 is polished to a value Ra <= 0.8. Suitable methods to achieve this Ra value are known to the person skilled in the art. For example, the inner surface 21 can be mechanically polished, electro-polished or chemically polished, etc.

[0170] As Figure 3B shown, the reaction vessel 2 comprises an outlet port 7 for exhaust gas or exhaust liquid. The outlet port 7 can be used, for example, for ventilation of the reaction vessel 2 during filling of the vessel. For this purpose, the outlet port 7 is arranged at the highest point of the reaction vessel 2. At the top of the reaction vessel 2, a first end 52 of a heat exchange channel 51 of the temperature element 5 is arranged. As Figure 11 shown, the outlet port 7 can be connected to at least one of an exhaust duct 73 and a waste channel 74. For example, the outlet port 7 can be connected to at least the exhaust duct 73 and the waste channel 74 by a multi-position valve. The outlet port 7 can allow the reception and discharge of one or more exhaust gases occurring within the reaction vessel 2. In the case of exhaust liquid or cleaning fluid, the outlet port 7 can be used to discharge the fluid from the reaction vessel 2. The outlet port, the exhaust duct and / or the waste channel can hold at least one device for measuring and / or regulating the pressure.

[0171] Furthermore, a medium port 6 is arranged at the lowest point of the reaction vessel 2 and can be further connected to a valve device 60 which guides the supply or discharge of components (inFigure 3A The reaction vessel 2 comprises two legs 25, 26, which can vertically support the reaction vessel 2. Furthermore, each leg 25, 26 comprises a pipe 251, 261 extending through the leg 25, 26. Thus, the first leg 25 comprises a first pipe 251 arranged in fluid communication with the valve device 60 and the second leg 26 comprises a second pipe 261 arranged in fluid communication with the second end 53 of the heat exchange channel 51 of the temperature element 5 (see Fig. 2). Figure 7B and Figure 7C ).

[0172] Figure 4 A preferred embodiment of the magnet unit 3 is shown. The magnet unit 3 is formed as a star comprising a magnetic ring 31 and a plurality of rods 32. The magnetic ring 31 and the rods 32 can be made of a plurality of magnetizable laminated electrical sheets, thus forming a laminated stack for shielding of peripheral components from the magnetic field. The magnetic ring 31 is designed to surround the reaction vessel 2. In other words, the reaction vessel 2 can be positioned in the center 33 of the magnetic ring 31.

[0173] The magnetic ring 31 comprises a first rod 320 and a second rod 322, which extend from an inner circumference 34 of the magnetic ring 31 to the center 33 of the magnetic ring 31, such that free ends 321, 323 of the first and second rods 320, 322 face each other. The free end 321 of the first rod 320 comprises a magnet having an N-pole, while the free end 323 of the second rod 322 comprises a magnet having an S-pole. However, as shown, the number and length of the rods 32 can vary. The rods 32 are arranged at the inner circumferential surface 34 of the magnetic ring 31 and extend in a direction towards the center 33 of the magnetic ring 31. The plurality of rods 32 is arranged in a star shape and uniformly spaced apart from each other, such that the magnetic ring 31 is formed symmetrically. At each free end of the rods 32, a magnet having an N-pole and a magnet having an S-pole are arranged alternately. Figure 5

[0174] Optionally, as shown, the magnet unit 3 comprises a magnet ring 31 comprising a plurality of guide plates 350 and electrical coils 351. The star-shaped guide plates 350 extend from an inner circumference 34 of the magnetic ring 31 to the center 33 of the magnetic ring 31. Each guide plate 350 comprises an electrical coil 351 for generating a magnetic field. The magnet ring 31 is surrounded by a housing with a cooling device 352. The cooling device 352 is integrated in the housing of the magnetic ring along the circumference of the magnetic ring 31 to take away the heat generated by the high current through the electrical coils. The cooling device 352 can be a cooling channel in which a cooling medium such as water is provided. Figure 6

[0175] Figures 7A to 7C ​​A further preferred embodiment of the bioreactor 1 is shown. The reaction vessel 2 can be made of a solid material and comprises an inner surface 21 and an outer surface 23. Integrated between the inner surface 21 and the outer surface 23 is a temperature element 5 for regulating the temperature of the reaction vessel 2. The temperature element 5 comprises a heat exchange channel 51 spirally surrounding the reaction vessel 2 in a radial direction with respect to the longitudinal axis of the reaction vessel 2. In order to facilitate the manufacturing of such a reaction vessel 2 with a complex geometry, the reaction vessel 2 can be manufactured by means of additive manufacturing.

[0176] The heat exchange channel 51 comprises a first end 52 and a second end 53, which are fluidically connected to the second duct 261 in the second leg 26. The first end 52 is arranged at the top of the reaction vessel 2, however, the first end 52 is positioned offset from the uppermost top or outlet port 7 to ensure reliable access to the outlet port 7. The second end 53 of the heat exchange channel is arranged at the bottom of the reaction vessel 2, however, the second end 53 is positioned offset from the bottommost or medium port 6 to ensure reliable access to the medium port 6. Through the first end 52 or the second end 53, a heat exchange medium such as water can be supplied into the heat exchange channel 51 for heating or cooling components inside the reaction vessel 2.

[0177] Figure 8A and Figure 8B An alternative embodiment of the temperature element 5 is shown. The temperature element 5 comprises a heating wire 54 spirally surrounding the reaction vessel 2 at least partially, preferably completely, in a radial direction with respect to the longitudinal axis of the reaction vessel 2. The heating wire 54 is at least partially integrated in the outer surface 23 of the reaction vessel 2 (in Figure 8A Additionally or alternatively, the outer surface 23 of the reaction vessel 2 can be coated with a thermal insulation material 55 and the heating wire 54 is at least partially retracted into the thermal insulation material 55 (in Figure 8B

[0178] With reference to Figure 7B and Figure 7C The reaction vessel 2 comprises at least one, preferably two, flow breakers 24 arranged at least partially along the inner surface 21 of the reaction vessel 2 in the longitudinal direction of the reaction vessel 2. The flow breakers 24 can disturb the uniform flow of components in the reaction vessel 2 and can thereby improve mixing. Furthermore, the flow breakers 24 can prevent the deposition of magnet particles when the magnet unit 3 is stopped from rotating and / or changes the direction of rotation. The two flow breakers 24 are spaced apart from each other in a radial direction with respect to the longitudinal axis of the reaction vessel 2.

[0179] As Figures 9A to 9H ​As shown, the flow interrupter 24 can be rib-like and protrude along the longitudinal direction of the reaction vessel 2 from the inner surface 21 of the reaction vessel 2. In another embodiment, the flow interrupter 24 is arc-shaped and comprises a T-shaped cross-section (in Figure 9A and Figure 9B ) or an L-shaped cross-section (in Figure 9E and Figure 9F ). In yet another embodiment, the flow interrupter 24 is corrugated or wavy (in Figure 9C and Figure 9D ). Optionally, the flow interrupter 24 comprises a plurality of semicircular protrusions spaced apart from each other at the inner surface 21 of the reaction vessel 2 in the longitudinal direction (in Figure 9G and Figure 9H ).

[0180] It is noted that the elements and features of the bioreactor 1 of the present application mentioned in the context of Figures 10 to 1 2 can equally be part of the reactor shown in Figure 1 to Fig. 9, even if not explicitly mentioned herein. Thus, the bioreactor 1 as shown in Figure 1 to Fig. 9 can further comprise at least one selected from the group consisting of a magnetic trap 61, a Hall sensor 63, a flow cell 64, a temperature sensor 91, an additional sensor 92 or a specific fill level 27 or a maximum fluid amplitude 28.

[0181] Figure 10 Another embodiment of the bioreactor 1 is shown. Figure 10The bioreactor 1 in the RNA in vitro transcription process comprises an electromagnet array 3 positioned on the outer surface of the reaction vessel. The electromagnet array 3 allows mixing of the reaction (by circulation of magnetic particles or DNA magnetic particles in the reaction), which is induced by the periodic activation of said electromagnet array 3. This enables contactless mixing of the reaction containing magnetic particles or DNA magnetic particles, which means that no mixing device has to be implemented in the mixing process, which is an advantageous feature in case of sufficient cleanability of the bioreactor 1 (e.g. in RNA pharmaceutical production). Furthermore, mixing of the RNA in vitro reaction can be performed without rotating / shaking the bioreactor 1. This is particularly advantageous, because rotation or vibration would be strongly impaired due to the different flow inlet and outlet ports that have to be mounted on the bioreactor 1. Furthermore, said electromagnet array 3 can be used to capture the DNA magnetic particles before starting another RNA in vitro transcription cycle, allowing repeated batch RNA in vitro transcription (IVT) on the same DNA template, which significantly reduces the total RNA production cost. Furthermore, said electromagnet array 3 can be used to remove the DNA magnetic particles for final cleaning or sterilization of the bioreactor 1. Thus, DNA can be removed without the need for enzymatic DNAse treatment, which (i) reduces costs, because no such enzyme is needed, (ii) reduces the risk of contaminating the final RNA product with additional components (i.e. DNAse), and (iii) reduces the risk of contaminating the final RNA product with DNA fragments or partially digested DNA fragments.

[0182] Figure 10 The filling level 27 of the fluid container in the reaction vessel 2 is also shown in the RNA in vitro transcription process. In addition, the maximum fluid amplitude 28 is shown by the dashed line. Thus, the maximum fluid amplitude 28 is to be understood as the amplitude reached by the fluid contained in the reaction vessel 2 and reaching the maximum shaking or rotation movement on the inner surface 21 of the reaction vessel 2. The bioreactor 1 further comprises a flow inlet port 8 arranged at the reaction vessel, which allows filling of the culture medium into the reaction vessel 2. As Figure 11As shown, the inlet port 8 is arranged laterally on the reaction vessel 2 and below the level of the maximum fluid amplitude 28 on the inner surface 21 of the reaction vessel 2. This configuration can help to prevent, for example, protein residue deposition and hardening on the inner surface 21 of the reaction vessel 2, which can be the case if the inlet port is arranged above the maximum fluid amplitude. In the latter case, residue from the fill of the reaction vessel 2 can deposit at and / or around the inlet port, for example. Furthermore, the lateral position of the inlet port 8 close to the fill level 27 allows the fill medium to enter the reaction vessel without undesirably forming splashes that can lead to residue deposition and hardening on the inner surface 21 of the reaction vessel. Upstream of the inlet port 8, an inlet tube 83 for guiding the fill medium towards the inlet port 8 and into the reaction vessel 2 is arranged. The bioreactor 1 further comprises a waste port 7 for waste gas or waste liquid. The waste port 7 can be used, for example, for ventilation of the reaction vessel 2 during filling of the vessel. To this end, the waste port 7 is arranged at the highest point of the reaction vessel 2. Downstream of the waste port 7, a waste channel 74 is arranged that allows the uptake of waste gas or waste liquid leaving the vessel 2 through the waste port 7. Furthermore, an outlet port 6 is arranged at the lowest point of the reaction vessel 2, thereby allowing for easy piping or discharge of fluids through the outlet port 6 for further guiding of these fluids through an outlet tube 66.

[0183] Figure 11 Another preferred embodiment of a bioreactor 1 according to the present application is shown. In addition to the components already shown in Figures 1 to 10 Fig. 1 - i.e. the reaction vessel 2 and the magnet unit 3, the waste port 7, the waste channel 74, the outlet port 6, the outlet tube 66 and the inlet port 8, the inlet tube 83 and the fill level 27, which refers to the line of contact of the fluid surface at the inner surface of the reaction vessel 2 and the maximum fluid amplitude 28, Figure 11Embodiments in the middle further comprise a magnetic trap 61 positioned at the flow outlet port 6 to minimize the risk of DNA magnetic particles and / or DNA magnetic particles contaminating the RNA product. This means that when the produced RNA is discharged from the reaction vessel 2 through the flow outlet port 6, the magnetic trap 61 helps to keep magnetic particles and / or DNA magnetic particles within the reaction vessel 2. The magnetic trap 61 can for example at least partially surround the flow outlet port 6 or a flow outlet tube 66 downstream adjoining the flow outlet port 6. Preferably, the magnetic trap 61 can be a ring magnet, for example an electromagnet in ring form. Downstream of the flow outlet port 6 and the magnetic trap 61 a multi-position valve 62 is arranged. The multi-position valve 62 connects the flow outlet port 6 or a flow outlet tube 66 downstream connected to the flow outlet port 6 with three further lines. A first of the three lines is used to transport the RNA containing fluid component after the RNA in vitro transcription reaction has successfully taken place. In order to monitor that no magnetic particles and / or DNA magnetic particles are contained in this component, a Hall sensor 63 is arranged downstream of the multi-position valve 62 at the first line. Thus, the Hall sensor 63 is set up to detect unwanted magnetic fields in the RNA product. A second line connected to the flow outlet port 6 is used as a waste channel 67 for example for cleaning fluids. For monitoring purposes, a flow cell 64 is arranged at this second line. A third line connected to the multi-position valve 62 can transport process gas or cleaning gas, for example N2 or a synthetic solution, in the direction indicated by arrow 65. The process gas or cleaning gas can be cyclically directed by the multi-position valve 62 in the direction of the flow outlet port 6. Thereby, a deposition of magnetic particles and / or DNA magnetic particles at the flow outlet port can be prevented, leading to a clogging of the port.

[0184] Furthermore, the bioreactor 1 comprises temperature elements, for example Peltier elements 9, to allow heating or cooling of the bioreactor 1 °C at 37°C, which is the optimal temperature for RNA in vitro transcription, and heating of the bioreactor 1 °C at 80°C, which is the optimal temperature for cleaning / sterilizing the bioreactor 1. A temperature sensor 91 is further arranged at the reaction vessel 2 for monitoring the temperature in the reaction vessel 2. Further temperature sensors can be positioned at the inner surface 21 of the reaction vessel and / or in the vicinity of the reaction vessel, for example at the flow inlet port or the flow outlet port. For example, additional sensors 92 can be located within the reaction vessel 2 for measuring for example the temperature, the pH value or the salt concentration.

[0185] Still referring to Figure 11The bioreactor 1 further comprises a multi-position valve 71 arranged downstream of the waste port 7 and a waste channel 74 adjoining the waste port 7. Via the multi-position valve 71, the waste port 7 and the waste channel 74 are connected to a line for waste liquid, wherein a waste flow chamber 72 is arranged at the line for monitoring the flow of waste liquid. Furthermore, the multi-position valve 71 connects the waste port 7 and the waste channel 74 to an exhaust duct 73 for waste gas, which can occur for example during filling or cleaning of the reaction vessel 2. Optionally, a pressure sensor 76 can be provided at the waste port or the waste channel 74 for measuring the pressure at the waste port 7 and / or in the waste channel 74. Upstream of the flow inlet port 8, a heater 81, exemplarily shown as a heating coil, is arranged around the flow inlet tube 83. Upstream of the tube section with the heater 81, a heating flow chamber is arranged for monitoring the feed of components into the reaction vessel 2. The heater 81 can be used to adjust the medium filled into the reactor to the desired optimal temperature (e.g. 37°C for RNA in vitro transcription).

[0186] Figure 12A and Figure 12B and Figure 13 An alternative design of the magnet unit of the bioreactor 1 according to the present application is shown. Reference is made to Figure 12A and Figure 12B An embodiment of the bioreactor 1 is shown, which comprises a reaction vessel 2 with a flow outlet port 6, a waste port 7 and a flow inlet port 8 and a magnet unit 3. It is worth noting that the elements mentioned in the context of the bioreactor specified in Figure 11 can equally be part of the reactor shown in Figure 12A , Figure 12B (e.g. temperature sensor 91, Hall sensor 63, flow chamber 64, egg shape, etc.), even if this is not explicitly mentioned here. The magnet unit 3 is realized in the form of a magnet, preferably an electromagnet or a permanent magnet, which can be moved along the transversal axis of the reaction vessel 2 towards and away from the reaction vessel 2, as indicated by the arrows 363 or a controllable Helmholtz coil. Furthermore, the magnet unit 3 can be moved up and down along the longitudinal axis of the reaction vessel 2, as indicated by the arrows 362. To this end, the magnet unit 3 is mounted on a movable support 361, which allows the above-mentioned movements of the magnet unit 3. In addition, as further shown in Figure 12A and Figure 12B in this embodiment, the reaction vessel 2 can be rotated around its vertical axis. Optionally, the reaction vessel 2 can be mounted on a movable support (not shown), which allows the above-mentioned movements of the reaction vessel 2 relative to the magnet unit 3 (the magnet unit 3 can not be mounted on a movable support 361). In addition, as further shown in Figure 12A and Figure 12BAs further shown in the figure, in this embodiment, the reaction vessel 2 can rotate about its vertical axis.

[0187] Figure 12A The bioreactor 1 is shown in the state where the magnet unit 3 has been laterally removed from the reaction vessel 2, while Figure 12B The diagram shows a configuration in which the magnet unit 3 is located laterally closest to the reaction vessel 2.

[0188] Figure 13 An embodiment of a bioreactor 1 is shown, comprising a magnetic unit 3 consisting of at least two magnetic coils, which are rotatable about the reaction vessel as indicated by arrow 111 and rotatably arranged at a horizontal rod 11 of a support 10. The horizontal rod 11 can move upward and downward, as indicated by arrow 110, allowing the magnetic field position of the magnetic coils 3 at the reaction vessel 2 to be further altered. It is noteworthy that, in Figures 1 to 11 The elements mentioned in the context of the bioreactor specified in the document can also be Figure 13 Parts of the reactor shown (e.g., temperature sensor 91, Hall sensor 63, flow chamber 64, oval shape, etc.), even though not explicitly mentioned here.

[0189] exist Figure 14A and Figure 14B The embodiment of bioreactor 1 is shown, which allows for the introduction of the device by mechanical motion and the mixing or stirring of the components contained in the reaction vessel by additionally guiding process gas or process fluid into the reaction vessel or by the cooperation of magnetic particles and magnet units.

[0190] Besides already Figure 11 In addition to the components described in the context, Figure 14A A bioreactor 1 is shown, in which a reaction vessel 2 is positioned on an orbital vibrator OS. The orbital vibrator OS allows for 3D motion of the reaction vessel, preferably with a small amplitude, due to the connections for fluids, gases, and sensors used in the bioreactor, and should not be damaged by the motion of the reaction vessel 2. To induce motion of the reaction vessel 2 via the orbital vibrator OS, the reaction vessel 2 is placed on top of the orbital vibrator OS. The reaction vessel 2 is at least partially enclosed laterally and can therefore be held by a support member 20. The support member 20 includes a Peltier element 9 for heating and / or cooling the reaction vessel. Figure 14A The outlet port 6 and outlet pipe 66 allow process gas (preferably N2) or process fluid to be introduced into the reaction vessel 2, thereby introducing additional motion for mixing / stirring the components contained in the reaction vessel 2. The outlet port 6 and outlet pipe 66 also allow culture medium (e.g., generated RNA) to flow out of the reaction vessel. Culture medium can be filled into the reaction vessel through the inlet pipe 83 and inlet port 8. Furthermore,Figure 14A The waste port 7 and the waste channel arranged at the highest point of the reaction vessel are shown.

[0191] In Figure 14B , an embodiment of the bioreactor 1 is shown, which allows mixing or stirring of the components contained in the vessel 2 by cooperation of the direction of the process gas or process fluid into the reaction vessel through the Helmholtz coil and the magnetic particles, the orbital shaker OS and the magnetic particles. For this purpose, the orbital shaker OS is connected with the reaction vessel 2 via the horizontal support S, which holds the orbital shaker OS and is positioned in the middle of the magnet unit 3. The magnet unit is here realized in the form of a Helmholtz coil. A part of the support holding the reaction vessel 2 contains a recess, in which the Peltier element 9 is positioned. In order to effectively heat and / or cool the vessel, the Peltier element is positioned close to or even in contact with the reaction vessel. In addition to the above-mentioned components, Figure 14B the inflow port 8 and the inflow tube 83, the waste port 7 and the waste channel 74, as well as the outflow port 6 and the outflow tube 66 are also shown, the elements of the latter being similar to those described in the context of Figure 11 and Figure 14A .

[0192] In Figure 15 , an embodiment of a module for transcribing DNA to RNA is shown. It comprises a unit for preparing an IVT premix 12, also called a premixer. As indicated by the arrow into the unit for preparing an IVT premix 12, this unit 12 can be filled with an IVT buffer (HEPES, Tris), a nucleotide mix (including nucleotides and optionally modified nucleotides), a DNA-dependent RNA polymerase, a cap analog, an RNAse inhibitor, a pyrophosphatase, MgCl2, an antioxidant (DTT), betaine, citrate.

[0193] The individual components can be provided by a media supply rack (not shown). The resulting IVT premix is guided from the unit 12 for preparing an IVT premix via a line 121 into the bioreactor 1 according to the application. In addition to the IVT premix, DNA is provided to the bioreactor 1 via a feed in a line 122.

[0194] In addition, the bioreactor 1 can be filled with a wash buffer by a feed in a line 123. It is to be understood that the filling of the bioreactor 1 takes place through the inflow port 8 of the bioreactor 1, which is exemplarily shown in Figures 12 to Figure 15 and discussed in the context of Figures 12 to Figure 15 . Further reference is made to Figure 5The crude RNA product is directed via line 124 to a conditioner 13, for example by tangential flow filtration. After conditioning, the RNA is introduced into a device for RNA purification 14, for example an RP-FIPLC using the method disclosed in WO 2008 / 077592; The device for RNA purification 14 is preferably an RP-FIPLC device for automated purification and fractionation of the raw RNA. The device for RNA purification 14 can additionally or alternatively comprise an oligo dT purification device for automated purification and fractionation of the raw RNA. As indicated by the dashed arrow, the RNA can subsequently be directed to a further device, for example a further device for RNA treatment, for example by tangential flow filtration, and / or a device for sterile filtration of the RNA.

[0195] In the context of the present application, as a suitable environment for carrying out the process, a process chamber or housing can be provided. The process chamber or housing can be separate from the control system required to control and / or monitor the process. In the process chamber, laboratory equipment can be provided. For example, the front of the process chamber can be opened by a sliding door. The base frame of the process chamber can consist of modular equipment, which can be divided into three parts. As an example, the three modules can consist of a one-meter module, a two-meter module and a backpack with a total length of 3.5 meters and a height of about 2 meters. In addition, an exhaust system can be included, which can require additional space. The medium supply can be located in the one-meter module and should be physically separated from the actual process chamber located in the two-meter module by a separation wall. The separation wall can be achieved, for example, by a glass partition and a PVC curtain behind the sliding door.

[0196] The internal process chamber can be optionally connected to an exhaust system. The liquids that can be desired to be processed by the process require further safety measures. This includes explosion protection and / or other biological and chemical safety measures, which can be included in the process chamber.

[0197] Figure 16A flow chart for a method for RNA in vitro transcription according to an embodiment of the application is shown. The method comprises a step S1 of providing magnetic particles, a DNA template, a DNA immobilization buffer and an IVT premix in the reaction vessel of a bioreactor according to an embodiment of the application. In step S2 the magnetic particles, the DNA template and the DNA immobilization buffer are mixed by cooperation of the magnetic particles and the magnet unit of the bioreactor to obtain DNA magnetic particles, which are DNA templates immobilized on freely floating magnetic particles. In method step S3 the DNA magnetic particles are mixed with the IVT premix by cooperation of the DNA magnetic particles and the magnet unit to obtain RNA. After step S3 the method can comprise a step S4 comprising capturing the DNA magnetic particles by the magnet unit and collecting / harvesting the in vitro transcribed RNA obtained e.g. through a flow outlet port (S4a), providing fresh IVT premix in the reaction vessel of the bioreactor of the first aspect (S4b), releasing the captured DNA magnetic particles to provide freely floating DNA magnetic particles (S4c), mixing the freely floating DNA magnetic particles with the IVT premix by cooperation of the DNA magnetic particles and the magnet unit to obtain RNA (S4d), and finally removing the DNA magnetic particles from the RNA to obtain DNA free in vitro transcribed RNA. It is worth noting that S4 can be performed multiple times.

[0198] In addition to the above described steps, a step ST of tempering the reaction vessel of the bioreactor can be performed between steps S1 and S2 and / or between steps S2 and S3. A cleaning or sterilization step SC can also be performed after step S3, in which the reaction vessel is cleaned with a cleaning fluid and / or a cleaning gas.

[0199] Figure 17 and Figure 18 Embodiments relating to an automated device for RNA manufacturing according to the application are described. In Figure 17In the middle, embodiments of modules of the automated device and elements for each module are shown. The device comprises a module for DNA synthesis ("template generator") T, a module for transcription of DNA into RNA M, and a module for RNA formulation and filling and finishing F. The module for DNA synthesis comprises a pre-mixer 40, which is a unit for preparing a PCR premix 41, which is directed to a unit for preparative PCR 42. The obtained raw DNA template is then directed to a DNA processing unit 43. The dotted lines and dashed boxes indicate that the processed DNA template can then be directed to further units, e.g. a unit for purification (e.g. comprising RP-HPLC and / or oligo dT). The purified DNA can then be released as indicated by the horizontal dotted arrow pointing out of module T. However, the purified DNA can also be provided to module M, in particular to element 1 (bioreactor) of module N. As additional input, the bioreactor 1 obtains an IVT premix from a unit for preparing an IVT premix 12. The raw RNA obtained by the RNA in vitro transcription reaction within the bioreactor 1 is directed to a unit for processing the raw RNA (e.g. comprising TFF) 13, and is then directed to a unit for RNA purification 14 (e.g. comprising RP-HPLC and / or oligo dT). As indicated by the dotted lines and dashed boxes, the obtained RNA can then be further processed and / or refined (e.g. RNA capping module for adding cap0 or cap1 structures to the in vitro transcribed RNA, RNA polyadenylation module, RNA mixing module, RNA spray-drying module, RNA lyophilization module). After said steps, the RNA is provided to module F. In this module, e.g. LNP-encapsulated RNA can be produced by a combination of different units, including at least one of a unit for mixing, a unit for processing (e.g. by TFF), a unit for sterile filtration, and a unit for filling the obtained drug product.

[0200] Figure 18 An overview of method steps comprising DNA synthesis, DNA purification, and RNA in vitro transcription performed in the context of the following Example 1 is shown.

[0201] Example

[0202] The following examples are merely illustrative and the present application will be further described in further manner. The examples should not be construed as limiting the application thereto.

[0203] Example: Model batch

[0204] As an illustrative example of the processes and methods described in the context of the present application, a model batch process has been performed manually in a laboratory. In the context of the model batch process, the following steps were performed: Figure 18The respective method steps are described in the middle. During the first step, i.e. the DNA template generation step, the sub-steps of PCR (Polymerase Chain Reaction) T1 and DNA purification (using RP-HPLC) T2 and AXP purification (using Agencourt AMPure XP) are performed. Thus, the last sub-step T3 should not be performed in the final and automated process according to the embodiments of the present application and is only required for model batches for manual handling as in the example. In the next step, the RNA in vitro transcription is performed, wherein this step comprises the following sub-steps: As first sub-step, DNA immobilization M1, wherein the DNA template is immobilized on freely floating magnetic beads. The second sub-step M2 refers to the RNA in vitro transcription reaction. As next sub-step (not shown in Fig. 20), the AXP purification is performed, wherein again this purification step should not be performed in the final automated process but only in model batches for manual handling. In sub-step M3, the produced crude RNA is purified. Sub-step M4 refers to ultrafiltration (UF) / diafiltration (DF), e.g. using TFF, and as sub-step M5, sterile filtration is performed. This example is non-limiting and in order to emphasize the fact that further method steps can be performed, the dashed box with reference sign M5 indicates that further sub-steps can be present within the RNA in vitro transcription step. As third step, the produced crude RNA is formulated. For this, in sub-step F1 an inline mixing is performed. As next step, not shown in Fig. 20, a dialysis is performed, wherein this sub-step is also intended to be omitted in case of the final automated process and is only required for model batches for manual handling. The next sub-step F2 involves UF / DF, followed by a cryoprotection step, which is also not shown in Fig. 20, as this step is only required for model batches for manual handling. The last three sub-steps can also be combined in a single UF / DF step. In sub-step F3, sterile filtration is performed. The dashed box with reference sign F4 indicates that additional sub-steps can be incorporated into the method according to the present application. However, in case of the embodiments, no further sub-steps are performed.

[0205] The repeated batch RNA in vitro transcription performed in the example comprises a PCR template generation step and a DNA template purification step, both of which are performed in the template generator. In the next step of RNA production, a template immobilization is performed in a first sub-step, followed by a repeated batch RNA in vitro transcription reaction step. This is then followed by a repeated batch HPLC sub-step, and finally a single batch TFF sub-step.

[0206] The results of the recovered, i.e. repeated, RNA in vitro transcription reactions are summarized in Figure 19The same immobilized DNA template was used in three in vitro RNA transcription reactions. The results showed stable performance in all three cycles of in vitro RNA transcription, both quantitatively and qualitatively.

[0207] exist Figure 20A and Figure 20B The diagram shows an RNA potency assay of the resulting drug substance, namely RNA (HPLC purified) expressed in HepG2 cells (RAVG mRNA), demonstrating that repeated in vitro RNA transcription reactions appropriately performed in the bioreactor of the present invention can produce high-quality RNA in a robust and reliable manner.

[0208] It should be noted that embodiments of the invention have been described with reference to different subject matter. In particular, some embodiments are described with reference to method-type claims, while other embodiments are described with reference to apparatus-type claims. However, those skilled in the art will understand from the above and below description that, unless otherwise notified, any combination of features related to different subject matter is also considered to be disclosed by this application, except for any combination of features belonging to one type of subject matter. However, all features can be combined to provide more synergistic effects than a simple summation of features.

[0209] Although the invention has been described and illustrated in detail in the accompanying drawings and the foregoing description, such description and illustration are to be regarded as illustrative or exemplary rather than restrictive. The invention is not limited to the disclosed embodiments. Other variations of the disclosed embodiments can be understood and implemented by those skilled in the art in carrying out the claimed invention, based on a study of the drawings, the disclosure, and the dependent claims.

[0210] In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" does not exclude a plurality. The fact that certain measures are recited merely in mutually different dependent claims does not indicate that a combination of those measures cannot be used advantageously. Any reference numerals in the claims should not be construed as limiting the scope.

[0211] List of reference numerals

[0212] 1. Bioreactor

[0213] 10 Support components

[0214] 11 Horizontal bar

[0215] 110 arrows

[0216] 111 Arrow

[0217] 2. Reaction Vessel

[0218] 21 inner surface of the reaction vessel

[0219] 23 outer surface of the reaction vessel

[0220] 24 interrupter

[0221] 25 first leg of the reaction vessel

[0222] 251 first duct

[0223] 26 second leg of the reaction vessel

[0224] 261 second duct

[0225] 27 filling level

[0226] 28 maximum fluid amplitude

[0227] 3 magnet unit

[0228] 31 magnetic ring

[0229] 32 rod

[0230] 320 first rod

[0231] 321 free end of the first rod

[0232] 322 second rod

[0233] 323 free end of the second rod

[0234] 33 center of the magnet unit

[0235] 34 inner circumference of the magnetic ring

[0236] 350 guide plate

[0237] 351 electric coil

[0238] 352 cooling device

[0239] 36 spindle axis

[0240] 37 arm

[0241] 38 rotary drive

[0242] 39 drive

[0243] 361 movable support

[0244] 362 arrow

[0245] 363 arrow

[0246] 5 temperature element

[0247] 51 heat exchange channel

[0248] 52 first end heat exchange channel

[0249] 53 second end heat exchange channel

[0250] 54 heating wire

[0251] 55 thermal insulation material

[0252] 6 media port / outlet port

[0253] 60 valve arrangement

[0254] 61 magnetic trap

[0255] 62 multi-position valve

[0256] 63 hall sensor

[0257] 64 flow chamber

[0258] 65 arrow

[0259] 66 media tube / outlet tube

[0260] 67 waste channel

[0261] 7 outlet port / waste port

[0262] 71 multi-position valve

[0263] 72 waste flow chamber

[0264] 73 exhaust tube

[0265] 74 waste channel

[0266] 76 pressure sensor

[0267] 8 inlet port

[0268] 81 heater

[0269] 83 inlet tube

[0270] 91 temperature sensor

[0271] 92 additional sensor

[0272] 12 IVT premix

[0273] 121 line into bioreactor

[0274] 122 line

[0275] 123 line

[0276] 124 line

[0277] 13 processor

[0278] 14 RNA purification

[0279] 40 premixer

[0280] 41 PCR premix

[0281] 42 preparative PCR

[0282] 43 unit for DNA treatment.

Claims

1. A bioreactor (1) for in vitro transcription of RNA, comprising: (a) a reaction vessel (2) suitable for containing magnetic particles, DNA template, DNA immobilization buffer, DNA magnetic particles and IVT premix, wherein the DNA magnetic particles are DNA templates immobilized on freely floating magnetic particles, wherein the IVT premix comprises ribonucleotide triphosphates and a DNA-dependent RNA polymerase; and (b) a magnet unit (3) located at the reaction vessel, wherein the magnet unit (3) is arranged to capture the magnetic particles and the DNA magnetic particles or to introduce motion to the magnetic particles and the DNA magnetic particles, thereby initiating mixing or stirring of the magnetic particles, the DNA template, the DNA immobilization buffer, the DNA magnetic particles and the IVT premix; wherein the magnet unit (3) comprises a magnetic ring (31) designed to surround the reaction vessel (2), wherein: the magnet unit (3) is a permanent magnet or an electromagnet movable in a longitudinal direction (362) along a longitudinal axis of the reaction vessel (2) and / or in a lateral direction (363) towards and away from the reaction vessel (2); the magnet unit (3) is arranged to rotate around the longitudinal axis of the reaction vessel (2), and wherein the direction of rotation of the magnet unit (3) is switchable during mixing; and the magnet unit (3) further comprises first drive means (36) arranged to rotate the magnetic ring (31) and second drive means (37) arranged to move the magnetic ring (31) in a perpendicular direction.

2. The bioreactor (1) according to claim 1, wherein The inner surface of the reaction vessel (2) has an ellipsoidal, ovoid or ovoid inner geometry.

3. The bioreactor (1) according to claim 2, wherein The inner surface of the reaction vessel (2) has a shape without edges.

4. The bioreactor (1) according to claim 1 or 2, wherein The motion of the magnetic particles and / or the DNA magnetic particles is arranged to avoid sedimentation of the particles and / or to keep the particles freely floating.

5. The bioreactor (1) according to any one of claims 1 to 3, wherein, The magnet unit (3) is an array of electromagnets positioned on or near the outer surface of the reaction vessel.

6. The bioreactor (1) according to claim 1, wherein The magnet unit (3) is an electromagnet.

7. The bioreactor (1) according to claim 1, wherein The magnetic ring (31) comprises at least a first rod (320) and a second rod (322) extending from an inner circumference (34) of the magnetic ring (31) to a center (33) of the magnetic ring (31) such that a free end (321) of the first rod (320) and a free end (323) of the second rod (322) face each other.

8. The bioreactor (1) according to claim 7, wherein The free end (321) of the first rod (320) comprises a magnet with an N-pole, while the free end (323) of the second rod (322) comprises a magnet with an S-pole.

9. The bioreactor (1) according to claim 1, wherein The magnetic ring (31) comprises a plurality of poles (320, 322), wherein the plurality of poles (320, 322) extend from an inner circumference (34) of the magnetic ring (31) to a center (33) of the magnetic ring (31) and are arranged in a star shape at a uniform distance from each other, and wherein magnets having a N-pole and magnets having a S-pole are arranged alternately at the free ends of each pole.

10. The bioreactor (1) according to any one of claims 7 to 9, wherein The magnetic ring (31) and the poles (320, 322) are arranged to form a laminate stack for shielding peripheral components from a magnetic field.

11. The bioreactor (1) according to claim 1, wherein The magnetic ring (31) comprises a plurality of guide plates (350) extending from an inner circumference (34) of the magnetic ring (31) to a center of the magnetic ring (31), and wherein each guide plate (350) comprises an electrical coil (351) arranged for generating a magnetic field.

12. The bioreactor (1) according to claim 11, wherein The magnetic ring (31) is arranged in a housing (352) having a cooling device.

13. The bioreactor (1) according to claim 1, wherein The reaction vessel (2) is paramagnetic or is arranged to allow penetration of a magnetic field to hold magnetic particles and DNA magnetic particles on the reaction vessel wall.

14. The bioreactor (1) according to claim 1, wherein The magnet unit (3) is arranged to be activated periodically to mix the magnetic particles or the DNA magnetic particles.

15. The bioreactor (1) according to claim 1, wherein The magnet unit (3) is arranged to be activated to capture the DNA magnetic particles between two subsequent RNA in vitro transcriptions on the same DNA template.

16. The bioreactor (1) according to claim 1, wherein The magnet unit (3) is arranged to be activated to remove the DNA magnetic particles, thereby cleaning the reaction vessel.

17. The bioreactor (1) according to claim 1, wherein There is no mechanical movement introduction device for the DNA magnetic particles and / or the reaction vessel (2).

18. The bioreactor (1) according to claim 1, wherein The mechanical movement of the reaction vessel is introduced by a track vibrator.

19. The bioreactor (1) according to claim 1, wherein The reaction vessel (2) comprises at least one flow breaker (4) arranged at least partially along an inner surface (21) of the reaction vessel (2) in a longitudinal direction of the reaction vessel (2).

20. The bioreactor (1) according to claim 19, wherein The reaction vessel (2) comprises two flow breakers (4) spaced apart from each other in a radial direction of the reaction vessel (2).

21. The bioreactor (1) according to claim 19 or 20, wherein The flow breaker (4) is rib-shaped.

22. The bioreactor (1) according to claim 21, wherein The rib-shaped flow breaker (4) comprises a T-shaped or L-shaped cross-section.

23. Bioreactor (1) according to claim 19 or 20, wherein The flow breaker (4) is corrugated.

24. The bioreactor (1) according to claim 19 or 20, wherein The flow breaker (4) comprises a plurality of protrusions.

25. The bioreactor (1) according to claim 1, wherein A temperature element (5) is located between an inner surface (21) and an outer surface (23) of the reaction vessel (2) for adjusting the temperature of the reaction vessel (2).

26. The bioreactor (1) according to claim 25, wherein The temperature element (5) comprises a heat exchange channel (51) at least partially helically around the reaction vessel (2) in a radial direction of the reaction vessel (2).

27. The bioreactor (1) according to claim 26, wherein The heat exchange channel (51) comprises a first end (52) and a second end (53), wherein the first end (52) is arranged at a top of the reaction vessel (2) and the second end (53) is positioned at a bottom of the reaction vessel (2).

28. Bioreactor (1) according to claim 26 or 27, wherein The heat exchange channel (51) and / or the reaction vessel (2) are manufactured by an additive manufacturing process.

29. The bioreactor (1) according to claim 1, wherein The reaction vessel (2) further comprises a temperature element (5) comprising a heating wire (54) at least partially helically around the reaction vessel (2) in a radial direction of the reaction vessel (2).

30. Bioreactor (1) according to claim 29, wherein The heating wire (54) is at least partially integrated on or at least partially coated on the outer surface of the reaction vessel (2).

31. The bioreactor (1) according to claim 1, wherein The reaction vessel (2) is configured to absorb at least 20 ml of fluid.

32. The bioreactor (1) according to claim 1, wherein The reaction vessel (2) is configured to absorb 20 ml to 100 ml of fluid.

33. The bioreactor (1) according to claim 1, wherein The reaction vessel (2) is configured to absorb 20 ml to 50 ml of fluid.

34. The bioreactor (1) according to claim 1, wherein The DNA immobilization buffer comprises a DNA template and a salt-containing buffer.

35. The bioreactor (1) according to claim 1, wherein The DNA template is a linear double-stranded DNA template.

36. The bioreactor (1) according to claim 1, wherein The magnetic particles are magnetic beads.

37. The bioreactor (1) according to claim 1, wherein The inner surface of the reaction vessel (2) has a Ra value of Ra < 0.

8.

38. The bioreactor (1) according to claim 1, wherein The inner surface of the reaction vessel (2) has a Ra value of Ra < 0.

6.

39. The bioreactor (1) according to claim 37, wherein The reaction vessel (2) comprises a port (24) at the bottom of the reaction vessel (2) for supplying and / or removing culture medium into and / or from the reaction vessel (2), and wherein the port (24) is connectable to a valve device (60).

40. Bioreactor (1) according to claim 39, wherein The valve device (60) comprises a magnetic trap (61), and wherein the magnetic trap (61) is configured to capture magnetic particles and DNA magnetic particles.

41. Bioreactor (1) according to claim 40, wherein The magnetic trap (61) comprises an electromagnet or magnetizable sphere or magnetizable ring and / or a semipermeable filter.

42. The bioreactor (1) according to claim 40 or 41, wherein The magnetic trap (61) is controllable to prevent magnetic particles and DNA magnetic particles from escaping from the reaction vessel.

43. Bioreactor (1) according to claim 40 or 41, wherein The magnetic trap (61) is located outside the reaction vessel (2), at least partially surrounding a culture medium tube (66) which adjoins the port (24) downstream.

44. Bioreactor (1) according to claim 43, wherein The port (24) is located at the lowest point of the reaction vessel (2).

45. The bioreactor (1) according to any one of claims 40, 41 or 44, further comprising a multi-position valve (62) positioned downstream of the magnetic trap and configured to direct a cleaning gas or cleaning fluid through the port (24) to remove magnetic particles and DNA magnetic particles from the port (24).

46. The bioreactor (1) according to claim 45, wherein The multi-position valve (62) is configured to direct a process gas or process fluid into the reaction vessel (2) to mix the DNA magnetic particles.

47. The bioreactor (1) according to claim 39 or 40, wherein A temperature element (5) is located between the inner surface (21) and the outer surface (23) of the reaction vessel (2) for regulating the temperature of the reaction vessel (2), the temperature element (5) comprising a heat exchange channel (51) at least partially spirally around the reaction vessel (2) in the radial direction of the reaction vessel (2), wherein the heat exchange channel (51) comprises a first end (52) and a second end (53), wherein the first end (52) is arranged at the top of the reaction vessel (2) and the second end (53) is positioned at the bottom of the reaction vessel (2).

48. The bioreactor (1) according to claim 47, wherein The bioreactor comprises at least a first leg (25) and a second leg (26) vertically supporting the bioreactor, wherein the first leg (25) comprises a first conduit (251) and the second leg (26) comprises a second conduit (261), wherein the first conduit (251) is arranged in fluid communication with the valve device (60) and the second conduit (261) is arranged in fluid communication with the second end (53) of the heat exchange channel (51) of the temperature element (5).

49. The bioreactor (1) according to claim 1, further comprising an outlet port (7) connected to at least one of an exhaust duct (73) and a waste channel (74), and optionally comprising an outlet flow chamber (72) arranged downstream of the outlet port (7).

50. The bioreactor (1) according to any one of claims 40, 41, 44 or 46, further comprising a Hall sensor (63) positioned downstream of the magnetic trap (61) and arranged to detect a magnetic field emerging from magnetic particles or DNA magnetic particles.

51. The bioreactor (1) according to claim 1, wherein The reaction vessel (2) comprises a titan.

52. The bioreactor (1) according to claim 39, wherein The bioreactor (1) further comprises a filter element at the port (24) for retaining the magnetic particles in the reaction vessel (2).

53. The bioreactor (1) according to any one of claims 25 to 27, wherein The temperature element (5) is arranged to regulate the temperature of the reaction vessel to a transcription temperature of 20°C to 37°C.

54. The bioreactor (1) according to claim 39, wherein The valve device (60) further comprises a flow chamber (64) arranged downstream of the port (24).

55. The bioreactor (1) according to claim 1, wherein The reaction vessel (2) is further arranged to contain at least one of the following ingredients: a buffer suitable for in vitro transcription of RNA, a cap analog, a modified ribonucleotide triphosphate, a ribonuclease inhibitor, a pyrophosphatase, MgCl2, an antioxidant, a polyamine, and a solution for cleaning and / or sterilization.

56. The bioreactor (1) according to claim 1, wherein The reaction vessel (2) is further arranged to retain at least one device for measuring and / or regulating pH, salt concentration, magnesium concentration, phosphate concentration, temperature, pressure, flow rate, RNA concentration, and / or ribonucleotide triphosphate concentration.

57. The bioreactor (1) according to claim 1, wherein The bioreactor is operated in a batch, semi-batch or repeated batch mode or in a semi-continuous or continuous mode.

58. The bioreactor (1) according to claim 39, further comprising a rotation device for rotating the reaction vessel to prevent deposition of magnetic particles at the port.

59. A method for in vitro transcription of RNA, wherein, The method comprises the following steps: - providing DNA magnetic particles and an IVT pre-mix in a reaction vessel of a bioreactor (1) according to any one of claims 1 to 58, - mixing free-floating DNA magnetic particles with the IVT pre-mix by cooperation of the DNA magnetic particles and the magnet unit to obtain RNA (S3).

60. The method according to claim 59, further comprising the following steps: - providing magnetic particles, DNA templates, DNA immobilization buffer (S1) in the reaction vessel of the bioreactor (1) according to any one of claims 1 to 58, - mixing the magnetic particles, the DNA templates and the DNA immobilization buffer by cooperation of the magnetic particles and the magnet unit of the bioreactor to obtain DNA magnetic particles, i.e. DNA templates immobilized on freely floating magnetic particles (S2), wherein steps S1 and S2 are performed prior to the steps defined in claim 59.

61. The method according to claim 60, further comprising the following steps: - capturing DNA magnetic particles by the magnet unit and collecting / harvesting the RNA obtained in step S3 (S4a), - providing fresh IVT-premix in the reaction vessel of the bioreactor (1) (S4b), - releasing the captured DNA magnetic particles to provide freely floating DNA magnetic particles (S4c), - mixing the freely floating DNA magnetic particles with the IVT-premix by cooperation of the DNA magnetic particles and the magnet unit to obtain RNA (S4d), wherein steps S4a to S4d are performed after the steps defined in claim 59.

62. The method according to any one of claims 59 to 61, further comprising the following step: - removing the DNA magnetic particles from the reaction vessel (2) through the port (24).

63. The method according to any one of claims 59 to 61, further comprising the following step: - tempering the reaction vessel (2) to a temperature between 20°C and 37°C (ST).

64. The method according to any one of claims 59 to 61, further comprising the following step: - cleaning the reaction vessel (2) with a cleaning gas and / or a cleaning fluid (SC).

65. The method of claim 61, wherein, Step S4 is performed at least 2 times.

66. Use of the bioreactor (1) according to any one of claims 1 to 58 in the method according to any one of claims 59 to 65.

67. A module (15) for transcribing DNA templates into RNA, the module (15) comprising a bioreactor (1) according to any one of claims 1 to 58, the module further comprising at least one of: a unit (12) for preparing an IVT-premix, a unit for preparing an immobilization buffer, a device (13) for handling the obtained RNA product, a device (14) for purifying the obtained RNA product, a device for RNA handling and / or a device for RNA sterile filtration.

68. The module (15) according to claim 67, the module (15) further comprising a media supply unit, the media supply unit supplying components of the IVT-premix to the unit (12) for preparing the IVT-premix.

69. The module (15) according to claim 67 or 68, wherein The DNA templates are end-modified or end-functionalized PCR-generated DNA templates.

70. The module (15) according to claim 69, wherein The DNA template is a biotinylated PCR generated DNA template, end-modified or unmodified linearized plasmid DNA or end-modified or unmodified linearized dog bone DNA.

71. An automated device for RNA manufacturing, comprising a bioreactor (1) according to any one of claims 1 to 58, the device further comprising at least one of: - a DNA synthesis module (T), and - an RNA formulation module (F).

72. The device of claim 71, wherein, The RNA formulation module is configured to produce LNP encapsulated RNA.

73. The device of claim 71 or 72, wherein, The device is arranged in a closed container, the container having a unit for generating a laminar air flow.

74. The device according to claim 71 or 72, further comprising at least one of: a DNA immobilization module, a DNA linearization module, an RNA capping module for adding a capO or capl structure to the in vitro transcribed RNA, an RNA polyadenylation module, an RNA mixing module, an RNA spray-drying module, an RNA lyophilization module and / or a module for final product storage.

75. The device of claim 71 or 72, wherein, The RNA formulation module is configured to produce protamine complexed RNA or polyethylene glycol / peptide polymer complexed RNA.

76. The device according to claim 71 or 72, further comprising at least one of: a next generation sequencing module, a mass spectrometry module, a capillary electrophoresis module, a ddPCR module, a media supply rack or media supply module, a documentation recording module and / or a module for computer-aided control of all processing steps.

Images