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WO2024141622A1 - Procédé et dispositif de mise en œuvre en continu d'une réaction biochimique liquide - Google Patents

Procédé et dispositif de mise en œuvre en continu d'une réaction biochimique liquide Download PDF

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Publication number
WO2024141622A1
WO2024141622A1 PCT/EP2023/087955 EP2023087955W WO2024141622A1 WO 2024141622 A1 WO2024141622 A1 WO 2024141622A1 EP 2023087955 W EP2023087955 W EP 2023087955W WO 2024141622 A1 WO2024141622 A1 WO 2024141622A1
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WIPO (PCT)
Prior art keywords
chamber
chambers
reaction
liquid
module
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PCT/EP2023/087955
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English (en)
Inventor
Anne DE LAMOTTE
Mairesse BASTIEN
José CASTILLO
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Quantoom Biosciences SA
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Quantoom Biosciences SA
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0848—Specific forms of parts of containers
    • B01L2300/0858—Side walls
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/087—Multiple sequential chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/08—Regulating or influencing the flow resistance
    • B01L2400/084—Passive control of flow resistance
    • B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/52—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips
    • B01L9/523—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips for multisample carriers, e.g. used for microtitration plates

Definitions

  • the present invention relates to a method and a device for continuously performing a liquid biochemical reaction. More specifically, the invention relates to a continuous reaction module and related methods, wherein the module comprises a plurality of chambers, which are fluidly connected with a preceding and/or further chamber in said module allowing a liquid transfer from one chamber to a further chamber, whilst performing a biochemical reaction.
  • Flow reactors (sometimes called continuous reactors) are known from the state of the art and are in their simplest form tubes along which a fluid is able to flow and within which a reaction is able to take place. Flow reactors promote the use of continuous processing.
  • reactor configurations in current research efforts are largely ad hoc and focused on specific conditions of (for example) temperature and pressure. Because of this specificity, the configurations in question do not easily permit access to a wide range of operating conditions or provide support for studies relating to optimal synthetic route selection and reaction optimisation, without redesign and assembly. Additional apparatus is also required for monitoring, probing, measuring, data collection etc. that must be integrated into the reactor, which typically raises the cost and requires additional effort for integration and calibration.
  • replication of reported reactions across laboratories is not easy as it requires considerable effort to replicate the reported configuration with substantial consequent trial and error. As such, flow reactors are not easily scalable and do not allow a seamless transition from discovery to industrial scale production.
  • a particular aim of the present invention is to provide a configurable and integrated flow system for the preparation of nucleic acid-based vaccines.
  • the present invention and embodiments thereof serve to provide a solution to one or more of above-mentioned disadvantages.
  • said transfer of at least a portion of said reaction liquid occurs via an overflow section configured to be disposed between one chamber and a further chamber that is fluidly connected to said one chamber.
  • the method further comprises homogenizing the liquid content of said chambers during said biochemical reaction, by preference by dynamic mixing or static mixing.
  • said liquid biochemical reaction is an in vitro transcription (IVT) of RIMA.
  • the method comprises halting the IVT reaction by the addition of one or more reaction-quenching reagents in at least one of said plurality of chambers.
  • the invention in a second aspect, relates to a continuous reaction module, said reaction module comprising: a plurality of chambers, wherein a chamber of said plurality of chambers is capable of containing a reaction liquid; and an inlet configured to be disposed within a first chamber of said plurality of chambers, wherein one or more reagents are able to be introduced continuously into said reaction module via said inlet; and wherein a chamber of said plurality of chambers is fluidly connected with a preceding and/or a further chamber in said reaction module; and whereby at least a part of said plurality of chambers is capable of transferring said reaction liquid from a chamber to a further chamber that is fluidly connected with said chamber, and thereby said reaction module is capable of transferring at least a portion of said reaction liquid to said further chamber, whilst continuously performing said liquid biochemical reaction in said reaction module.
  • the continuous reaction module further comprises one or more outlets, wherein said one or more outlets are fluidly connected to one or more chambers of said plurality of chambers respectively, and wherein said one or more outlets are capable of removing one or more reagents and/or reaction products.
  • the continuous reaction module is configured to homogenize a liquid content of a chamber of said plurality of chambers.
  • said plurality of chambers are configured to be disposed on a single cartridge or on a plurality of cartridges.
  • the continuous reaction module further comprises one or more conduits configured to be disposed between chambers of said plurality of chambers, wherein said one or more conduits are capable of connecting a chamber with its preceding and/or its further chamber fluidly and transferring liquid between said chamber with its preceding chamber and/or its further chamber.
  • the continuous reaction module further comprises an overflow section between a chamber and a further chamber that is fluidly connected to said chamber.
  • the continuous reaction module further comprises one or more valves configured to connect fluidly with a chamber of said plurality of chambers.
  • Figures 1A and IB show schematic representations of a premixing device for premixing reagents before being continuously introduced into a continuous reaction module according to an embodiment of the invention.
  • Figures 6-7 show schematic representations of a continuous reaction module according to an embodiment of the invention, wherein the module comprises one cartridge comprising conduits connecting one chamber with a further chamber, a heater for temperature control and flow-dispersive structures for static mixing when no gas phase is present in the chambers.
  • Figure 8 shows a schematic representation of a continuous reaction module according to an embodiment of the invention, wherein the module comprises one IVT cartridge and one cartridge for terminating the IVT reaction, both cartridges comprising overflow sections between one chamber and a further chamber and a heater-shaker for homogenization and temperature control in the chambers when a gas phase is present.
  • Figure 10 shows a schematic representation of a continuous reaction module according to an embodiment of the invention, wherein the cartridge of the module is placed inside a heater-shaker.
  • the present invention concerns a method and a device for continuously performing a liquid biochemical reaction such as a cell-free biochemical reaction, said method comprising: introducing one or more reagents into a first chamber of a reaction module thereby initiating said liquid biochemical reaction, wherein the reaction module comprises a plurality of chambers, and a chamber of said plurality of chambers is fluidly connected with a preceding and/or further chamber in said reaction module and whereby at least a part of said plurality of chambers are designed to allow transfer of a reaction liquid from one chamber to a further chamber that is fluidly connected with said one chamber, thereby allowing transfer of at least a portion of said reaction liquid to said further chamber, whilst continuously performing said liquid biochemical reaction in said reaction module.
  • in vitro is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained.
  • In vitro reactions can encompass cellbased reactions in which living or dead cells are employed.
  • In vitro reactions can also encompass a cell-free reaction in which no intact cells are employed.
  • the in vitro liquid biochemical reaction is an in vitro cell-free biochemical reaction.
  • said biochemical reaction is a cell-free biochemical reaction.
  • said biochemical reaction comprises DNA amplification.
  • said biochemical reaction comprises DNA enzymatic synthesis.
  • said biochemical reaction comprises RNA transcription.
  • said biochemical reaction comprises RNA capping.
  • said biochemical reaction comprises protein synthesis.
  • said biochemical reaction comprises fatty acid synthesis.
  • said reaction is an in vitro transcription of RNA.
  • the biochemical reaction comprises DNA synthesis and the continuous reaction module as disclosed herein may be used for DNA synthesis.
  • the biochemical reaction comprises downstream processing of DNA after synthesis and the continuous reaction module may be used for downstream processing of DNA after synthesis.
  • the method may comprise a DNA synthesis method comprising thermocycling and the continuous reaction module may be used for DNA synthesis using thermocycling.
  • the continuous reaction module in conjunction with the DNA-preparation method for plasmid amplification may be use.
  • DNA synthesis is based on chemical processes (for instance technologies based on the phosphoramidite approach).
  • DNA synthesis is based on enzymatic processes, such as template-independent synthesis using thermostable enzymes.
  • Common components of a cell-free reaction include a cell extract, an energy source, a supply of amino acids, cofactors such as magnesium, and the DNA with the desired genes.
  • a cell extract is obtained by lysing the cell of interest and centrifuging out the cell walls, DNA genome, and other debris. The remains are the necessary cell machinery including ribosomes, aminoacyl-tRNA synthetases, translation initiation and elongation factors, nucleases, etc.
  • the cell extract used is made from E. coli (ECE).
  • EAE E. coli
  • the cell extract used is made from rabbit reticulocytes (RRL).
  • the cell extract used is made from wheat germ (WGE).
  • the cell extract used is made from insect cells (ICE).
  • the cross-section of the body portion comprises a polygonal shape.
  • the polygonal shape comprises a trigonal, tetragonal, pentagonal, or hexagonal shape.
  • the cross-section of the body portion comprises a hexagonal shape.
  • each chamber of a plurality of chambers comprise a hexagonal shaped cross section.
  • the plurality of chambers, each chamber comprising the hexagonal shaped cross section comprise a hexagonally packed arrangement in a cartridge.
  • each chamber of the plurality comprising a hexagonally shaped cross-section may be arranged in a honey-comb shaped structure in the cartridge.
  • said liquid transfer occurs by means of overflow from one chamber to said further chamber.
  • said overflow section allowing the overflow comprises a connection between two adjacent walls of two subsequent chambers, said connection being positioned at the upper half of the respective chambers, more preferably at the upper one fifth of the wall height of the body portion of the chamber.
  • said connection comprises a modification to one or more of the walls of the body portion of the chambers between which said connection is made.
  • said overflow section has a slope, being more highly positioned on the wall of a first chamber than on the wall of the subsequent chamber, allowing liquid transfer to occur by means of gravitational force.
  • said valves may comprise one or more inlets and/or outlets enabling one or more reagents to be added to or removed from said one or more chambers.
  • the valves may be programmable to introduce or remove a component from the system. For example, when the introduction of a reagent is desired, one or more valves may receive the instruction to open. Similarly, the one or more valves may also be instructed to close once the desired reagent is added in the desired amount.
  • said liquid content of said chambers is homogenized during said biochemical reaction, by preference by dynamic mixing or by the presence of one or more flow-dispersive structures in said chambers.
  • the liquid content is homogenized by means of a pressurized system. In some embodiments, the liquid content is homogenized by means of an ultrasonic system. In some embodiments, a gas phase is present in the chambers and the liquid content is homogenized by agitating the chambers of the continuous reaction module. In some embodiments, the agitating comprises rotating the chambers of the module. In some embodiments, the agitating comprises the use of an agitating device, such as a heater-shaker. In some embodiments, one or more chambers of the module comprise a baffle, a magnetic object, an impeller, or a bead for homogenizing the reagents.
  • the chambers do not comprise a gas phase and the liquid content is homogenized by dynamic mixing, said dynamic mixing comprises agitating the chambers on an agitating device, such as a heater-shaker, whilst providing the chambers with external solid elements, and as such agitating the chambers with the external solid elements comprised therein.
  • the chambers do not comprise a gas phase and the liquid content is homogenized by static mixing, said static mixing comprising providing the chambers with flow-dispersive structures and creating a dispersive flow in said chambers.
  • Flow-dispersive structures can be any structure known in the prior art, and include for instance a packed-bed or a fixed-bed as known from cell culture bioreactors.
  • the reaction module comprises at least a plurality of chambers, wherein the plurality of chambers is sufficient for the liquid to flow through the reaction module at a speed to complete at least one biochemical reaction at a pre-defined reaction speed.
  • a computer system operating a purposely constructed software code enables the directed control of the entire set of process parameters within the flow system. These include mixing conditions, temperature, pH, reagent concentrations, monitoring, residence times, purity profile, yield etc.
  • the reagents comprise a DNA template containing a promoter, water (H 2 O), ribonucleotide triphosphates (rNTPs), a buffer system that includes DTT and magnesium ions, and an appropriate RNA polymerase.
  • the RNA polymerase During transcription, the RNA polymerase 'reads' the DNA template and catalyzes the synthesis of the corresponding RNA molecule.
  • the DNA dependent RNA polymerases comprise at least one of a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, a RNA polymerase I, a RNA polymerase II, a RNA polymerase III, a RNA polymerase IV, a RNA polymerase V, and a single subunit RNA polymerase.
  • the RNA polymerase comprises T7 polymerase.
  • the ribonucleotide triphosphate comprises any one or a combination of an adenosine triphosphate (ATP); cytidine triphosphate (CTP); guanosine triphosphate (GTP); uridine-triphosphate (UTP).
  • ATP adenosine triphosphate
  • CTP cytidine triphosphate
  • GTP guanosine triphosphate
  • UDP uridine-triphosphate
  • the DNA template is circular. In some embodiments, the DNA template is linear. In an embodiment, the DNA is plasmid DNA.
  • cloning vectors DNA are applied as template for the generation of RNA transcripts following linearization of circular plasmid DNA molecule. These cloning vectors are generally designated as transcription vector.
  • RNA may be obtained by DNA dependent in vitro transcription of an appropriate DNA template.
  • a promoter for controlling RNA /n vitro transcription can be any promoter for any DNA dependent RNA polymerase.
  • a viral promoter binds a viral RNA polymerase and is at least one promoter selected from the list consisting of T7, T3, T7lac, SP6, pL, CMV, SV40, and CaMV35S.
  • the nucleic acid fragment comprising promoter sequence comprises a bacterial promoter.
  • the bacterial promoter binds a bacterial RNA polymerase and is at least one promoter selected from the list consisting of araBAD, trp, lac, and Ptac.
  • the nucleic acid fragment comprising promoter sequence comprises a eukaryotic promoter.
  • the eukaryotic promoter binds a eukaryotic RNA polymerase and is at least one promoter selected from the list consisting of EFla, PGK1, Ubc, beta actin, CAG, TRE, UAS, Ac5, Polyhedrin, CaMKIIa, ALB, GALI, GAL10, TEF1, GDS, ADH1, Ubi, Hl, and U6.
  • the eukaryotic promoter is at least one promoter selected from the list consisting of an RNA pol I promoter, an RNA pol II promoter and an RNA pol III promoter.
  • the DNA template relates to a DNA molecule comprising a nucleic acid sequence encoding the RNA sequence.
  • the template DNA is used as a template for RNA in vitro transcription in order to produce the RNA encoded by the template DNA. Therefore, the template DNA comprises all elements necessary for RNA /n vitro transcription, particularly a promoter element for binding of a DNA dependent RNA polymerase as e.g. T3, T7 and SP6 RNA polymerases 5' of the DNA sequence encoding the target RNA sequence.
  • the poly(A) tail can be either encoded into the DNA template or added enzymatically to RNA in a separate step after in vitro transcription.
  • the template DNA comprises primer binding sites 5' and/or 3' of the DNA sequence encoding the target RNA sequence to determine the identity of the DNA sequence encoding the target RNA sequence e.g. by PCR or DNA sequencing.
  • the DNA template comprises a DNA vector, such as a plasmid DNA, which comprises a nucleic acid sequence encoding the RNA sequence.
  • the DNA template comprises a linear or a circular DNA molecule.
  • a DNA template encodes a different RIMA molecule species.
  • the DNA template contains a sub-genomic promoter and a large ORF encoding for non-structural proteins which, following delivery of the biopharmaceutical into the cytosol, are transcribed in four functional components (nsPl, nsP2, nsP3, and nsp4) by the encoded RNA-dependent RNA polymerase (RDRP).
  • RDRP than produces a negative-sense copy of the genome which serves as a template for two positive-strand RNA molecules: the genomic mRNA and a shorter sub-genomic mRNA. This sub-genomic mRNA is transcribed at very high levels, allowing the amplification of mRNA encoding the antigen of choice.
  • the pathogen is selected from the group that causes human disease which includes but are not limited to, Bacillus anthracis (anthrax), Clostridium botulinum toxin (botulism), Yersinia pestis (plague).
  • Variola major (smallpox) and other related pox viruses Francisella tularensis (tularemia), Viral hemorrhagic fevers, Arenaviruses, (e.g., Junin, Machupo, Guanarito, Chapare, Lassa, and/or Lujo), Bunyaviruses (e.g., Hantaviruses causing Hanta Pulmonary syn-drome, Rift Valley Fever, and/or Crimean Congo Hemorrhagic Fever), Flaviviruses, Dengue, Filoviruses (e.g., Ebola and Marburg viruses), Burkholderia pseudomallei (melioidosis), Coxiella burnetii (Q fever), Brucella species (brucellosis), Burkholderia mallei (glanders), Chlamydia psittaci (Psittacosis), Ricin toxin (Ricinus communis), Epsilon toxin (Clostri
  • coli Pathogenic Vibrios, Shigella species, Salmonella, Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica, Caliciviruses, Hepatitis A, Cryptosporidium parvum, Cyclospora cayatanensis, Giardia lamblia, Entamoeba histolytica , Toxo-plasma gondii, Naegleria fowleri, Balamuthia mandrillaris, Fungi, Microsporidia, Mosquito-borne viruses (e.g., West Nile virus (WNV), LaCrosse encephalitis (LACV), California encephalitis, Venezuelan equine encephalitis (VEE), Eastern equine encephalitis (EEE), Western equine en-cephalitis (WEE), Japanese encephalitis virus (JE), St.
  • WNV West Nile virus
  • LACV LaCrosse encephalitis
  • VEE Venezuelan
  • SLEV Louis encephalitis virus
  • YFV Yellow fever virus
  • ZFV Yellow fever virus
  • Chikungunya virus Zika virus
  • Nipah and Hendra viruses Additional hanta-viruses
  • Tickborne hemorrhagic fever viruses Bunyaviruses
  • Severe Fever with Thrombocytopenia Syndrome virus SFTSV
  • Heartland virus Flaviviruses (e.g., Omsk Hemorrhagic Fever virus, Alkhurma virus, Kyasanur Forest virus), Tickborne encephalitis complex flaviviruses, Tickborne encephalitis viruses, Powassan/Deer Tick virus, Tuberculosis, including drug-resistant Tuberculosis, Influenza virus, Prions, Streptococcus, Pseudomonas, Shigella, Campylobacter, Salmonella, Clostridium, Escherichia, Hepatitis C, papillomavirus,
  • the virus is a respiratory virus that primarily results in respiratory symptoms including, without limitation, coronaviruses, influenza viruses, adenoviruses, rhinoviruses, coxsackieviruses, and metapneumoviruseses.
  • the virus is an enteric virus that primarily results in digestive symptoms including, without limitation, enteroviruses, noroviruses, heptoviruses, reoviruses, rotaviruses, parvoviruses, toroviruses, and mastadenovirus.
  • the virus is a hemorrhagic fever virus including, without limitation, Ebola virus, Marburg virus, dengue fever virus, yellow fever virus, Rift valley fever virus, hanta virus, and Lassa fever virus.
  • a DNA template encodes a different RNA molecule species.
  • different RNA molecule species comprises RNA constructs representing an engineered (non-natural) variant of a protein (e.g., chimeric protein), or fragment thereof.
  • a DNA template encodes different whole proteins that are separated by a linker.
  • different RNA molecule species encodes a different pathogenic antigen or a fragment or variant thereof.
  • the pathogen-associated antigen is from an influenza virus.
  • the pathogen-associated antigen is from an influenza A virus, such as the H5N1 strain.
  • the pathogen-associated antigen is from an influenza B virus.
  • the pathogen-associated antigen is an influenza matrix Ml protein or a fragment thereof. In certain cases, the pathogen-associated antigen is an influenza neuraminidase or a fragment thereof. In certain cases, the pathogen- associated antigen is an influenza hemagglutinin or a fragment thereof.
  • the pathogen-associated antigen may comprise an entire hemagglutinin, an HA1 domain, an HA2 domain or any antigenic portion thereof.
  • the pathogen-associated antigen is a Coronaviridae antigen.
  • the Coronaviridae exhibits human tropism.
  • the Coronaviridae is selected from the list consisting of SARS Coronavirus (SARS-CoV- 1), COVID-19 (SARS-CoV-2), MERS-coronavirus (MERS-CoV), or any combination thereof.
  • the Coronaviridae comprises SARS Coronavirus (SARS-CoV- 1).
  • the Coronaviridae comprises COVID-19 (SARS-CoV-2).
  • the Coronaviridae comprises MERS-coronavirus (MERS-CoV).
  • the Coronaviridae antigen comprises a spike protein, an envelope protein, a nucleocapsid protein, a membrane protein, a membrane glycoprotein, or a non- structural protein.
  • the Coronaviridae antigen comprises a spike protein, an envelope small membrane protein, a membrane protein, a non-structural protein 6 (NSP6), a nucleoprotein, an ORFIO protein, Protein 3a, Protein7a, Protein 9b, structural protein 8, uncharacterized protein 4, or any combination thereof.
  • a DNA template, ribonucleotide triphosphates, water and a buffer system are premixed.
  • a DNA template, ribonucleotide triphosphates, water, capping reagents and a buffer system are premixed.
  • a premixing step of all the required reagents except the enzyme mix comprising the DNA dependent RNA polymerase is performed.
  • premixing is accompanied with a heating ramp to reach the temperature identified as optimum for the RNA synthesis.
  • the outlet of this premix is directly put in contact and mixed with the enzymes mix (which includes the DNA dependent RNA polymerases, for instance T7 RNAP) via a mixing tee at the inlet of the RNA production module.
  • the reaction relates to in vitro transcription of RNA from a DNA template with or without ensuing post-transcriptional reactions, such as enzymatic capping and/or poly(A)-tail addition.
  • capping reagents are added to provide the RNA with a capping structure at its 5' end by either co-transcriptional capping (whereby specific reagents are introduced into the reaction mix at the start of the IVT reaction) or post-transcriptional capping (addition of a cap-structure to the starting nucleotide of the already formed RNA molecule by enzymatic action, with or without further enzymatically catalyzed methylation of the first nucleotide to obtain a ca pl -structure) (whereby capping reagents are introduced after completion of the in vitro transcription reaction).
  • one or more enzymes could be immobilized on a particulate support, e.g. magnetic or non-magnetic beads.
  • a particulate flow can be introduced into one or more chambers.
  • the beads can be filtered via pressure- driven filtration, for instance in the case of non-magnetic beads, via continuous vaccuum filtration machine or a self-cleaning filter.
  • the beads can be filtered by means of magnetic filtration.
  • the macromolecules comprise a reagent or component of the liquid.
  • the macromolecules may serve as a scaffold for reagents, enzymes, tags, or other components of the liquid.
  • Reagents or other components may comprise a protein, a modified protein, an enzyme, a polymerase, a reverse transcriptase, a nucleic acid, a modified nucleic acid, a nucleic acid analogue, an RNA molecule, a DNA molecule, any other reagent relevant to an RNA production process as described herein, or any combination thereof.
  • the resulting reaction product is further purified.
  • purification comprises magnetic purification.
  • purification comprises chromatographic purification.
  • said chromatographic purification is based on a bind-elute mode. In bind-elute mode, the target molecule binds to the ligand coupled to the resin through interactions. Changes in the buffer composition and pH release the molecule from the resin to allow collection (the molecule "elutes" with the buffer).
  • said chromatographic purification is based on a flow-through mode. In flow-through mode, impurities bind to the resin while the target molecule is collected in the chromatography flowthrough.
  • the purified reaction product may be further formulated in a formulation unit.
  • said formulation unit comprises an appropriate microfluidic mixing device.
  • the formulated reaction product can be filled in vials and sealed.
  • the formulated reaction product may comprise a liposome, a lipid nanoparticle, a nanostructured lipid carrier, a cubosome, a polyplex, a cochleate, a virosome, an immune-stimulating complex, a microparticle, a microsphere, a nanosphere, a unilamellar vesicle, a multilamellar vesicle, an oil-in water emulsion, a water-in-oil emulsion, an emulsome, or a cationic nano-emulsion.
  • the invention provides reaction products prepared by the above method.
  • the invention provides DNA prepared by the above method.
  • the invention provides peptides prepared by the above method.
  • the invention provides proteins prepared by the above method.
  • the invention provides fatty acids prepared by the above method.
  • the invention provides RIMA prepared by the above method.
  • a base modification in connection with the present disclosure is a chemical modification of the base moiety of the nucleotides of the RNA molecule.
  • nucleotide analogues or modifications are selected from nucleotide analogues, which are applicable for transcription and/or translation.
  • a cartridge of the plurality of cartridges may comprise at least one chamber. In some embodiments, each cartridge of the plurality of cartridges comprises at least one chamber. In some embodiments, a first cartridge of the plurality of cartridges comprises the same number of chamber(s) as a second cartridge of the plurality of cartridges. In some embodiments, the first cartridge of the plurality of cartridges comprises a different number of chamber(s) as the second cartridge of the plurality of cartridges. In some embodiments, the cartridge comprises at least one aperture. In some embodiments, the aperture is configured to engage with a gripping mechanism. In some embodiments, a robotic arm or handling device comprises the gripping mechanism. In some embodiments, the cartridge comprises at least two apertures. In some embodiments, the apertures are positioned at the upper surface of the cartridge.
  • said plurality of chambers are comprised on a single cartridge or a plurality of cartridges. In an embodiment, said plurality of chambers are comprised on a single cartridge. In an embodiment, said plurality of chambers are comprised on two or more cartridges, such as 3, 4, 5, 6, 7, 8, 9 or 10 cartridges.
  • the cartridge may be provided with means for agitating the plurality of chambers. Agitation may comprise rocking, rotating, shaking or any other suitable moving of the cartridge. By allowing the cartridge to rotate around an axis, or to perform a rocking motion, sedimentation of the contents of the chambers may be avoided. Additionally or alternatively, agitation of the cartridge/the chambers may permit mixing of the contents to facilitate an incubation such as a reaction within the cartridge/chambers.
  • the sample taken from the chamber through the at least one opening in the lid is transferred to, for example but not limited to, laser-based analyzers such as a flow cytometry and cell sorting devices.
  • said premixing device is configured to comprise a volume of at least about 1 pL, about 2 pL, about 5 pL, about 10 pL, about 20 pL, about 25 pL, 50 pL, about 100 pL, about 0.2 mL, about 0.5 mL, 1 ml, about 2 mL, about 5 mL, about 10 mL, about 20 mL, about 50 mL, about 100 mL, about 200 mL, about 400 mL, about 500 mL, about 800 mL, about 1 L, about 2 L, about 4 L, about 5 L, about 8 L, or about 10 L.
  • said premixing device may be configured to comprise a volume of not more than about 10 L, about 8 L, about 5 L, about 4 L, about 2 L, about 1 L, about 800 mL, about 500 mL, about 400 mL, about 200, mL, about 100 mL, about 50 mL, about 20 mL, about 10 mL, about 5 mL, about 2 mL, about 1 mL, about 0.5 mL, about 0.2 mL, about 100 pL, about 50 pL, about 20 pL, about 10 pL, about 5 pL, about 2 pL, or about 1 pL.
  • said device comprises agitation or rotation means, allowing for instance intermittent rotation or orbital shaking.
  • said device comprises means for temperature regulation, such as a heating mat or heating block.
  • a specific objective is to provide a system and method integrating various cartridges including for instance one or more IVT cartridges and one or more cartridges for terminating the IVT reaction interconnected via respective conduits/channels to create a continuous fluid flow pathway that may be operated continuously and may be automated using electronic and/or software control utilities involving the use of membranes, component actuators such as pumps, valves, gates, delivery ports, outlet ports, syringe pumps, sensors etc. as will be appreciated.
  • the continuous reaction module may be designed and operated so that only limited handling is required. Limited handling may avoid contamination and disturbance of the process conditions. If irregularities are observed, an operator may manipulate the process via one or more control devices present inside or outside the system and/or modules. These control devices may control (parts of) the process taking place in the system.
  • each chamber may be coupled to one or more control devices that are configured to perform multivariate analysis, automatically control operation of the processes, and optionally, communicate with components remotely (using, for example, network protocols) in order to control operation in the chamber.
  • a valve or valves may be coupled to one or more such control devices.
  • control unit comprises a control module comprising any one or a combination of the following: software; electronic components; a data storage utility; wired or wireless communication modules and/or ports; a visual display output; a user interface; at least one actuator to actuate any one or a combination of the at least one pump, valve, fluid flow gate, fluid flow port, heating element, fluid storage or retention reservoir and the sensors.
  • the CPU can be part of a circuit, such as an integrated circuit.
  • a circuit such as an integrated circuit.
  • One or more other components of the system can be included in the circuit.
  • the circuit is an application specific integrated circuit (ASIC).
  • ASIC application specific integrated circuit
  • inlet 104.1 can be for instance used for the introduction of water
  • inlet 104.2 can be for instance used for the introduction of buffer
  • inlet 104.3 can be for instance used for the introduction of the DNA template
  • inlet 104.4 can be for instance used for the introduction of NTPs and capping reagent.
  • Said premixing device 100 further comprises a rotator 107 for rotating the premixing tank 101 and the reagents comprised therein. In order to heat the reagents and obtain the desired temperature, a heating mat 102 is placed around the premixing tank 101. A temperature sensor 105 monitors the temperature of the liquid inside the premixing tank.
  • the continuous reaction module 200 further comprises a heater-shaker 207 to heat and homogenize the liquid 219 comprised in the chambers 201 of the cartridge 218. Presence of a gas phase 211 allows to properly homogenize the liquid comprised in the chambers 201 of the cartridge 218 by means of agitation.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne un procédé de mise en œuvre en continu d'une réaction biochimique liquide telle qu'une réaction biochimique acellulaire, ledit procédé comprenant les étapes suivantes : introduction d'un ou de plusieurs réactifs dans une première chambre d'un module de réaction, initiant ainsi ladite réaction biochimique liquide, le module de réaction comprenant une pluralité de chambres, et une chambre de ladite pluralité de chambres étant en communication fluidique avec une chambre précédente et/ou supplémentaire dans ledit module de réaction et grâce à quoi au moins une partie de ladite pluralité de chambres est conçue pour permettre le transfert d'un liquide de réaction d'une chambre à une chambre supplémentaire qui est en communication fluidique avec ladite chambre, permettant ainsi le transfert d'au moins une partie dudit liquide de réaction vers ladite chambre supplémentaire, tout en mettant en œuvre en continu ladite réaction biochimique liquide dans ledit module de réaction.
PCT/EP2023/087955 2022-12-30 2023-12-29 Procédé et dispositif de mise en œuvre en continu d'une réaction biochimique liquide Ceased WO2024141622A1 (fr)

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EP22217265.2 2022-12-30

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2125184A1 (fr) * 2006-12-06 2009-12-02 Ashe Morris Limited Réacteur à écoulement perfectionné
WO2018187745A1 (fr) * 2017-04-06 2018-10-11 Sri International Systèmes modulaires destinés à la réalisation de réactions chimiques à étapes, et procédés d'utilisation associés
WO2022266513A2 (fr) * 2021-06-17 2022-12-22 Mammoth Biosciences, Inc. Dispositifs, systèmes et procédés d'analyse d'acides nucléiques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2125184A1 (fr) * 2006-12-06 2009-12-02 Ashe Morris Limited Réacteur à écoulement perfectionné
WO2018187745A1 (fr) * 2017-04-06 2018-10-11 Sri International Systèmes modulaires destinés à la réalisation de réactions chimiques à étapes, et procédés d'utilisation associés
WO2022266513A2 (fr) * 2021-06-17 2022-12-22 Mammoth Biosciences, Inc. Dispositifs, systèmes et procédés d'analyse d'acides nucléiques

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