BIOREACTOR FOR RNA IN VITRO TRANSCRIPTION

Abstract

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 apparatus for RNA manufacturing. Further, 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, said RNA in vitro transcription reactor allows repetitive use of DNA template for various RNA in vitro transcription reactions. Further, the invention relates to an apparatus for RNA manufacturing comprising (a) a module for template DNA synthesis, (b) a module for transcribing DNA into RNA comprising said RNA in vitro transcription reactor, and, optionally, (c) a module for RNA formulation.

Claims

1. A bioreactor (1) for RNA in vitro transcription comprising: (a) a reaction vessel (2) suitable to hold magnetic particles, DNA templates, a DNA immobilization buffer, DNA magnetic particles and an IVT master mix, wherein the DNA magnetic particles are DNA templates immobilized on the free-floating magnetic particles, and (b) a magnet unit (3) positioned at the reaction vessel, wherein the magnet unit is configured to capture or to introduce a movement of the magnetic particles and the DNA magnetic particles.

2. Bioreactor (1) according to claim 1, wherein an inner surface of the reaction vessel (2) has an ellipsoid, an oval inner geometry or an egg-shape inner geometry.

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

4. Bioreactor (1) according to one of the preceding claims, wherein the movement of the magnetic particles and/or the DNA magnetic particles is configured to avoid sedimentation of the particles and/or to keep the particles free-floating.

5. Bioreactor (1) according to one of the claims 1 to 4, wherein the magnet unit (3) is an array of electromagnets positioned on or in proximity to an outer surface of the reaction vessel.

6. Bioreactor (1) according to one of the claim 1 or 4, 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 a transversal direction (363) towards and apart from the reaction vessel (2).

7. Bioreactor (1) according to one of the claim 1 or 4, wherein the magnet unit (3) is an electromagnet and preferably at least an induction coil or a pair of Helmholtz coils movable in a longitudinal direction (110) along a longitudinal axis of the reaction vessel (2) and rotatable (111) around a vertical axis of the reaction vessel (2).

8. Bioreactor (1) according to one of the preceding claims, wherein the magnet unit (3) is configured to rotate around the longitudinal axis of the reaction vessel (2), and wherein a rotation direction of the magnet unit (3) is switchable during mixing.

9. Bioreactor (1) according to one of the preceding claims, wherein the magnet unit (3) comprises a magnetic ring (31), and wherein the magnetic ring (31) is designed to surround the reaction vessel (2).

10. Bioreactor (1) according to the preceding claim, 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 centre (33) of the magnetic ring (31), so that free ends (321, 323) of the first and second rod (320, 322) face each other.

11. Bioreactor (1) according to the preceding claim, wherein the free end (321) of first rod (320) comprises a magnet with an N pole and the free end (323) of the second rod (322) comprises a magnet with an S pole.

12. Bioreactor (1) according to claim 8 or 9, wherein the magnetic ring (31) comprises a plurality of rods (320, 322), wherein the plurality of the rods (320, 322) extend from an inner circumference (34) of the magnetic ring (31) to a centre (33) of the magnetic ring (31) and are arranged in a star shape evenly spaced apart from each other, and wherein a magnet with an N pole and a magnet with an S pole are arranged alternately at a free end of each rod.

13. Bioreactor (1) according to one of the preceding claims 8 to 12, wherein the magnetic ring (31) and the rods (320, 322) are configured to form a laminated stack for shielding periphery components from a magnet field.

14. Bioreactor (1) according to claim 9, 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 centre of the magnetic ring (31), and wherein each guide plate (350) comprises an electric coil (351) configured for generating a magnetic field.

15. Bioreactor (1) according to the preceding claim, wherein the magnetic ring (31) is arranged in a housing (352) having cooling means.

16. Bioreactor (1) according to one of the preceding claims, wherein the magnet unit (3) further comprises a first driving means (36) configured to rotate the magnetic ring (31) and a second driving means (37) configured to move the magnetic ring (31) in the vertical direction.

17. Bioreactor (1) according to one of the preceding claims, wherein the reaction vessel (2) is paramagnetic or is configured to allow penetration of a magnetic field for withholding magnetic particles and DNA magnetic particles on the reaction vessel wall.

18. Bioreactor (1) according to one of the preceding claims, wherein the magnet unit (3) is configured to be periodically activated to mix the magnetic particles or the DNA magnetic particles.

19. Bioreactor (1) according to one of the preceding claims, wherein the magnet unit (3) is configured to be activated to capture the DNA magnetic particles between two subsequent RNA in vitro transcriptions on the same DNA templates.

20. Bioreactor (1) according to one of the preceding claims, wherein the magnet unit (3) is configured to be activated to remove the DNA magnetic particles to clean the reaction vessel.

21. Bioreactor (1) according to one of the preceding claims, wherein there are no mechanical motion introducing means for the DNA magnetic particles and/or the reaction vessel (2).

22. Bioreactor (1) according to one of the preceding claims apart from claim 21, wherein a mechanical motion for the reaction vessel is introduced by an orbital shaker.

23. Bioreactor (1) according to one of the preceding claims, 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).

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

25. Bioreactor (1) according claim 23 or 24, wherein the flow breaker (4) is rib-shaped.

26. Bioreactor (1) according to the preceding claim, wherein the rib-shaped flow breaker (4) comprises a T- or L shaped cross section.

27. Bioreactor (1) according to claim 23 or 24, wherein the flow breaker (4) is corrugated.

28. Bioreactor (1) according to claim 23 or 24, wherein the flow breaker (4) comprises a plurality of protrusions, and wherein the protrusions are preferably spaced apart from each other.

29. Bioreactor (1) according to one of the preceding claims, wherein a temperature element (5) is positioned between the inner surface (21) and the outer surface (23) of the reaction vessel (2) for adjusting a temperature of the reaction vessel (2).

30. Bioreactor (1) according to the preceding claim, wherein the temperature element (5) comprises a heat exchange channel (51) at least partially helically surrounding the reaction vessel (2) in a radial direction of the reaction vessel (2).

31. Bioreactor (1) according to the preceding claim, 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 portion of the reaction vessel (2) and the second end (53) is positioned at a bottom portion of the reaction vessel (2).

32. Bioreactor (1) according to one of the claim 30 or 31, wherein the heat exchange channel (51) and/or the reaction vessel (2) is manufactured by means of an additive manufacturing process.

33. Bioreactor (1) according to one of the preceding claims 1 to 28, wherein the reaction vessel (2) further comprises a temperature element (5), which comprises a heating wire (54) at least partially helically surrounding the reaction vessel (2) in a radial direction of the reaction vessel (2).

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

35. Bioreactor (1) according to one of the preceding claims, wherein the reaction vessel (2) is configured for an uptake of at least 20 ml of fluid, preferably 20 ml to 100 ml or 20 ml to 50 ml of fluid.

36. Bioreactor (1) according to one of the preceding claims, wherein the IVT master mix comprises ribonucleoside triphosphates and DNA dependent RNA polymerase.

37. Bioreactor (1) according to one of the preceding claims, wherein the DNA immobilization buffer comprises DNA templates and salt containing buffers.

38. Bioreactor (1) according to one of the preceding claims, wherein the DNA templates are linear double stranded DNA templates and preferably PCR amplified DNA templates.

39. Bioreactor (1) according to one of the preceding claims, wherein the magnetic particles are magnetic beads and preferably streptavidin magnetic beads or chemically functionalized magnetic beads.

40. Bioreactor (1) according to one of the preceding claims, wherein an inner surface of the reaction vessel (2) has a Ra value of Ra<=0.8 and preferably Ra<=0.6.

41. Bioreactor (1) according to the preceding claim, wherein the reaction vessel (2) comprises a port (24) at a bottom of the reaction vessel (2) for supplying and/or removing medium into/out of the reaction vessel (2), and wherein the port (24) is connectable to a valve means (60).

42. Bioreactor (1) according to the preceding claim, wherein the valve means (60) comprises a magnetic trap (61), and wherein the magnetic trap (61) is configured to catch magnetic particles and DNA magnetic particles.

43. Bioreactor (1) according to the preceding claim, wherein the magnetic trap (61) comprises an electromagnet or magnetisable spheres or a magnetisable ring and/or semi-permeable filters.

44. Bioreactor (1) according to one of the claim 42 or 43, wherein the magnetic trap (61) is controllable to prevent an escape of magnetic particles and DNA magnetic particles from the reaction vessel.

45. Bioreactor (1) according to one of claims 42 to 44, wherein the magnetic trap (61) is positioned outside the reaction vessel (2) at least partially surrounding an medium pipe (66), which downstream abuts the port (24).

46. Bioreactor (1) according to the preceding claim, wherein the port (24) is positioned at the lowermost point of the reaction vessel (2).

47. Bioreactor (1) according to one of the preceding claims, further comprising a multi position valve (62) positioned downstream 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).

48. Bioreactor (1) according to the preceding claim, 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.

49. Bioreactor (1) according to one of the preceding claims, 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 configured to be in fluid communication with the valve means (60) and the second conduit (261) is configured to be in fluid communication with the second end (53) of the heat exchange channel (51) of the temperature element (5).

50. Bioreactor (1) according to one of the preceding claims, further comprising an exit port (7) connected to at least one of an exhaust duct (73) and a waste channel (74), and, optionally, an exit flow cell (72) arranged downstream the exit port (7).

51. Bioreactor (1) according to one of the preceding claims, further comprising a Hall sensor (63) positioned downstream the magnetic trap (61) and configured to detect magnetic fields emerging from magnetic particles or DNA magnetic particles.

52. Bioreactor (1) according to one of the preceding claims, wherein the reaction vessel (2) comprises Titan.

53. Bioreactor (1) according to one of the preceding claims, further comprising a filter element, preferably a single use filter, at the port (24) for withholding the magnetic particles in the reaction vessel (2), wherein the pores of the filter element are, preferably, of the order of 1 μm, or more preferably, have sub-micron size between 0.1 μm and 0.9 μm.

54. Bioreactor (1) according to one of the preceding claims, wherein the temperature element (5) is configured to adjust the reaction vessel temperature to a transcription temperature of 20 to 37° C. and preferably also to a cleaning temperature of 75 to 85° C.

55. Bioreactor (1) according to one of the preceding claims, wherein the valve means (60) further comprises a flow cell (64) arranged downstream the port (24).

56. Bioreactor (1) according to one of the preceding claims, wherein the reaction vessel (2) is further configured to hold at least one of the following elements: a buffer suitable for RNA in vitro transcription, a cap analogue, modified ribonucleoside triphosphates, a ribonuclease inhibitor, a pyrophosphatase, MgCl2, an antioxidant, a polyamine and a solution for cleaning and/or sanitizing.

57. Bioreactor (1) according to one of the preceding claims, wherein the reaction vessel (2) is further configured to hold at least one means for measuring and/or adjusting pH, salt concentration, magnesium concentration, phosphate concentration, temperature, pressure, flow velocity, RNA concentration and/or ribonucleotide triphosphate concentration.

58. Bioreactor (1) according to one of the preceding claims, wherein the bioreactor operates in batch, semi batch or in a repeated batch mode or in a semi-continuous or continuous mode.

59. Bioreactor (1) according to one of the preceding claims, besides claim 21, further comprising rotation means for rotating the reaction vessel in order to prevent sedimentation of magnetic particles at the port.

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

61. Method according to claim 60, further comprising the steps providing magnetic particles, DNA templates, a DNA immobilisation buffer in a reaction vessel of a bioreactor (1) according to any one of claims 1 to 59 (S1), mixing the magnetic particles, the DNA templates and the DNA immobilisation buffer by means of a cooperation of the magnetic particles and a magnet unit of the bioreactor to obtain DNA magnetic particles, which are the DNA templates immobilized on the free-floating magnetic particles (S2), wherein steps S1 and S2 are performed prior to the steps defined in claim 60.

62. Method according to claim 61, further comprising the steps capturing DNA magnetic particles by means of the magnet unit and collecting/harvesting obtained RNA from step S3 (S4a), providing fresh IVT master mix in a reaction vessel of a bioreactor (1) (S4b), releasing captured DNA magnetic particles to provide free-floating DNA magnetic particles (S4c), mixing the free-floating DNA magnetic particles with the IVT master mix by means of a cooperation of the DNA magnetic particles and the magnet unit to obtain RNA (S4d) wherein steps S4a-S4d are performed after the steps defined in claim 60.

63. Method according to one of the claims 60 to 62, further comprising the step: removing the DNA magnetic particles from the reaction vessel (2) by means of an port (24).

64. Method according to one of the claims 60 to 62, further comprising the step: tempering the reaction vessel (2) to a temperature between 20° and 37° C. (ST).

65. Method according to any one of claims 62 to 62, further comprising the step: cleaning the reaction vessel (2) by a cleaning gas and/or a cleaning fluid (SC).

66. Method according to one of claims 60 and 65, wherein the step S4 is performed at least 2 times.

67. Use of a bioreactor (1) according to any one of claims 1 to 59 in a method according to any one of claims 60 to 66.

68. A module (15) for transcribing DNA template into RNA comprising a bioreactor (1) according to any one of claims 1 to 59, the module further comprising at least one of a unit for preparing an IVT master mix (12), a unit for preparing an immobilization buffer, a device for conditioning an obtained RNA product (13), a device for purifying an obtained RNA product (14), a device for RNA conditioning and/or a device for RNA sterile filtration.

69. Module (15) according to claim 68, further comprising a media supply unit supplying components of the IVT master mix to the unit for preparing the IVT master mix (12).

70. Module (15) according to one of claims 68 and 69, wherein the DNA template is an end-modified or end-functionalized PCR-generated DNA template, preferably a biotinylated PCR-generated DNA template, an end-modified or non-modified linearized plasmid DNA or an end-modified or non-modified linearized doggy bone DNA.

71. An automated apparatus for RNA manufacturing, comprising a bioreactor (1) according to any one of claims 1 to 59, the apparatus further comprising at least one of: a module for DNA synthesis (T), and a module for RNA formulation (F).

72. Apparatus according to claim 71, wherein the module for RNA formulation is configured to generate LNP encapsulated RNA.

73. Apparatus according to claim 71 or 72, wherein the apparatus is arranged in a closed container, preferably a single container, with a unit for laminar airflow generation.

74. Apparatus according to any of claims 71 to 73, further comprising at least one of a DNA immobilization module, a DNA linearization module, an RNA capping module for adding a cap0 or cap1 structure to 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 end-product storage.

75. Apparatus according to any of claims 71 to 74, wherein the module for RNA formulation is configured to generate a Protamine complexed RNA or a polyethylene glycol/peptide polymer complexed RNA.

76. Apparatus according to any of claims 71 to 75, further comprising at least one of an NGS module, an MS module, a capillary electrophoresis module, a ddPCR module, a media supply rack or a media supply module, a documentation module and/or a module for computer assisted control for all processing steps.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0120] The Figures shown in the following are merely illustrative and shall describe the present invention in a further way. These figures shall not be construed to limit the present invention thereto.

[0121] FIG. 1 shows a schematic view of a bioreactor according to an embodiment of the present invention.

[0122] FIG. 2 shows a schematic view of a reaction vessel according to an embodiment of the present invention.

[0123] FIGS. 3A, B show schematic views of a reaction vessel according to an embodiment of the present invention.

[0124] FIG. 4 shows a schematic view of a magnet unit according to an embodiment of the present invention.

[0125] FIG. 5 shows a schematic view of magnet rings according to another embodiment of the present invention.

[0126] FIG. 6 shows a schematic view of a magnet unit according to another embodiment of the present invention.

[0127] FIG. 7A-C show schematic views of a bioreactor according to another embodiment of the present invention.

[0128] FIG. 8A-C show schematic views of a bioreactor according to another embodiment of the present invention.

[0129] FIG. 9A-H show schematic views of a bioreactor according to another embodiment of the present invention.

[0130] FIG. 10 shows a schematic view of a bioreactor according to another embodiment of the present invention.

[0131] FIG. 11 shows a schematic view of a bioreactor according to another embodiment of the present invention.

[0132] FIGS. 12A, B show schematic views of a bioreactor with a movable magnet unit according to an embodiment of the invention.

[0133] FIG. 13 shows a schematic view of a bioreactor with a rotatable magnet unit according to an embodiment of the invention.

[0134] FIGS. 14A, B show schematic views of a bioreactor with an orbital shaker

[0135] FIG. 15 shows exemplary components of a module for transcribing DNA into RNA.

[0136] FIG. 16 shows an example of a method for transcribing DNA into RNA according to an embodiment.

[0137] FIG. 17 shows an exemplary apparatus for automated RNA production according to an embodiment.

[0138] FIG. 18 shows an exemplary process overview for RNA production according to an embodiment.

[0139] FIG. 19 show the result of a repeated batch RNA in vitro transcription using the same immobilized DNA template over 3 IVT reactions. The experiment was performed as described in Example 1.

[0140] FIG. 20A, B shows the potency on the produced RNA expressed in HepG2 cells (RAVG mRNA). The experiment was performed as described in Example 1.

DEFINITIONS

[0141] For the sake of clarity and readability the following definitions are provided. Any technical feature mentioned for these definitions may be read on each and every embodiment of the invention. Additional definitions and explanations may be specifically provided in the context of these embodiments.

Doggybone, Doggy Bone DNA

[0142] The term “Doggybone™” (dbDNA) as used herein denotes a minimal, closed-linear DNA vector enzymatically developed by Touchlight Genetics Ltd. The linear DNA is rapidly produced, plasmid-free and synthesized through an enzymatic process that yields a vector cassette containing only the encoded sequence of interest, promoter, e.g. poly A tail and telomeric ends.

Mixing

[0143] In the context of the invention, “mixing” is typically a process that involves manipulation of a heterogeneous physical system with the intent to make it more homogeneous. Mixing is performed to allow mass transfer to occur between one or more streams, components or phases. Mixing is fundamentally the evolution in time of spatially dependent concentrations toward a more homogeneous state.

[0144] In the context of the present invention, a magnet unit is used, which allows in cooperation with magnetic particles and/or DNA magnetic particles for an improved mixing of components contained in the reaction vessel as defined herein, preferably without exerting any mechanical stress (such as shear stress) on said components. In particular, conventional mixing means that are known to induce mechanical stress on the components to be mixed are preferably avoided according to the present invention. For example, the mixing of fluids is preferably performed without shaking and/or agitating the reaction vessel. Instead, the magnet unit is configured to generate appropriate magnetic fields which lead to forces acting on magnetic particles and/or DNA magnetic particles, such that the latter particles start a movement within the reaction vessel, thereby leading to a mixing of the components contained in the reaction vessel.

[0145] The induced movement of the magnetic particles and or DNA magnetic particles may introduce turbulences in the components contained in the reaction vessel that are not caused by shaking or vibrating which allows for an improved mixing of the components in the reaction vessel to generate a homogeneous composition.

RNA In Vitro Transcription

[0146] The term “RNA in vitro transcription” relates to a process wherein RNA is synthesized in a cell-free system. RNA may be obtained by DNA-dependent RNA in vitro transcription of an appropriate DNA template, which according to the present invention may be a linearized plasmid DNA template or a PCR-amplified DNA template. The promoter for controlling RNA in vitro transcription can be any promoter for any DNA-dependent RNA polymerase. Particular examples of DNA-dependent RNA polymerases are the T7, T3, SP6, or Syn5 RNA polymerases.

[0147] The DNA template (e.g., plasmid DNA, doggy bone DNA) may be linearized with a suitable restriction enzyme and immobilized on magnetic beads (e.g. as described in PCT/EP2017/084264 or PCT/EP2018/086684) before it is subjected to RNA in vitro transcription. Alternatively, the DNA template may 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).

[0148] Reagents used in RNA in vitro transcription typically include: a DNA template (linearized DNA or linear PCR product) with a promoter sequence that has a high binding affinity for its respective RNA polymerase such as bacteriophage-encoded RNA polymerases (T7, T3, SP6, or Syn5); ribonucleotide triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil); optionally, a cap analogue (e.g. m7G(5′)ppp(5′)G (m7G) or a cap analogue derivable from the structure disclosed in claim 1-5 of WO2017/053297 or any cap structures derivable from the structure defined in claim 1 or claim 21 of WO2018075827); optionally, further modified nucleotides as defined herein; a DNA-dependent RNA polymerase capable of binding to the promoter sequence within the DNA template (e.g. T7, T3, SP6, or Syn5 RNA polymerase); optionally, a ribonuclease (RNase) inhibitor to inactivate any potentially contaminating RNase; optionally, a pyrophosphatase to degrade pyrophosphate (inhibitor of RNA synthesis); MgCl2, which supplies Mg2+ ions as a co-factor for the polymerase; a buffer (TRIS or HEPES) to maintain a suitable pH value, which can also contain antioxidants (e.g. DTT), and/or polyamines such as spermidine at optimal concentrations, e.g. a buffer system comprising Citrate and/or betaine as disclosed in WO2017/109161.

[0149] The nucleotide mixture used in RNA in vitro transcription may additionally contain modified nucleotides as defined herein. In that context, preferred modified nucleotides comprise pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine, and 5-methoxyuridine. The nucleotide mixture (i.e. the fraction of each nucleotide in the mixture) used for RNA in vitro transcription reactions may be optimized for the given RNA sequence, preferably as described in WO2015188933.

RNA In Vitro Transcription Master Mix, IVT Master Mix

[0150] An RNA in vitro transcription (IVT) master mix may comprise the components necessary for performing an RNA in vitro transcription reaction as defined above. Accordingly, an IVT master mix may comprise at least one of the components selected from a nucleotide mixture, a cap analogue, a DNA-dependent RNA polymerase, an RNAse inhibitor, a pyrophosphatase, MgCl2, a buffer, an antioxidant, betaine, Citrate.

Semi-Permeable Filter

[0151] 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 transmission of particles larger than the filter material pore size.

[0152] If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also meant to encompass a group which preferably consists of these embodiments only.

[0153] As used in the specification and the claims, the singular forms of “a” and “an” also include the corresponding plurals unless the context clearly dictates otherwise.

[0154] It needs to be understood that the term “comprising” is not limiting. For the purposes of the present invention, the term “consisting of” is considered to be a preferred embodiment of the term “comprising of”.

DETAILED DESCRIPTION OF THE FINDINGS UNDERLYING THE PRESENT INVENTION

[0155] The invention relates to a bioreactor for RNA in vitro transcription configured to be operable in an automated manner under GMP-compliant conditions. A schematic drawing of a bioreactor for RNA in vitro transcription according to an embodiment of the invention is provided i.a. in FIGS. 1 and 7.

[0156] The bioreactor 1 comprises a reaction vessel 2 for holding magnetic particles, DNA templates, a DNA immobilisation buffer, DNA magnetic particles and an IVT master mix. The inner surface 21 of the reaction vessel 2 has an egg-shape inner geometry. Alternatively, the inner surface 21 of the reaction vessel 2 according to the present invention may be ellipsoidal or oval. In any case, it is preferred that the inner surface 21 of the reaction vessel 2 has a shape without edges. This may be particularly important for the mixing properties of the bioreactor 1. Moreover, said ellipsoid, oval or egg shape, in particular the absence of edges, is advantageous for cleanability (important for GMP compatibility) and reduces the risk of formation of unwanted precipitations in the bioreactor. Moreover, the egg shape has the advantage over e.g. flat round shape that the fluids (e.g. the RNA product) may easily flow off the bioreactor 1 via a medium port 6 into a medium pipe 66 (see also FIG. 11).

[0157] Further, the above described inner geometries help to prevent sticking and drying out of e.g. proteinaceus residues at the inner surfaces, as generally a shape without edges, and more particularly an ellipsoidal, oval or egg shape supports a good drain off of fluids. In addition, the ellipsoidal, oval or egg shape has the advantage over e.g. a “cone shape” that the risk is minimized that DNA magnetic particles assemble at the bottom of the reactor which may reduce the yield of the RNA in vitro transcription (e.g. those DNA templates would not be accessible for RNA polymerases) or clog the medium port 6. To further prevent clogging of the medium port 6 liquid may be flushed in regular intervals through the medium port 6 into the bioreactor 1 during transcription reaction. Those flushes may additionally improve the mixing properties of the biochemical reaction in the bioreactor (e.g. IVT reaction, DNA immobilization reaction).

[0158] The bioreactor 1 is configured to allow repetitive RNA in vitro transcription reactions on DNA templates that are immobilized on free-floating magnetic particles (“DNA magnetic particles”). For example, DNA templates may be provided as PCR amplified DNA that is immobilized on magnetic beads (using biotinylated primers for PCR-based DNA template amplification and subsequent immobilization on streptavidin magnetic beads) or linearized plasmid DNA that is immobilized on magnetic beads (e.g. as described in PCT/EP2017/084264 or PCT/EP2018/086684).

[0159] The bioreactor 1 further comprises a magnet unit 3 positioned at the reaction vessel 2. The magnet unit 3 enables contactless mixing of the reaction containing magnetic particles or DNA magnetic particles, implying that no mixing means have to be implemented in the mixing process, which is an advantageous feature in the context of sufficient cleanability of the bioreactor 1 e.g. in pharmaceutical production of RNA. Moreover, mixing of the RNA in vitro reaction may be performed without rotation/shaking of the bioreactor 1. This is particularly advantageous as rotation or shaking would be strongly impaired due to different inlet and outlet ports that have to be mounted on the bioreactor 1.

[0160] Further, the magnet unit 3 may be used for capturing DNA magnetic particles before starting another cycle of RNA in vitro transcription thereby allowing repeated batch RNA in vitro transcription (IVT) on the same DNA template which dramatically reduces overall RNA production costs. Further, the magnet unit 3 may be used for removing DNA magnetic particles for final cleaning or sanitizing of the bioreactor 1. Accordingly, DNA may be removed without the need of enzymatic DNAse treatment which (i) reduces costs as no such enzyme is needed, (ii) reduces the risk of contaminating the final RNA product with a further component (that is DNAse), and (iii) reduces the risk of contaminating the final RNA product with DNA fragments or partially digested DNA fragments.

[0161] In FIG. 1, the magnet unit 3 is formed in a ring shape (see also FIG. 4) and receives the reaction vessel 2 in a centre 33 of the magnet unit 3 such that the magnet unit 3 may rotate 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 may move the magnet unit 3 in a vertical direction. By vertically moving the magnet unit 3, a magnet field can be generated along a longitudinal direction of the reaction vessel 2. Accordingly, a homogeneous mixing of components in the reaction vessel 2 may be realised by inducing the magnetic particles both in a radial direction and in a longitudinal direction. A rotation driving means 38 for the magnet unit 3 is arranged on the arm 37 directly above the reaction vessel 2 and a driving means 39 to operate the spindle axis 36 is arranged directly at the spindle axis 36.

[0162] FIG. 2 shows the reaction vessel 2 in a perspective view, FIG. 3A shows a bottom view of the reaction vessel 2 and FIG. 3B shows a top view of the reaction vessel 2. The reaction vessel 2 may be made of a material such as titan, which is chemically resistant, resistant to extreme temperatures, extreme pH values, mechanical forces and/or corrosion.

[0163] In all embodiments of the bioreactor 1 according to the present invention, the inner surface 21 of the reaction vessel 2 has a shape without edges, preferably an ellipsoid, oval or egg shape. It is further preferred that the inner surface 21 of the reaction vessel 2 is polished with a value Ra<=0.8. A suitable way to obtain such Ra values is known to the skilled in the art. For instance, the inner surface 21 may be mechanically polished, electro polished, or chemically polished or the like.

[0164] As shown in FIG. 3B, the reaction vessel 2 comprises an exit port 7 for exhaust gas or waste fluids. The exit port 7 may e.g. be used for venting of the reaction vessel 2 during filling of the vessel. To this end, the exit port 7 is arranged at the uppermost point of the reaction vessel 2. At a top portion of the reaction vessel 2, a first end 52 of a heat exchange channel 51 of a temperature element 5 is arranged. As shown in FIG. 11, the exit port 7 may be connected to at least one of an exhaust duct 73 and a waste channel 74. For instance, the exit port 7 may be connected to at least both, the exhaust duct 73 and the waste channel 74, by a multi position valve. The exit port 7 may allow for receiving and venting exhaust gas or exhaust gases emerging within the reaction vessel 2. In case of a waste fluid or a cleaning fluid, the exit port 7 may serve for draining the fluid out of the reaction vessel 2. The exit port, the exhaust duct and/or the waste channel may hold at least one means for measuring and/or adjusting pressure.

[0165] Further, a medium port 6 is arranged at a lowermost point of the reaction vessel 2 and may be further connected to a valve means 60 guiding a supplying or draining of components (in FIG. 3A). The reaction vessel 2 comprises two legs 25, 26, which may support the reaction vessel 2 vertically. Further, each leg 25, 26 comprises a conduit 251, 261 extending through the legs 25, 26. Accordingly, the first leg 25 comprises a first conduit 251 configured to be in a fluid communication with the valve means 60 and the second leg 26 comprises a second conduit 261 configured to be in a fluid communication with a second end 53 of a heat exchange channel 51 of a temperature element 5 (see FIGS. 7B and 7C).

[0166] FIG. 4 shows a preferred embodiment of the magnet unit 3. The magnet unit 3 is formed in a star-shape comprising a magnetic ring 31 and a plurality of rods 32. The magnetic ring 31 and the rods 32 may be made of a plurality of magnetisable laminated electrical sheets, thus form a laminated stack for shielding periphery components from the magnet field. The magnet ring 31 is designed to surround the reaction vessel 2. In other words, the reaction vessel 2 can be positioned in the centre 33 of the magnetic ring 31.

[0167] The magnetic ring 31 comprises a first rod 320 and a second rod 322 extending from an inner circumference 34 of the magnetic ring 31 to a centre 33 of the magnetic ring 31, so that free ends 321, 323 of the first and second rod 320, 322 face each other. The free end 321 of the first rod 320 comprises a magnet with an N pole and the free end 323 the second rod 322 comprises a magnet with an S pole. As shown in FIG. 5, however, the number and length of rods 32 can vary. The rods 32 are arranged at the inner circumference 34 of the magnetic ring 31 and extend in a direction to the centre 33 of the magnetic ring 31. The plurality of the rods 32 are arranged in a star shape and evenly spaced apart from each other, such that the magnetic ring 31 is formed symmetrically. At each free end of the rods 32, a magnet with an N pole and a magnet with an S pole is alternately arranged.

[0168] Alternatively, as shown in FIG. 6, the magnet unit 3 comprises a magnet ring 31 including a plurality of guide plates 350 and electric coils 351. The star-shaped guide plates 350 extend from the inner circumference 34 of the magnetic ring 31 to the centre 33 of the magnetic ring 31. Each guide plate 350 comprises an electric coil 351 for generating a magnetic field. The magnet ring 31 is surrounded by a housing having cooling means 352. The cooling means 352 are integrated in the housing of the magnetic ring along the circumference of the magnetic ring 31 to carry away heat generated by the high current passing through the electromagnetic coils. The cooling means 352 may be a cooling channel in which a cooling medium such as water is provided.

[0169] FIGS. 7A to 7C show another preferred embodiment of a bioreactor 1. The reaction vessel 2 may be made of a solid material and comprises an inner surface 21 and an outer surface 23. Between the inner surface 21 and the outer surface 23 a temperature element 5 is integrated for adjusting a temperature of the reaction vessel 2. The temperature element 5 comprises a heat exchange channel 51 helically surrounding the reaction vessel 2 in the radial direction relative to a longitudinal axis of the reaction vessel 2. To facilitate manufacturing of such reaction vessel 2 with a complex geometry, the reaction vessel 2 may be manufactured by means of additive manufacturing.

[0170] The heat exchange channel 51 comprises a first end 52 and a second end 53 fluidly connected to the second conduit 261 in the second leg 26. The first end 52 is arranged at the top portion of the reaction vessel 2, however, positioned offset from the uppermost top or the exit port 7 to secure a reliable accessibility of the exit port 7. The second end 53 of the heat exchange channel is arranged at the bottom portion of the reaction vessel 2, however, positioned offset from the lowermost bottom or the medium port 6 to secure a reliable accessibility of the medium port 6. Through the first end 52 or second end 53 a heat exchange medium such as water can be supplied into the heat exchange channel 51 for heating or cooling the components inside the reaction vessel 2.

[0171] FIGS. 8A and 8B show an alternative embodiment of the temperature element 5. The temperature element 5 comprises a heating wire 54 at least partially, preferably completely, helically surrounding the reaction vessel 2 in a radial direction relative to the longitudinal axis of the reaction vessel 2. The heating wire 54 is at least partially integrated in an outer surface 23 of the reaction vessel 2 (in FIG. 8A). Additionally or alternatively, the outer surface 23 of the reaction vessel 2 may be coated with a heat isolation material 55 and the heating wire 54 is at least partially retracted in the heat isolation material 55 (in FIG. 8B).

[0172] Referring to FIGS. 7B and 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 a longitudinal direction of the reaction vessel 2. The flow breaker 24 may disturb a uniform flow of the components in the reaction vessel 2 and can thereby improve mixing. Moreover, the flow breaker 24 may prevent sedimentation of the magnet particles when the magnet unit 3 stops rotating and/or changes rotation direction. Two flow breakers 24 are spaced apart from each other in a radial direction relative to the longitudinal axis of the reaction vessel 2.

[0173] As shown in FIGS. 9A to 9H, the flow breaker 24 may be rib-shaped and protrude from the inner surface 21 of the reaction vessel 2 along the longitudinal direction of the reaction vessel 2. In another embodiment, the flow breaker 24 is arc-shaped and comprises a T-shaped cross section (in FIGS. 9A and 9B) or a L-shaped cross section (in FIGS. 9E and 9F). In yet another embodiment, the flow breaker 24 is corrugated or wave-shaped (in FIGS. 9C and 9D). Alternatively, the flow breaker 24 comprises a plurality of protrusions in a semi-circle shape spaced apart from each other at the inner surface 21 of the reaction vessel 2 along the longitudinal direction (in FIGS. 9G and 9H).

[0174] Notably, elements and features of the bioreactor 1 of the invention mentioned in the context of FIGS. 10 to 12 may likewise be part of the reactor shown in FIGS. 1 to 9 even if not explicitly mentioned herein. Accordingly, the bioreactor 1 as illustrated in FIGS. 1 to 9 may also comprise at least one selected from magnetic trap 61, Hall sensor 63, flow cell 64, temperature sensor 91, additional sensor 92, or a specific filling level 27 or a maximal fluid amplitude 28.

[0175] FIG. 10 shows another embodiment of the bioreactor 1. The bioreactor 1 in FIG. 10 comprises an array of electromagnets 3 positioned on the outer surface of the reaction vessel. The array of electromagnets 3 allows for mixing of the reaction (by circulation of magnetic particles or DNA magnetic particles in the reaction) which is caused by periodic activation of said array of electromagnets 3. This enables contactless mixing of the reaction containing magnetic particles or DNA magnetic particles, implying that no mixing means have to be implemented in the mixing process, which is an advantageous feature in the context of sufficient cleanability of the bioreactor 1 e.g. in pharmaceutical production of RNA. Moreover, mixing of the RNA in vitro reaction may be performed without rotation/shaking of the bioreactor 1. This is particularly advantageous as rotation or shaking would be strongly impaired due to different inlet and outlet ports that have to be mounted on the bioreactor 1. Further, said array of electromagnets 3 may be used for capturing DNA magnetic particles before starting another cycle of RNA in vitro transcription thereby allowing repeated batch RNA in vitro transcription (IVT) on the same DNA template which dramatically reduces overall RNA production costs. Further, said array of electromagnets 3 may be used for removing DNA magnetic particles for final cleaning or sanitizing of the bioreactor 1. Accordingly, DNA may be removed without the need of enzymatic DNAse treatment which (i) reduces costs as no such enzyme is needed, (ii) reduces the risk of contaminating the final RNA product with a further component (that is DNAse), and (iii) reduces the risk of contaminating the final RNA product with DNA fragments or partially digested DNA fragments.

[0176] Further shown in FIG. 10 is a filling level 27 of a fluid hold in the reaction vessel 2. Additionally, the dashed line shows a maximal fluid amplitude 28. Thereby, the maximal fluid amplitude 28 is understood to be the amplitude a fluid contained in the reaction vessel 2 and brought into a shaking or rotational movement maximally reaches on the inner surface 21 of the reaction vessel 2. The bioreactor 1 further comprises an inlet port 8 arranged at the reaction vessel which allows for filling media into the reaction vessel 2. As can be seen in FIG. 11, the inlet port 8 is arranged laterally on the reaction vessel 2 and below the level of a maximal fluid amplitude 28 on the inner surface 21 of the reaction vessel 2. This configuration may help to prevent that e.g. protein residues deposit and harden on the inner surface 21 of the reaction vessel 2, which might be the case of the inlet port is arranged above a maximal fluid amplitude. In the latter case, residues from a filling of the reaction vessel 2 might deposit e.g. at and/or around the inlet port. Moreover, the lateral position of the inlet port 8 close to the filling level 27 allows a filling media into the reaction vessel without unwanted formation of splashes that may form residues deposit and harden on the inner surface 21 of the reaction vessel. Upstream the inlet port 8, an inlet pipe 83 for guiding filling media towards the inlet port 8 and into the reaction vessel 2 is arranged. The bioreactor 1 further comprises a waste port 7 for exhaust gas or waste fluids. The waste port 7 may e.g. used for venting of the reaction vessel 2 during filling of the vessel. To this end, the waste port 7 is arranged at the uppermost point of the reaction vessel 2. Downstream the waste port 7, a waste channel 74 is arranged which allows to uptake exhaust gas or waste fluids which leave the vessel 2 through the waste port 7. Further, an outlet port 6 is arranged at the lowermost point of the reaction vessel 2, thereby allowing a convenient duct or drain of fluids through the outlet port 6 in order to further guide these fluids through an outlet pipe 66.

[0177] FIG. 11 shows another preferred embodiment of a bioreactor 1 according to the present invention. Apart from the components already shown in FIG. 1-10—namely e.g. a reaction vessel 2 and a magnet unit 3, a waste port 7, a waste channel 74, an outlet port 6, an outlet pipe 66 an inlet port 8, an inlet pipe 83 as well as a filling level 27 referring to a contact line of a fluid surface at the inner surface of the reaction vessel 2 and a maximal fluid amplitude 28—the embodiment in FIG. 11 additionally comprises a magnetic trap 61 positioned at the outlet port 6 to minimize the risk of contaminating the RNA product with DNA magnetic particles and/or DNA magnetic particles. This implies that the magnetic trap 61 helps to retain the magnetic particles and/or the DNA magnetic particles within the reaction vessel 2 when draining a produced RNA out of the reaction vessel 2 through the outlet port 6. The magnetic trap 61 may, for instance, at least partially surround the outlet port 6 or the outlet pipe 66 downstream abutting the outlet port 6. Preferably, the magnetic trap 61 may be a ring magnet, e.g. an electromagnet in form of a ring. Downstream the outlet port 6 and the magnetic trap 61, a multi position valve 62 is arranged. The multi position valve 62 connects the outlet port 6, or the outlet pipe 66 downstream connected to the outlet port 6, with three further lines. The first out of the three lines serves for ducting the RNA containing fluid component after the RNA in vitro transcription reaction successfully has 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 the multi position valve 62 at the first line. Accordingly, the Hall sensor 63 is configured for detecting unwanted magnetic fields in the RNA product. A second line connected to the outlet port 6 serves as a waste channel 67 for e.g. cleaning fluids. For monitoring purposes, a flow cell 64 is arranged at this second line. The third line connected to the multi position valve 62 may duct a process gas or a cleaning gas, e.g. N2 or a synthetic solution, in the direction indicated with arrow 65. The process gas or cleaning gas may be cyclically directed by the multi position valve 62 in direction of the outlet port 6. Thereby, a sedimentation of magnetic particles and/or DNA magnetic particles at the outlet port, leading to a clogging of the port, may be prevented.

[0178] Further, the bioreactor 1 comprises temperature elements, e.g. Peltier elements 9 to allow heating or cooling of the bioreactor 1° C. at 37° C., which is an optimal temperature for RNA in vitro transcription, and heating of the bioreactor 1° C. at 80° C., which is an optimal temperature for cleaning/sanitizing of 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 may be positioned at the inner surface 21 of the reaction vessel and/or in proximity to the reaction vessel (e.g., at the inlet port or outlet port). For instance, an additional sensor 92 may be positioned inside the reaction vessel 2 for measuring, for example, the temperature, the pH value or the salt concentration.

[0179] Still referring to FIG. 11, the bioreactor 1 further comprises a multi position valve 71 arranged downstream the waste port 7 and the waste channel 74 abutting the waste port 7. Via the multi position valve 71, the waste port 7 and waste channel 74 are connected to a line for waste fluid with a waste flow cell 72 arranged at this line for monitoring the flow of waste fluids. Further, the multi position valve 71 connects the waste port 7 and waste channel 74 to an exhaust duct 73 for exhaust gases, which may, e.g. emerge during filling or cleaning of the reaction vessel 2. Optionally, there may be a pressure sensor 76 arranged 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. At the inlet pipe 83 upstream the inlet port 8, a heating 81, exemplarily shown as a heating coil, is arranged around the inlet pipe. Upstream the pipe section with the heating 81, a heating flow cell is arranged for monitoring the feed of the components into the reaction vessel 2. Said heating 81 may be used for adjusting the media filled into the reactor to the desired optimal temperature (e.g., 37° C. for RNA in vitro transcription).

[0180] FIGS. 12A and 12B as well as FIG. 13 shows the alternative designs of the magnet unit of a bioreactor 1 according to the present invention. Referring to FIGS. 12A and 12B, an embodiment of a bioreactor 1 is shown, comprising a reaction vessel 2 with outlet port 6, waste port 7, and inlet port 8 as well as a magnet unit 3. Notably, elements mentioned in the context of the bioreactor as specified in FIG. 11 may likewise be part of the reactor shown in FIGS. 12A, 12B (e.g., temperature sensor 91, hall sensor 63, flow cells 64, egg shape, etc.) even if not explicitly mentioned herein. The magnet unit 3 is realised in form of a magnet, preferably an electromagnet, or a permanent magnet, which can be moved towards and apart the reaction vessel 2 along a transversal axis of the reaction vessel 2, as indicated by the arrows 363 or controllable Helmholtz Coils. Further, the magnet unit 3 can be moved upwardly and downwardly along a 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 described movement of the magnet unit 3. Additionally, as further indicated in FIGS. 12A and 12B, the reaction vessel 2 may, in this embodiment, be rotatable around its vertical axis. Alternatively, the reaction vessel 2 may be mounted on a movable support (not shown), which allows the above described movement of the reaction vessel 2 relative to the magnet unit 3 (which may not be mounted on a movable support 361) . Additionally, as further indicated in FIGS. 12A and 12B, the reaction vessel 2 may, in this embodiment, be rotatable around its vertical axis.

[0181] FIG. 12A shows the bioreactor 1 in a state where the magnet unit 3 is laterally removed from the reaction vessel 2, whereas in FIG. 12B a configuration is shown, where the magnet unit 3 is laterally in the closest position to the reaction vessel 2.

[0182] FIG. 13 shows an embodiment of the bioreactor 1 with a magnet unit 3 realized by at least two magnetic coils, which are rotatable around the reaction vessel as indicated by arrows 111 and rotatable arranged at horizontal bar 11 of a support 10. The horizontal bar 11 can be moved upwardly and downwardly, indicated by arrows 110, such that the position of the magnetic fields of the magnetic coils 3 at the reaction vessel 2 can be additionally varied. Notably, elements mentioned in the context of the bioreactor as specified in FIG. 1-11 may likewise be part of the reactor shown in FIG. 13 (e.g., temperature sensor 91, hall sensor 63, flow cells 64, egg shape, etc.) even if not explicitly mentioned herein.

[0183] In FIGS. 14A and 14B embodiments of the bioreactor 1 are shown, which allow for a mixing or stirring of the components hold in the reaction vessel by mechanical motion introducing means as well as by either additionally directing a process gas or a process fluid into the reaction vessel or by a cooperation of the magnetic particles and a magnet unit.

[0184] Apart from the components already described in context of FIG. 11, FIG. 14A shows a bioreactor 1 with reaction vessel 2 positioned on an orbital shaker OS. Orbital shaker OS allows for a 3 dimensional movement of the reaction vessel, preferably with small amplitudes due to the connections for fluids, gas and sensors of the bioreactor, which shall not be damaged by a movement of the reaction vessel 2. For inducing a movement of the reaction vessel 2 by means of the orbital shaker OS, the former is placed on top of the orbital shaker OS. The reaction vessel 2 is laterally at least partially surrounded and may thereby be hold by a support 20. The support 20 contains Peltier elements 9 for heating and/or cooling the reaction vessel. Through the outlet port 6 and outlet pipe 66 in FIG. 14A, a process gas, preferably N.sub.2, or alternatively a process fluid, may be guided into the reaction vessel 2, in order to introduce an additional movement for mixing/stirring the components hold in the reaction vessel 2. Outlet port 6 and outlet pipe 66 also serve to outlet media, e.g. the produced RNA, out of the reaction vessel. Through inlet pipe 83 and inlet port 8, media can be filled into the reaction vessel. Further, FIG. 14A shows a waste port 7 and waste channel arranged at the uppermost point of the reaction vessel.

[0185] In FIG. 14B, an embodiment of a bioreactor 1 is shown, which allows for mixing or stirring of the components hold in the vessel 2 by a cooperation of a Helmholtz coil and the magnetic particles, an orbital shaker OS and a direction of process gas or process fluid into the reaction vessel. To this end, an orbital shaker OS is connected via a horizontal support S with the reaction vessel 2, which is hold by the support S and which is positioned in the middle of a magnet unit 3. The magnet unit is here realized in form of a Helmholtz coil. Part of the support which holds the reaction vessel 2 contains recesses in which Peltier elements 9 are positioned. The Peltier elements are positioned close to or even touch the reaction vessel for efficient heating and/or cooling of the vessel. In addition to the aforementioned components, FIG. 14B shows an inlet port 8 and an inlet pipe 83, a waste port 7 and a waste channel 74, as well as an outlet port 6 and an outlet pipe 66, which latter elements are similar to those described in context of FIG. 11 and FIG. 14A.

[0186] In FIG. 15, an embodiment of the module for transcribing DNA into RNA is shown. It comprises a unit for preparing an IVT master mix 12, also referred to as pre-mixer. As indicated by the arrows incoming at the unit for preparing an IVT master mix 12, this unit 12 may be filled with an IVT buffer (HEPES, Tris), a nucleotide mixture (comprising nucleotides and, optionally, modified nucleotides), a DNA-dependent RNA polymerase, a cap analogue, an RNAse inhibitor, Pyrophosphatase, MgCl2, an antioxidant (DTT), betaine, Citrate.

[0187] The respective components may be provided by a media supply rack (not shown). The produced IVT master mix is guided from the unit for preparing an IVT master mix 12 via line 121 into the bioreactor 1 according to the present invention. Apart from the IVT master mix, DNA is provided to the bioreactor 1 via feed in line 122.

[0188] Additionally, the bioreactor 1 may be filled with a wash buffer via feed in line 123. It shall be understood, that filling of the bioreactor 1 processes through the inlet port 8 of the bioreactor 1, which is exemplarily shown in and discussed in context of FIGS. 12-15. With further reference to FIG. 5, a raw RNA product is directed via line 124 to a conditioner 13, e.g. working by tangential flow filtration. Following the conditioning, the RNA is directed to a device for RNA purification 14 (e.g. RP-HPLC, using a method disclosed in WO2008/077592; PureMessenger®). The device for RNA purification 14 is preferably a RP-HPLC device for automated purification and fractionation of the raw RNA. The device for RNA purification 14 may, additionally, or alternatively comprise an oligo dT purification device for automated purification and fractionation of the raw RNA. As indicated by the dotted arrow, the RNA may be subsequently directed to further devices, e.g. another device for RNA conditioning, e.g. by tangential flow filtration, and/or a device for RNA sterile filtration.

[0189] As a suitable environment for preforming a process in context of the present invention, a process room or housing may be provided. The process room or housing may be separated from the control systems needed to control and/or monitor the process. In the process room, the experimental set-up may be located. The front of the process room may, for instance, be opened by sliding doors. The base frame of the process room may consist of a modular setup that may be divided into three parts. As an example, the three modules may consist of a one meter module, a two meter module and a backpack with a total length of 3.5 meter and a height of about 2 meter. Further, an exhaust system may be included, which may require additional space. The media supply may be located in the one meter module and shall be physically separated from the actual process room located in the two meter module by a separation wall. A separation wall may, for instance, be realised by a glass divider and also a PVC curtain located behind the sliding doors.

[0190] The inner process room may be optionally connected to an exhaust system. It may be desirable, that the liquids which are being processed require further safety measures. This includes explosion protection and/or further biological and chemical safety measures, which may be included in the process room.

[0191] FIG. 16 shows a flow diagram for a method for RNA in vitro transcription according to an embodiment of the present invention. The method comprises the step S1, providing magnetic particles, DNA templates, a DNA immobilisation buffer and an IVT master mix in a reaction vessel of a bioreactor according to an embodiment of the present invention. In a step S2 the magnetic particles, the DNA templates and the DNA immobilisation buffer are mixed by means of a cooperation of the magnetic particles and a magnet unit of the bioreactor in order to obtain DNA magnetic particles, which are the DNA templates immobilized on the free-floating magnetic particles. In a method step S3, the DNA magnetic particles are mixed with the IVT master mix by means of a cooperation of the DNA magnetic particles and the magnet unit to obtain RNA. After step S3, the method may comprise step S4, comprising capturing DNA magnetic particles by means of the magnet unit and collecting/harvesting obtained in vitro transcribed RNA e.g. through the outlet port (S4a), providing fresh IVT master mix in a reaction vessel of a bioreactor of the first aspect (S4b), releasing captured DNA magnetic particles to provide free-floating DNA magnetic particles (S4c), mixing the free-floating DNA magnetic particles with the IVT master mix by means of a 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. Notably, S4 may be performed several times.

[0192] In addition to the above steps, a step ST of tempering the reaction vessel of the bioreactor can be performed between steps S1 and S2 or/and between steps S2 and S3. A cleaning or sanitizing step SC, where the reaction vessel is cleaned with a cleaning fluid and/or cleaning gas, may in addition follow step S3.

[0193] FIGS. 17 and 18 refer to embodiments of an automated apparatus for RNA manufacturing according to the present invention. In FIG. 17, an example with modules of the automated apparatus and elements for each module is shown. The apparatus comprises a module for DNA synthesis (“template generator”), T, a module for transcribing DNA into RNA, M, and a module for RNA formulation and fill and finish, F. The module for DNA synthesis comprises a pre-mixer 40, which is a unit for preparing PCR master mix 41, which is guided to a unit for preparative PCR 42. The obtained raw DNA template is subsequently guided to a unit for DNA conditioning 43. The dotted line as well as the dotted box indicate, that the conditioned DNA template may be subsequently guided to additional units, such as a unit for purification (e.g. comprising RP-HPLC and/or oligo dT). A purified DNA may then be released as indicated by the dashed arrow pointing horizontally out of module T. However, purified DNA may also be provided to module M, in particular to element 1, a bioreactor, of module N. As an additional input, the bioreactor 1 obtains an IVT master mix from the unit for preparing an IVT master mix 12. The raw RNA obtained by an RNA in vitro transcription reaction within the bioreactor 1 is guided to a unit for conditioning the raw RNA (e.g. comprising a TFF), 13, and subsequently to a unit for RNA purification 14 (e.g. comprising RP-HPLC and/or oligo dT). As indicated by the dotted line and dotted box, additional units may follow that further process and/or refine the obtained RNA (e.g. an RNA capping module for adding a cap0 or cap1 structure to in vitro transcribed RNA, an RNA polyadenylation module, an RNA mixing module, an RNA spray drying module, an RNA lyophilization module). After the described steps, the RNA is provided to module F. In this module, e.g. LNP encapsulated RNA may be produced by a combination of different units comprising at least one of a unit for mixing, a unit for conditioning (e.g. via TFF), a unit for sterile filtration and a unit for filling the obtained drug product.

[0194] FIG. 18 shows an overview of method steps comprising DNA synthesis, DNA purification and RNA in vitro transcription as performed in context of Example 1 described below.

EXAMPLE

[0195] The following Example is merely illustrative and shall describe the present invention in a further way. The Example shall not be construed to limit the present invention thereto.

Example: Model Batch

[0196] As an illustrative example of the processes and methods described in context of the invention, an example model batch process has been performed manually in the laboratory. The respective method steps are depicted in FIG. 18. In course of a first step, a DNA template generation step, the sub steps of a PCR (polymerase chain reaction), T1, and DNA purification (using RP-HPLC), T2, as well as AXP Purification (using Agencourt AMPure XP) have been performed. Thereby, the last sub step T3 shall not be performed in the final and automated process according to embodiments of the present invention and is only required for a manually processed model batch as in the Example. In a next step, RNA in vitro transcription is performed, wherein this step comprises the following sub steps: as a first sub step DNA immobilisation, M1, wherein the DNA templates are immobilised on free-floating magnetic beads. The second sub step M2 refers to the RNA in vitro transcription reaction. As a next sub step (not indicated in FIG. 20), AXP purification is performed, wherein again, this purification step shall not be performed in the final and automated process, but is performed only in the manually processed model batch. In sub step M3, the produced raw 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. The example is non-limiting, and to highlight the fact, that additional method steps may be performed, the dashed box with reference sign M5 indicates that there may be additional sub steps within the RNA in vitro transcription step. As third step, formulation is performed on the produced raw RNA. To this end, in-line mixing was carried out in sub step F1. As a next step not indicated in the FIG. 20, a dialysis was carried out, wherein also this sub step is intended to be left out in case of the final and automated process and was only required for the manually processed model batch. The next sub step F2 refers to UF/DF, followed by a cryo-protection step also not indicated on FIG. 20, as this step is only required for the manually processed model batch. The last three sub steps may also be combined in a single UF/DF step. In sub step F3, a sterile filtration is performed. The dashed box with reference sign F4 indicates that additional sub steps may be incorporated into a method according to the invention. In case of the Example, however, no further sub steps were performed.

[0197] A repeated batch RNA in vitro transcription as performed within the Example comprises the steps of PCR template generation and DNA template purification, both performed in a template generator. Within the next step of RNA production, in a first sub step template immobilisation takes place, followed by a repeated batch RNA in vitro transcription reaction step. The latter is then followed by a repeated batch HPLC sub step and finally a single batch TFF sub step.

[0198] Results on the recycled, i.e. repeated RNA in vitro transcription reaction are collected in FIG. 19. The same immobilized DNA template was used over 3 RNA in vitro transcription reactions. The results show a stable performance over the three cycles of RNA in vitro transcriptions, both quantitatively and qualitatively.

[0199] In FIGS. 20A and B, an RNA potency assay of the produced drug substance, the produced (HPLC purified) RNA, expressed in HepG2 cells (RAVG mRNA) is shown, demonstrating that the repeated RNA in vitro transcription reaction that may be suitably performed in the bioreactor of the invention produces RNA of high quality in a robust and reliable manner.

[0200] It has to be noted that embodiments of the invention are described with reference to different subject matters. In particular, some embodiments are described with reference to method type claims whereas other embodiments are described with reference to the device type claims. However, a person skilled in the art will gather from the above and the following description that, unless otherwise notified, in addition to any combination of features belonging to one type of subject matter also any combination between features relating to different subject matters is considered to be disclosed with this application. However, all features can be combined providing synergetic effects that are more than the simple summation of the features.

[0201] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. The invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing a claimed invention, from a study of the drawings, the disclosure, and the dependent claims.

[0202] In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are re-cited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

LIST OF REFERENCES SIGNS

[0203] 1 bioreactor [0204] 10 support [0205] 11 horizontal bar [0206] 110 arrow [0207] 111 arrow [0208] 2 reaction vessel [0209] 21 inner surface of the reaction vessel [0210] 23 /outer surface of the reaction vessel [0211] 24 flow breaker [0212] 25 first leg of the reaction vessel [0213] 251 first conduit [0214] 26 second leg of the reaction vessel [0215] 261 second conduit [0216] 27 filling level [0217] 28 maximal fluid amplitude [0218] 3 magnet unit [0219] 31 magnetic ring [0220] 32 rod [0221] 320 first rod [0222] 321 free end of the first rod [0223] 322 second rod [0224] 323 free end of the second rod [0225] 33 centre of the magnet unit [0226] 34 inner circumference of the magnetic ring [0227] 350 guide plate [0228] 351 electric coil [0229] 352 cooling means [0230] 36 spindle axis [0231] 37 arm [0232] 38 rotation driving means [0233] 39 driving means [0234] 361 movable support [0235] 362 arrow [0236] 363 arrow [0237] 5 temperature element [0238] 51 heat exchange channel [0239] 52 first end heat exchange channel [0240] 53 second end heat exchange channel [0241] 54 heating wire [0242] 55 heat isolation material [0243] 6 medium port/outlet port [0244] 60 valve means [0245] 61 magnetic trap [0246] 62 multi position valve [0247] 63 hall sensor [0248] 64 flow cells [0249] 65 arrow [0250] 66 medium pipe/outlet pipe [0251] 67 waste channel [0252] 7 exit port/waste port [0253] 71 multi position valve [0254] 72 waste flow cell [0255] 73 exhaust duct [0256] 74 waste channel [0257] 76 pressure sensor [0258] 8 inlet port [0259] 81 heating [0260] 83 inlet pipe [0261] 91 temperature sensor [0262] 92 additional sensor [0263] 12 IVT master mix [0264] 121 line into the bioreactor [0265] 122 line [0266] 123 line [0267] 124 line [0268] 13 conditioner [0269] 14 RNA purification [0270] 40 pre-mixer [0271] 41 PCR master mix [0272] 42 preparative PCR [0273] 43 unit for DNA conditioning