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US-20260125628-A1 - DEVICE AND METHOD FOR IN VITRO MANUFACTURING AND PURIFICATION OF THERAPEUTIC MRNA

US20260125628A1US 20260125628 A1US20260125628 A1US 20260125628A1US-20260125628-A1

Abstract

The invention includes a device for manufacturing mRNA by a continuous-flow recombinant process. The device comprises a housing; a cassette engaged with the housing, the cassette having a reaction chamber therein that holds an input reaction mixture having a DNA template wherein the manufacturing occurs within the cassette. The device has a thermal assembly providing a source of heat or cooling for the cassette, a pump to engage at least one tube within the cassette to control fluid flow through the cassette during manufacturing, a valve assembly to control a direction of fluid flow through the cassette, and a software application enabling a user to selectively manage operation of the device. The invention includes the cassette and the device as separate sub-combinations. The software application includes a non-transitory computer-readable medium containing computer executable instructions and a coupled computer processor or computer to execute the method of the manufacturing of mRNA.

Inventors

  • Handy Yowanto
  • Max Franz Wehebrink
  • Nathan DUVAL
  • Juan Alberto Allende Cabrera
  • Eric Michael Benner
  • Julian Brandon-Jones
  • Martin Philip Hereford Hoole
  • Christopher William Rosier
  • Nicholas Warrington
  • Simon Ian Burnell

Assignees

  • NATURE'S TOOLBOX, INC.

Dates

Publication Date
20260507
Application Date
20251107

Claims (20)

  1. 1 . A device for the manufacturing of mRNA by a continuous-flow recombinant process, the device comprising: a housing; a cassette engaged with the housing, the cassette having at least one reaction chamber therein configured to hold an input reaction mixture having a DNA template, at least one continuous-flow conduit configured hold and circulate a feed solution and to further configured to be in fluid communication with the reaction chamber, and a plurality of tubes within the cassette that carry the feed solution to the reaction chamber and that deliver the manufactured mRNA to a location external to the cassette; a thermal assembly mounted within the housing for providing a source of heat or cooling to the cassette; at least one pump mounted within the housing and facing the engaged cassette, wherein the pump is selectively operated to engage at least one tube of the plurality of tubes thereby controlling fluid flow through the cassette during the manufacturing; a valve assembly mounted within the housing for controlling a direction of fluid flow through the cassette; at least one electronic controller mounted in the housing and communicating with components of the device for controlling the operation of the device during use; and a software application communicating with the electronic controller enabling a user to selectively manage operation of the device for manufacturing of mRNA.
  2. 2 . The device, as claimed in claim 1 , further including: at least one bubble sensor in the housing for monitoring the presence of liquid in the tubes and through the reaction chamber;
  3. 3 . The device, as claimed in claim 1 , further including: a cassette locking assembly mounted in the housing and communicating with the cassette for locking the cassette to the housing, the cassette locking assembly having a motor, a pulley communicating with the motor, a pulley, and at least one driven belt that enables drive of the pulley and actuation of an adjacent cam that engages the housing to lock the cassette to the housing.
  4. 4 . The device, as claimed in claim 1 , wherein the housing further includes: at least one thermal port communicating with the thermal assembly to enable heated or cooled air generated from the thermal assembly to enter the engaged cassette for desired heating or cooling.
  5. 5 . The device, as claimed in claim 1 , wherein the housing further includes: a loading plate extending from the housing that enables the cassette to be placed thereon.
  6. 6 . The device, as claimed in claim 1 , wherein the pump further includes: at least one pump head having a pump channel wheel that is oriented to face the engaged cassette and positioned to selectively engage at least one tube of said plurality of tubes that is exposed and mounted to the cassette.
  7. 7 . The device, as claimed in claim 1 , wherein the valve assembly further includes: a plurality of pinch valves oriented to face the engaged cassette and positioned to selectively engage at least one tube of said plurality of tubes that is exposed and mounted to the cassette to selectively contact the tube and to thereby close the flow of fluid through the tube.
  8. 8 . The device, as claimed in claim 7 , wherein: the plurality of pinch valves are positioned to selectively engage a corresponding number of the plurality of tubes.
  9. 9 . The device, as claimed in claim 7 , wherein: the plurality of pinch valves are operated by corresponding solenoids.
  10. 10 . The device, as claimed in claim 1 , further including: at least one pressure sensor mounted in line with at least tube of said plurality of tubes for measuring fluid pressure in said tube.
  11. 11 . The device, as claimed in claim 1 , further including: a bezel assembly mounted on a side of said housing facing said cassette for visually indicating a status of the mRNA being manufactured in the cassette,
  12. 12 . The device, as claimed in claim 1 , further including: a plurality of said devices connected to one another by at least one connecting tube for sequential manufacturing of mRNA in a desired sequence, the connecting tube interconnecting adjacent cassettes engaged with corresponding devices.
  13. 13 . The device, as claimed in claim 1 , wherein said software application includes at least one of: computer executable instructions for stopping, starting and control of a rate of fluid flow in the plurality of tubes; computer executable instructions for opening and closing valves to shift a direction of fluid through the tubes; computer executable instructions for controlling temperature zones for the bioreactor and fluid therein; computer executable instructions for monitoring pressure and bubbles in tubes during manufacturing; computer instructions for interfacing the device with the cassette mechanically, to thereby secure the cassette into position for manufacturing; and computer instructions for visually indicating a status of the manufacturing and cassette loading.
  14. 14 . The device, as claimed in claim 1 , wherein the thermal assembly further includes: at least one heater subassembly having an electric heating element therein for producing heat to warm air passing through an adjacent heated air duct; at least one cooling subassembly having an electric cooling element therein for cooling air passing through an adjacent cooling air duct; and an exhaust duct subassembly communicating with said heating and cooling subassemblies for evacuating air within said housing that may be warmed by operation of said subassemblies.
  15. 15 . A device for the manufacturing of mRNA by a continuous-flow recombinant process, the device comprising: a housing; a cassette engaged with the housing, the cassette having at least one reaction chamber therein configured to hold an input reaction mixture having a DNA template, at least one conduit configured hold and circulate a feed solution and to further configured to be in fluid communication with the reaction chamber, and a plurality of tubes within the cassette that carry the feed solution to the reaction chamber and that deliver the manufactured mRNA to a location external to the cassette; at least one pump mounted within the housing and facing the engaged cassette, wherein the pump is selectively operated to engage at least one tube of the plurality of tubes thereby controlling fluid flow through the cassette during the manufacturing; a valve assembly mounted within the housing for controlling a direction of fluid flow through the cassette; at least one electronic controller mounted in the housing and communicating with components of the device for controlling the operation of the device during use; and a software application communicating with the electronic controller enabling a user to selectively manage operation of the device for manufacturing of mRNA.
  16. 16 . The device of claim 14 further including: a thermal assembly mounted within the housing for providing a source of heat or cooling to the cassette.
  17. 17 . The device, as claimed in claim 14 , further including: at least one bubble sensor in the housing for monitoring the presence of liquid in the tubes and through the reaction chamber.
  18. 18 . The device, as claimed in claim 14 , further including: a cassette locking assembly mounted in the housing and communicating with the cassette for locking the cassette to the housing, the cassette locking assembly having a motor, a pulley communicating with the motor, a pulley, and at least one driven belt that enables drive of the pulley and actuation of an adjacent cam that engages the housing to lock the cassette to the housing.
  19. 19 . The device, as claimed in claim 14 , wherein the housing further includes: at least one thermal port communicating with the thermal assembly to enable heated or cooled air generated from the thermal assembly to enter the engaged cassette for desired heating or cooling.
  20. 20 . The device, as claimed in claim 14 , wherein the housing further includes: a loading plate extending from the housing that enables the cassette to be placed thereon.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This International application claims the benefit of and priority to both U.S. Provisional Application No. 63/717,881, filed Nov. 7, 2024 and U.S. Provisional Application No. 63/717,884, filed Nov. 7, 2024. The entire specifications and figures of the above-referenced applications are hereby incorporated, in their entirety by reference. FIELD OF THE INVENTION The invention generally relates to device and method for in vitro manufacturing of polynucleotides. More specifically, the invention relates to a device and method for the manufacturing of mRNA for use in therapeutic applications in which the device controls the manufacturing process through use of a disposable cassette assembly which is isolated from but communicates with the device so that the device does not have to be cleaned or sterilized between manufacturing runs. BACKGROUND OF THE INVENTION Messenger RNA (mRNA) is the template molecule that is transcribed from cellular DNA and is translated into an amino acid sequence, i.e. a protein, at ribosomes in the cells of an organism. In order to control the expression level of the encoded proteins, mRNAs possess untranslated regions (UTRs) flanking the actual open reading frame (ORF) which contains the genetic information encoding the amino acid sequence. Such UTRs, termed 5′-UTR and the 3′-UTR, respectively, are sections of the mRNA located before the start codon and after the stop codon. Further, mRNA contains a poly(A) tail region which is a long sequence of adenine nucleotides which promotes export of mRNA from the nucleus, translation and to some extent protects the mRNA from degradation. Scientific and technological advances of the recent years have made mRNA a promising candidate for a variety of uses, including diagnostic applications, and therapeutic products, like vaccines. Due to the increasing demands of the medical community to enable personalized medicine, many approaches have been developed for mRNA production at scale. Most current methods utilize fermentation to synthesize mRNA in culture from self-replicating DNA templates, then isolate the total RNA as raw material utilizing volatile organic solvents. These processes are costly, dangerous, produce hazardous waste streams that must be mediated, while the production rate is severely dependent on the performance of the producing strain and the ability to remove impurities from diverse tRNA, rRNA and host mRNA. There exist a long-felt need for an effective in vitro mRNA manufacturing process that does not require volatile organic solvents, produces no hazardous waste stream, and costs significantly less than its fermentation-based counterpart, while generating uniform pure mRNA fit for therapeutic applications. There also exists a long-felt need for a manufacturing device that can produce pure mRNA at scale in which the device can be controlled with a software application enabling the device to conduct the manufacturing of pure mRNA through different types of RNA transcription processes. The term “transcription” as used herein relates to any process or method of making an RNA copies of selected gene DNA sequences. The copies, messenger RNA (mRNA), carry the corresponding gene protein information encoded in DNA. SUMMARY OF THE INVENTION The invention relates to a device and method for the manufacturing of mRNA for use in therapeutic applications. The device is capable of handling all steps involved in the process of synthesizing and purifying RNA molecules. These steps may include in vitro transcription (IVT), capping, tailing, and purification. IVT may be generally described as a process that uses a DNA template, RNA polymerase, and other components to create single-stranded RNA molecules. The resulting RNA molecules are similar to natural eukaryotic mRNA and can regulate protein expression. Capping may be generally described as a process that adds a 5′ cap to an mRNA molecule. Capping can be achieved co-transcriptionally in which a cap analog is added to an IVT reaction mixture. Alternatively, capping can be achieved by using enzymes, such as guanylyltransferase and 2′-O-methyltransferase after IVT. Tailing may be generally described as a process that adds a non-template nucleotide to the 3′ end of a DNA molecule. Purification may be generally described as a process that removes residual molecules, enzymes, and unincorporated nucleotides from the RNA. With respect to the system of the present invention for producing messenger RNA (mRNA) polynucleotides in vitro and a method of the present invention for recombinant production of messenger RNA (mRNA), this provisional application incorporates by reference the pending US Application U.S. Ser. No. 17/913,392 filed on Sep. 21, 2022, which claims priority to U.S. Provisional Application Ser. No. 63/011,133 filed on Apr. 16, 2020. According to the invention in one general aspect, the invention provides a device and method of continuous, automated and sem