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WO2025040263A1 - Procédé de purification d'adn amplifié in vitro - Google Patents

Procédé de purification d'adn amplifié in vitro Download PDF

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Publication number
WO2025040263A1
WO2025040263A1 PCT/EP2023/073229 EP2023073229W WO2025040263A1 WO 2025040263 A1 WO2025040263 A1 WO 2025040263A1 EP 2023073229 W EP2023073229 W EP 2023073229W WO 2025040263 A1 WO2025040263 A1 WO 2025040263A1
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WIPO (PCT)
Prior art keywords
dna
pcr
vitro
rna
chromatography
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PCT/EP2023/073229
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English (en)
Inventor
Michael THOMMEN
Christina HEINZ
Andreas Sternecker
Robert MÜNCH
Drazen KOSUTIC
Felix BERTSCH
Tilmann Roos
Original Assignee
CureVac Manufacturing GmbH
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Filing date
Publication date
Application filed by CureVac Manufacturing GmbH filed Critical CureVac Manufacturing GmbH
Priority to PCT/EP2023/073229 priority Critical patent/WO2025040263A1/fr
Publication of WO2025040263A1 publication Critical patent/WO2025040263A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase

Definitions

  • RNA-based therapeutics belong to the most promising and quickly developing therapeutic fields in modem medicine.
  • Established manufacturing processes for RNA molecules implement many separate manufacturing steps, including the generation of a suitable DNA template for RNA in vitro transcription.
  • DNA templates are produced in bacteria in form of plasmid DNA (pDNA).
  • pDNA plasmid DNA
  • the production of pDNA and its subsequent purification is well established in the art.
  • pDNA production and purification is a substantial cost factor, is time consuming, and is based on cells which should ideally be avoided.
  • RNA manufacturing processes make use of in vitro processes to produce DNA.
  • in vitro DNA amplification such as PCR amplification is typically performed in small scale in molecular biology.
  • Such methods are typically performed in analytic assays or e.g. for cloning a certain amplified DNA element into a suitable DNA vector, and purification is normally carried out using commercially available kits to remove the enzymes and buffer components of the PCR reaction.
  • PCR- amplified DNA can be used as a template for RNA in vitro transcription to produce RNA-based medicaments.
  • RP-HPLC has been shown to be effective in purifying PCR amplified DNA in larger scales and in a sufficient quality (W02020002598 and WD2022112498) - however such a purification is time consuming, costly, and produces organic waste. Accordingly, further effective purification strategies for vitro amplified DNA are urgently needed to accelerate and economize the production of DNA which is of particular importance in the context of automated RNA production or RNA production for personalized medicaments.
  • an object of the present invention is to provide an effective purification for vitro amplified DNA such as PCR amplified DNA.
  • the purification method should not involve organic solvents, should be scalable, should be operatable in an automated and/or continuous manner, and should be operatable in a production process for personalized medicaments.
  • the present invention is inter alia directed to a method of purifying in vitro amplified DNA, in particular PCR amplified DNA, comprising least one step of core-bead flowthrough chromatography and at least one additional purification step, e.g. ion exchange chromatography or tangential flow filtration (TFF).
  • the invention provides methods of producing DNA and methods of producing RNA by RNA in vitro transcription using the purified in vitro amplified DNA as a template.
  • Further aspects relate to a system for purifying in vitro amplified DNA, a system for producing in vitro amplified DNA, and a system for producing RNA.
  • the inventors developed a purification process for in vitro amplified DNA (e.g. PCR amplified DNA) that is scalable and that can be inter alia used in automated DNA and RNA production.
  • the purification process can be operated using single-use equipment which simplifies the DNA purification or DNA production under GMP conditions.
  • the present invention provides a method of purifying in vitro amplified DNA, e.g. PCR amplified DNA, comprising core-bead flow through chromatography followed by at least one additional purification step.
  • the at least one additional purification step may be selected from ion exchange chromatography, HIC, orTFF.
  • the at least one additional purification step is selected from anion exchange chromatography.
  • the invention provides a method of producing in vitro amplified DNA, e.g. PCR amplified DNA, comprising the steps (i) generating a DNA preparation by in vitro DNA amplification step, and (ii) purifying the in vitro amplified DNA according to a purification method of the first aspect.
  • in vitro amplified DNA e.g. PCR amplified DNA
  • a fifth aspect of the invention relates to a system for producing in vitro amplified DNA comprising an in vitro DNA amplification unit and at least one DNA purification system according to the fourth aspect.
  • the system is configured to perform a method according to the second aspect.
  • a sixth aspect of the invention relates to an RNA production system comprising at least one RNA in vitro transcription unit and at least one DNA purification system of the fourth aspect, or at least one DNA production system of the fifth aspect.
  • the system is configured to perform a method according to the third aspect.
  • coding sequence or “coding region” and the abbreviation “cds” as used herein will be recognized and understood by the person of ordinary skill in the art, and are e.g., intended to refer to a sequence of several nucleotide triplets that may be translated into a peptide or protein.
  • a cds in the context of the present invention may be an RNA sequence consisting of a number of nucleotides that may be divided by three, which starts with a start codon and preferably terminates with a stop codon.
  • PCR Polymerase chain reaction
  • the DNA polymerase enzymatically assembles a new DNA strand from DNA building-blocks, the nucleotides, by using single-stranded DNA as a PCR template and DNA primers, which are required for initiation of DNA synthesis.
  • the vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample through a defined series of temperature steps.
  • PCR master mix may comprise the components necessary for performing a PCR. Accordingly, a PCR master mix may comprise at least one of the components selected from a nucleotide mixture, a DNA polymerase, a DNA to be amplified (amplification template; e.g. a plasmid DNA), and a buffer.
  • RNA The terms “RNA” and “mRNA” are e.g., intended to be a ribonucleic acid molecule, i.e., a polymer consisting of nucleotides. These nucleotides are usually adenosine-monophosphate, uridine-monophosphate, guanosine-monophosphate and cytidine-monophosphate monomers which are connected to each other along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar, i.e., ribose, of a first and a phosphate moiety of a second, adjacent monomer. The specific succession of the monomers is called the RNA-sequence.
  • the mRNA messenger RNA
  • the mRNA provides the nucleotide coding sequence that may be translated into an amino-acid sequence of a particular peptide or protein.
  • 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 (a DNA template for RNA in vitro transcription).
  • the promoter for controlling RNA in vitro transcription can be any promoter for any DNA-dependent RNA polymerase.
  • DNA-dependent RNA polymerases are the T7, T3, SP6, or Syn5 RNA polymerases.
  • RNA in vitro transcription takes place in an in vitro transcription buffer (IVT buffer) that comprises the components required to transcribe the DNA into RNA.
  • IVTT buffer in vitro transcription buffer
  • RNA polymerase T7, T3, SP6, or Syn5 RNA polymerase
  • RNase ribonuclease
  • MgCb which supplies Mg 2+ ions as a co-factorforthe polymerase
  • a buffer substance e.g. TRIS or HEPES
  • antioxidants e.g. DTT
  • polyamines such as spermidine.
  • the present invention provides a method of purifying a DNA.
  • the method of purifying DNA comprises at least one step of core-bead flow through chromatography (herein also referred to as step A).
  • the method of purifying DNA comprises at least one step of core-bead flow through chromatography (step A) and does not comprise an additional purification step for purifying DNA.
  • the method of purifying DNA comprises at least one step of core-bead flow through chromatography (step A) and at least one additional purification step (herein also referred to as step B).
  • the method of purifying DNA comprises at least one step of core-bead flow through chromatography (step A) to provide a purified (or pre-purified) flow through that is then subjected to at least one additional purification step B.
  • the method comprises the steps
  • step B purifying the flow through of step A by at least one additional purification step B to obtain a pure preparation of DNA, preferably in vitro amplified DNA.
  • step A Particularly suitable features and embodiments of “step A” are specified in paragraph “Step A: Core-bead flow through chromatography”. Particularly suitable features and embodiments of “step B” are specified herein in paragraph “Step B: Additional purification step”.
  • the target DNA is a DNA sequence having the target DNA:
  • target DNA The DNA intended to be purified by the method of the invention is herein also referred to as “target DNA”.
  • the method is for purifying in vitro amplified DNA, for example enzymatically amplified DNA.
  • the DNA that is intended to be purified by the method i.e. the target DNA
  • the DNA that is intended to be purified by the method is preferably an in vitro amplified DNA.
  • in vitro amplified DNA in the context of the invention has to be understood as a DNA that is amplified in a cell-free system in vitro. Accordingly, plasmid DNA that is amplified in bacteria (e.g. Escherichia coli) or cells has to be considered as “in vivo amplified DNA” and is hence not encompassed by the term “in vitro amplified DNA”.
  • In vitro amplified DNA may be generated by using enzymes that amplify a DNA in vitro. For example, DNA polymerases may be used to enzymatically amplify a DNA in vitro.
  • the DNA that is amplified by an in vitro amplification method is herein also referred to as DNA amplification template.
  • the DNA used for amplification via an in vitro amplification process may be selected from e.g. a de-novo synthetized DNA, an in vivo amplified DNA (e.g. plasmid DNA).
  • the DNA i.e. the target DNA
  • the DNA has not been prepared by bacterial amplification.
  • the DNA i.e. the target DNA
  • the DNA has been prepared by an in vitro DNA amplification process, preferably an enzymatic in vitro DNA amplification process, said in vitro DNA amplification process comprising or consisting of polymerase chain reaction (PCR), rolling circle amplification, or isothermal amplification.
  • PCR polymerase chain reaction
  • PCR Polymerase Chain Reaction
  • PCR is an enzymatic DNA amplification process and one of the fastest ways to prepare a DNA in vitro.
  • PCR can be performed in PCR devices. It is a technology in molecular biology to synthetically amplify a DNA amplification template (e.g. a DNA sequence comprised in a plasmid DNA) across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
  • PCR relies on thermal cycling comprising cycles of repeated heating and cooling for DNA melting and enzymatic replication of the DNA using thermostable DNA polymerases. The cycles may comprise a denaturation temperature, an annealing temperature, and an elongation temperature.
  • Specific PCR techniques comprise for example RT-PCR, Hot-Start PCR, Long-Range PCR, RT-qPCR, etc.
  • Rolling circle amplification is an enzymatic DNA amplification process to produce DNA in vitro that typically uses a circular DNA or doggy bone DNA as amplification template and random primers that anneal to the DNA amplification template.
  • Phi29, Bst, and Vent DNA polymerases are used for DNA amplification.
  • RCA can be conducted at a constant temperature (e.g. room temperature to 65°C) in both free solution and on top of immobilized targets (e.g. solid phase amplification).
  • a constant temperature e.g. room temperature to 65°C
  • immobilized targets e.g. solid phase amplification
  • Isothermal DNA amplification is an enzymatic DNA amplification process and is performed at constant temperature (isotherm) with a strand-transferring DNA polymerase, while PCR uses a thermostable DNA polymerase and heating to 95°C by a thermocycler for strand separation.
  • the strand-transferring DNA polymerase e.g. for example, 029 DNA polymerase from bacteriophage (p29, displaces an existing second strand of doublestranded DNA while using the first strand to make a new strand with the same sequence as the second strand.
  • the DNA i.e. the target DNA
  • PCR polymerase chain reaction
  • the DNA i.e. the target DNA
  • the DNA is selected from a linear DNA, circular DNA, single stranded DNA (ssDNA), double stranded DNA (dsDNA), or any combination thereof.
  • the DNA i.e. the target DNA
  • a linear dsDNA e.g. PCR amplified DNA
  • dbDNA doggy bone DNA
  • dbDNA is an enzymatically amplified DNA vector.
  • dbDNA are minimal, double stranded, and covalently closed DNA vectors amplified through an in vitro enzymatic RCA process.
  • the dbDNA may additionally be digested with a restriction endonuclease to produce a linearized doggy bone DNA.
  • the PCR amplified DNA may additionally be digested with a restriction endonuclease to trim the 3’ or 5’ terminus of the DNA (e.g. to produce a homopolymeric poly(A) terminus).
  • restriction endonucleases may represent a further impurity and the purification of the invention is capable of removing restriction endonucleases from the impure preparation.
  • the DNA i.e. the target DNA
  • the DNA is a PCR amplified DNA. Accordingly, in these embodiments, the DNA is in linear and double stranded form.
  • the DNA i.e. the target DNA
  • the DNA is not a plasmid DNA (or any other circular DNA) and not a virus DNA.
  • these DNA species may be part ofthe impurities as defined herein (e.g. if for example a plasmid DNA is used as a DNA amplification template).
  • the DNA i.e. the target DNA
  • the DNA has a size of at least 250 nucleotides or 500 nucleotides.
  • the DNA intended to be purified is larger than 500 nucleotides.
  • the DNA i.e. the target DNA
  • the DNA has a size ranging from at least about 500 nucleotides to 10000 nucleotides, preferably ranging from at least about 500 nucleotides to 7000 nucleotides, even more preferably ranging from at least about 1000 nucleotides to 5000 nucleotides.
  • a promoter for RNA in vitro transcription may be selected from a T7 RNA polymerase promoter, an SP6 RNA polymerase promoter, a T3 RNA polymerase promoter.
  • T7 RNA polymerase promoter is preferred.
  • the DNA template for RNA in vitro transcription may additionally comprise at least one UTR as defined herein and/or at least one histone stem loop sequence as defined herein.
  • the coding sequence encodes a therapeutic peptide or protein.
  • the at least one therapeutic peptide or protein is selected or derived from an antibody, an intrabody, a receptor, a receptor agonist, a receptor antagonist, a binding protein, a CRISPR- associated endonuclease, a chaperone, a transporter protein, an ion channel, a membrane protein, a secreted protein, a transcription factor, an enzyme, a peptide or protein hormone, a growth factor, a structural protein, a cytoplasmic protein, a cytoskeletal protein, a viral antigen or epitope, a bacterial antigen or epitope, a protozoan antigen or epitope, an allergen, a tumor antigen or epitope, or fragments, variants, or combinations of any of these.
  • the at least one therapeutic peptide or protein is selected or derived from an antigen or epitope of a pathogen (e.g. a viral antigen or epitope, a bacterial antigen or epitope, a protozoan antigen or epitope) or from an antigen or epitope of a tumor.
  • a pathogen e.g. a viral antigen or epitope, a bacterial antigen or epitope, a protozoan antigen or epitope
  • the DNA i.e. the target DNA
  • the DNA is a codon modified DNA.
  • the DNA may comprise a coding sequence with a codon modified sequence.
  • the term “codon modified coding sequence” relates to coding sequences that differ in at least one codon (triplets of nucleotides coding for one amino acid) compared to the corresponding wild type (or reference) coding sequence. Codon modifications in the broadest sense make use of the degeneracy of the genetic code wherein multiple codons may encode the same amino acid and may be used interchangeably to optimize/modify the coding sequence for in vivo applications as outlined herein.
  • the codon modified coding sequence is selected from a C maximized coding sequence, CAI maximized coding sequence, human codon usage adapted coding sequence, G/C content modified coding sequence, and G/C optimized coding sequence, or any combination thereof.
  • the at least one codon modified coding sequence is a G/C optimized coding sequence.
  • the DNA i.e. the target DNA
  • the DNA is a G/C optimized DNA (that is, the DNA comprises a GC optimized coding sequence as defined herein).
  • the DNA that is subjected to the purification may have a G/C content that is increased compared to a reference or wildtype DNA sequence.
  • the G/C enriched DNA may have a G/C content of more than 50%, e.g. 55%, 60%, 65%, 70%.
  • the DNA is biotinylated at the 3’ and/or 5’ terminus.
  • the DNA i.e. the target DNA
  • the in vitro amplified DNA is suitably produced by PCR using biotinylated DNA primers.
  • the target DNA is biotinylated because the biotinylation allows for a specific DNA capturing or DNA immobilization which can further reduce the impurities.
  • the biotinylated DNA may be immobilized on a solid support, and residual impurities may be washed off. The resulting immobilized DNA may be used in a downstream process (e.g. RNA in vitro transcription).
  • the impure preparation that is subjected to the purification method comprises the DNA intended to be purified (i.e. the target DNA) and at least one impurity.
  • the target DNA is preferably a DNA that has been produced by an in vitro DNA amplification (e.g. PCR) such as linear dsDNA.
  • the impure preparation comprises at least one impurity derived from the process that has been used to prepare the DNA.
  • the process that has been used to prepare the DNA preferably comprises an in vitro DNA amplification, for example an enzymatic vitro DNA amplification.
  • the at least one impurity is selected from peptides, proteins, enzymes, DNA primers, dNTPs, abortive DNA, elongated DNA, plasmid DNA, DNA concatemers, antibodies, BSA, betaine, gelatine, Tetramethylammonium chloride, ora fragment of any of these.
  • the at least one impurity is derived from a PCR process.
  • the impurities comprise proteins or enzymes (e.g. DNA polymerase), DNA primers (e.g. biotinylated DNA primers), dNTPs, and PCR buffer components (e.g. Tetramethylammonium chloride, betaine, gelatine, BSA).
  • the impure preparation comprising in vitro amplified DNA is a PCR reaction product comprising PCR-amplified DNA as a target DNA.
  • the impure preparation comprises or consists of a non-purified PCR reaction product.
  • a typical PCR reaction product may comprise
  • DNA Polymerase e.g. hot-start DNA Polymerase
  • a storage matrix for the DNA polymerase such as gelatine and/or BSA and/or glycerol
  • reaction buffer which typically comprises T ris
  • a DNA amplification template e.g. plasmid DNA
  • antibody or aptamer for hot-start PCR or “antibodies for hot-start function” or “antibodies or aptamers for mediating hot-start PCR” relates to agents that inactivate a DNA polymerase, e.g. a Taq DNA polymerase.
  • the antibodies or aptamers link and bind to the polymerase, preventing early DNA amplification which could occur at lower temperatures. Once the optimal annealing temperature is met, the antibodies or aptamers will begin to degrade and dissociate, releasing the Taq DNA polymerase into the reaction and allowing the amplification process to start.
  • the PCR reaction product comprises DNA Polymerase, forward DNA primers and reverse DNA primers, a plasmid DNA as DNA amplification template, the target DNA to be purified, dNTPs.
  • the PCR reaction product may comprise 20ng to 200ng/pL PCR amplified DNA (i.e. the target DNA); 10pg/pLto 30pg/pL DNA amplification template (e.g. plasmid DNA); 200nM to 400nM DNA primers; 0.1 mM to 0.4mM dNTPs; 1 ,5mM to 3mM MgCh; optionally, 0.5M to 1 M betaine; 0.01 U/pL to 0.03U/pL DNA polymerase (e.g.
  • KapaHifi Polymerase optionally, 0.05% to 0.1% gelatine; optionally, 0.001% to 0.003% blocking antibody for hot-start PCR; optionally, 0.25% to 1 .5% Tetramethylammonium chloride, optionally, 2.5% to 15% glycerol, 0.5% to 5% buffer, preferably Tris.
  • the PCR reaction product is directly subjected to step A without any further conditioning, re-buffering, or purification step.
  • the impure preparation comprises in vitro amplified DNA at a purity level (with reference to the target DNA) of below 70%, below 60% or below 50%.
  • the purity level of DNA may be determined by gel electrophoresis, capillary gel electrophoresis, analytical RP HPLC, cation and anion chromatography, or mass spectrometry.
  • the DNA i.e. the target DNA
  • a method of the invention can comprise one or more steps of core bead flow-through chromatography.
  • the inventors have found that this technique enables a fast, scalable purification process for obtaining pure DNA with high yield and is particularly advantageous for removing protein contaminants (e.g. DNA polymerase) and/or DNA primers from the desired target DNA.
  • the core bead flow-through chromatography can be performed using aqueous buffer systems and can be performed using single use materials.
  • the core bead flow-through chromatography is particularly suitable for purifying an impure preparation comprising PCR amplified DNA, in particular, for purifying a PCR reaction product.
  • the core-bead flowthrough chromatography is performed by contacting the DNA with a corebead flow through chromatography material.
  • the core-bead flowthrough chromatography is performed by contacting the impure preparation comprising DNA, preferably in vitro amplified DNA, more preferably an impure preparation comprising PCR amplified DNA, with a core-bead flowthrough chromatography material.
  • contacting the impure preparation with a core-bead flowthrough chromatography material is performed under conditions to allow size-exclusion of the DNA, preferably the in vitro amplified (target) DNA, and binding of impurities.
  • Core bead flow-through chromatography may be performed using a batch format or a column format.
  • a column format is preferred.
  • the column comprises the stationary phase.
  • the column format may include applying a DNA-containing impure preparation to the column, collecting the flow-through, and optionally passing elution buffer through the column (e.g. for analytical purpose).
  • the method may comprise additional steps such as wash steps e.g. after applying the sample to the column.
  • a buffer may also be applied to the column or bead and passed through the column or bead using gravitational force alone or by applying external pressure in orderto increase the rate at which the sample components pass through the column.
  • the core-bead flowthrough chromatography material comprises or consists of porous beads. Accordingly, the core bead flow-through chromatography material is selected from core bead flow- through chromatography beads that are comprised of a porous material (or matrix).
  • porous beads of the core-bead flow through chromatography material
  • the porous material of the beads comprises at least two layers, for example an inner layer (core) surrounded by an outer layer (shell).
  • the matrix may also comprise one or more additional (intermediate) layers between the inner layer and the outer layer.
  • the porous beads of the core-bead flowthrough chromatography material
  • have a defined pore size thereby preventing a proportion of molecules comprised in the impure preparation from entering the core based on the size of the molecules, which are collected in the column flow-through (flow- through mode).
  • Molecules that are able to pass through the porous beads enter the core, where they may be retained or bound, typically by binding to a ligand as further specified below.
  • Retained/Bound molecules may optionally be eluted from the beads using a suitable eluent (bind-elute mode, e.g. for analytical purpose).
  • the eluent is a solution comprising sodium hydroxide (NaOH) and/or a solvent.
  • the porosity of the beads (of the core-bead flowthrough chromatography material), in particular the porosity of the inner porous core and the outer porous shell, does not allow entering of the DNA, in particularthe in vitro amplified DNA, into the inner porous core of the beads.
  • the porosity of the beads in particular the porosity of the inner porous core and the outer porous shell, does allow entering of impurities (e.g. proteins (DNA polymerase), DNA primers) into the inner porous core of the beads.
  • impurities e.g. proteins (DNA polymerase), DNA primers
  • the pore size of the beads is usually stated in kDa and refers to the average molecular mass of the smallest particle the matrix is likely to reject (also referred to as MWCO).
  • the pore size of the beads can be stated in pm and refers to the diameter of the smallest particle the matrix is likely to reject.
  • the inventors have found thatforthe purification of the target DNA a certain MWCO/pore is useful.
  • the pore size as defined herein is selected so that the cut-off is below the target DNA size (e.g. larger than 500 nucleotides) but above protein (e.g. DNA polymerase) or DNA primer size.
  • DNA species can be purified that are larger than the molecular cut-off of the beads.
  • the target DNA species, in particularthe in vitro amplified DNA is the largest molecule in the impure preparation to be purified.
  • the porous beads (of the core-bead flowthrough chromatography material) have a molecular weight cut-off of below 700kDa, below 600kDa, or below 500kDa.
  • the porosity of the beads (of the core-bead flowthrough chromatography material) does allow entering of (globular) molecules smaller than about 700kDa, about 600kDa, about 500kDa.
  • the core-bead flowthrough chromatography material used in step A has a molecular weight cut-off of about 400kDa, in other words, the porosity of the beads (of the core-bead flow through chromatography material) does not allow entering of molecules larger than about 400kDa but does allow entering of molecules smaller than about 400kDa.
  • each matrix layer of the core-bead flow through chromatography material may be functionalised with at least one ligand, or it may not be functionalised.
  • the layers can be distinguished from each other by the presence or absence of at least one ligand.
  • the porous beads (of the core-bead flowthrough chromatography material) have an inner porous core that is functionalized with a ligand that allows binding of impurities that enter the inner porous core of the beads.
  • the ligand is a ligand for anion exchange and/or hydrophobicity interactions (with an impurity). Accordingly, at least one ligand is a ligand that has multiple functionalities, for example the ligand is both hydrophobic and positively charged.
  • the ligand is selected from an amine, preferably selected from a monoalkylamine, more preferably from a mono-(C1-C8)alkyl-amine, even more preferably wherein the ligand is octylamine (CH3(CH2)7NH2).
  • the porous beads (of the core-bead flowthrough chromatography material) have an outer porous shell that is not functionalized with a ligand as defined herein.
  • the porous beads comprise a synthetic (porous) polymer material, such as styrene or styrene derivatives, divinylbenzene, acrylamides, acrylate esters, methacrylate esters, vinyl esters and vinylamides, or a natural polymer material, such as carbohydrate material selected from agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan and alginate.
  • a synthetic (porous) polymer material such as styrene or styrene derivatives, divinylbenzene, acrylamides, acrylate esters, methacrylate esters, vinyl esters and vinylamides
  • a natural polymer material such as carbohydrate material selected from agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan and alginate.
  • the porous beads (of the core-bead flowthrough chromatography material) comprise agarose, preferably a cross- linked agarose, more preferably highly cross-linked agarose.
  • the impurities that enter the porous beads and bind to the functionalized porous core of the core-bead flow through chromatography material comprise proteins or enzymes, preferably DNA polymerase, DNA polymerase stabilizers i.e. BSA and gelatine, and DNA primers.
  • the target DNA in particular the in vitro amplified DNA, does not enter the pores, passes through the core-bead flowthrough chromatography material, and is eventually collected in the flowthrough.
  • the DNA-containing impure preparation may optionally be diluted before it is subjected to the core-bead flow through chromatography.
  • the DNA-containing impure preparation may be diluted with a diluent volume that corresponds to about 5-fold, about 4-fold, about 3-fold, about 2-fold, or about 1 -fold of sample volume.
  • Any suitable diluent may be used and will typically be a buffer (e.g. a TE buffer).
  • the core-bead flowthrough chromatography includes a step of priming or washing the core-bead flowthrough chromatography material using a buffer preferably comprising a chelating agent (e.g., EDTA) and a buffer agent (e.g., Tris) before subjecting the impure preparation.
  • a buffer preferably comprising a chelating agent (e.g., EDTA) and a buffer agent (e.g., Tris) before subjecting the impure preparation.
  • the impure preparation is subjected to the core-bead flow through chromatography subsequent to the priming or washing step.
  • the buffer used for priming or washing is also used for running the core-bead flow through chromatography.
  • the buffer comprises a buffer agent selected from Tris, Bis-tris-methane, triethanolamine (TEA), imidazole, histidine (e.g. histidine-HCI), citrate (e.g. sodium citrate), MES, MOPS, HEPES, sodium succinate, sodium malate, sodium carbonate, bis(2-hydroxyethyl)amino- tris(hydroxymethyl)methane (Bis-Tris-methane or BTM) and its protonated form, triethanolamine (TEA) and its protonated form, ethyldiethanolamine and its protonated form, 2-(diethylamino)ethan-1-ol and its protonated form, triethylamine and its protonated form, 2-[2-(diethylamino)ethoxy]ethan-1-ol and its protonated form, diethanolamine and its protonated form, N,N'-bis(2-hydroxyethyl)piperazine and its proton
  • the buffer agent is selected from Tris (Tris(hydroxy methyl) aminomethane; Trometamol), in particular, Tris-HCl.
  • the buffer comprises 1 mM to 1 M buffer agent, preferably 1 mM to 50mM buffer agent, more preferably, 10mM to 150mM buffer agent, in particular 10mM buffer agent.
  • the buffer comprises a chelating agent.
  • the chelating agent is selected from EDTA, or a EDTA derivatives.
  • the buffer comprises the chelating agent, preferably EDTA, at a concentration ranging from 0.1 mM to 10OmM, preferably ranging from 0.1 mM to 5mM, more preferably ranging from 0.5mM to 2mM, in particular 1mM.
  • the chelating agent preferably EDTA
  • the buffer has a pH value of between 7.0 and 9.0. In preferred embodiments, the wash buffer has a pH value of about 8.0.
  • the core-bead flowthrough chromatography buffer comprises at least one salt, preferably selected from NaCI, KCI, LiCI.
  • the buffer is a TE buffer comprising 10mM Tris-HCl, and 1 mM EDTA, pH 8.0.
  • the in vitro amplified DNA is a PCR amplified DNA
  • the PCR reaction product is directly subjected to the core-bead flow through chromatography, accordingly, the buffer used for the core-bead flow through chromatography corresponds to the buffer of the PCR reaction product.
  • the buffer used for the core-bead flowthrough chromatography for example the buffer contained in the PCR reaction product, can directly be applied (as a flowthrough) to a subsequent purification step, preferably, to an ion exchange chromatography step.
  • the buffer used for the core-bead flowthrough chromatography is suitably selected from a buffer that does not interfere with any subsequent purification or buffer exchange steps.
  • the buffer used for the core-bead flow through chromatography for example the buffer contained in the PCR reaction product, is compatible with ion exchange chromatography, TFF or HIC. “Compatible” in that context means that the buffer (in the flow through) can directly be used for then respective method or with small adjustments (e.g. adding of a salt or diluting).
  • the core-bead flowthrough chromatography is performed at a flow rate of at least 10pL/min, preferably at least 100pL/min, more preferably at least 1 mL/min.
  • the flow rate is a linear flow rate.
  • the flow rate is controlled using a pump and, optionally, using a flow sensor.
  • the pump is part of a liquid chromatography system (e.g. FPLC) or part of an upstream device (e.g. a pump used for carrying out a PCR step).
  • the core-bead flowthrough chromatography is performed at a pressure of less than 8 bar, less than 5 bar, preferably less than 2 bar.
  • the pressure is controlled using a pump and, optionally, using a pressure sensor.
  • the average diameter (particle size) of the beads of the core-bead flow through chromatography material will be selected so to enable efficient DNA purification with minimal operation time without significantly affecting DNA integrity due to excessive pressures required for performance. Larger particles and larger pores typically allow the use of lower pressures, but the separation efficiency may be reduced.
  • a particle size of about 50-150pm is preferable, wherein a particle size of about 60-120pm is more preferably, and wherein a particle size of about 80-100pm is even more preferable.
  • a particle size of about 90 pm is most preferred.
  • the core-bead flowthrough chromatography material is selected from commercially available core-bead flow through chromatography materials that include, among others, Capto Core 400 or Capto Core 700 (all registered trademark names), or any functional equivalent thereof.
  • the core-bead flowthrough chromatography material is selected from Capto Core 400, or a functional equivalent thereof.
  • a resin is used that is packed with the respective Capto Core material.
  • the flowthrough obtained after the core-bead flow through chromatography comprises a reduced amount of impurities as defined herein, preferably comprises a reduced amount of proteins or enzymes (e.g. DNA polymerase, BSA, gelatine, antibodies for mediating hot-start PCR) and DNA primers, compared to the impure preparation subjected to the core-bead flow through chromatography.
  • proteins or enzymes e.g. DNA polymerase, BSA, gelatine, antibodies for mediating hot-start PCR
  • the flowthrough obtained after the core-bead flow through chromatography comprises a reduced amount of DNA polymerase (or other proteins such as BSA, gelatine, antibodies for mediating hot-start PCR) and DNA primers, compared to the impure preparation (that is the PCR reaction product) subjected to the core-bead flow through chromatography.
  • DNA polymerase or other proteins such as BSA, gelatine, antibodies for mediating hot-start PCR
  • DNA primers compared to the impure preparation (that is the PCR reaction product) subjected to the core-bead flow through chromatography.
  • the flowthrough obtained after the core-bead flow through chromatography is essentially free of proteins or enzymes (e.g. DNA polymerase, BSA, gelatine, antibodies for mediating hot-start PCR) and DNA primers.
  • proteins or enzymes e.g. DNA polymerase, BSA, gelatine, antibodies for mediating hot-start PCR
  • the flowthrough obtained after the core-bead flow through chromatography is essentially free of DNA polymerase (or other proteins such as BSA, gelatine, antibodies for mediating hot-start PCR) and DNA primers, compared to the impure preparation (that is the PCR reaction product) subjected to the core-bead flow through chromatography.
  • DNA polymerase or other proteins such as BSA, gelatine, antibodies for mediating hot-start PCR
  • DNA primers compared to the impure preparation (that is the PCR reaction product) subjected to the core-bead flow through chromatography.
  • “Essentially free” in that context means that the flow through comprises less than DNA polymerase 0.01 U/pL or less than 5pg/mL proteins or enzymes, preferably less than 3pg/ml proteins or enzymes.
  • the flowthrough comprises the in vitro amplified DNA (i.e. the target DNA) in an amount that is not significantly reduced compared to the impure preparation. Accordingly, the purification via core-bead flowthrough chromatography as described herein does not lead to a substantial loss in target DNA.
  • the flow through comprises 80%, 85%, 90%, 95%, 98% or even almost 100% of the in vitro amplified DNA that has been subjected to the purification as part of the impure preparation.
  • the core-bead flowthrough chromatography allows fora purification of DNA (i.e. the target DNA) that has a size of at least 500 nucleotides.
  • the core-bead flow through chromatography allows for a purification of DNA (i.e. the target DNA) that has a size ranging from at least about 500 nucleotides to 10000 nucleotides, preferably ranging from at least about 500 nucleotides to 7000 nucleotides or ranging from at least about 1000 nucleotides to 5000 nucleotides.
  • the flow through obtained after step A has a purity level (with reference to the target DNA) of more than 70%, 80%, 90%. Purity may be determined using analytical RP HPLC, agarose gel electrophoresis, or capillary gel electrophoresis.
  • the vitro amplified DNA preferably the PCR amplified DNA, contained in the pure preparation obtained after step A has an integrity of more than 70%, 80%, 90%. Integrity of DNA may be determined using analytical RP HPLC or capillary gel electrophoresis.
  • step A is performed continuously, preferably wherein the impure preparation comprising in vitro amplified DNA is continuously subjected to the core-bead flow through chromatography.
  • the core-bead flow through chromatography step is flu idically connected with the upstream DNA amplification step.
  • the fluidic connection is preferably facilitated by tubes, channels, or microchannels (depending on the desired scale of the method).
  • the core-bead flow through chromatography of the invention is particularly suitable for purifying in vitro amplified DNA, in particular PCR amplified DNA, to efficiently remove DNA polymerase and DNA primers.
  • the method can be performed in a continuous manner wherein e.g. continuously produced DNA (e.g. PCR product) can be subjected to step A to continuously purify the PCR product.
  • the method can be performed with single use materials which simplifies the method in the context GMP compliance.
  • the core-bead flow through chromatography was able to effectively remove protein contaminants as defined herein and DNA primers.
  • the core-bead flow through chromatography was not able to completely remove dNTPs, even though dNTPs are small, and these impurities should accordingly enter the pores of the core-bead flow through chromatography material.
  • a fraction of dNTPs has been found in the flow-through fraction after performing the core-bead flowthrough chromatography. Accordingly, for some downstream applications, a further step of removing dNTPs via an additional purification step may be needed in particular in embodiments where e.g.
  • PCR amplified DNA is to be purified and/or in embodiments where a complete removal of dNTPs is desired.
  • an additional purification step may be required for the removal of small impurities such as buffer components (betaine, Tetramethylammonium chloride, salts, buffering agents).
  • Such an additional purification step may most efficiently be implemented after the core-bead flow through chromatography step A by subjecting the pre-purified flow through to an additional purification step (herein referred to as step B).
  • Step B Additional purification step
  • the DNA i.e. the target DNA
  • the DNA may be purified using at least one additional purification step B.
  • a method of the invention can comprise one or more additional purification steps.
  • the inventors have found that the implementation of at least one additional purification step enables a fast, scalable purification process for obtaining pure DNA with high yield and is particularly advantageous for removing dNTPs from the desired target DNA along with other potential impurities such as betaine, Tetramethylammonium chloride, salts, buffering agents.
  • the at least one additional purification step B is configured to bind or retain the target DNA contained in the purified flow through of step A and does not bind or retain impurities such as dNTPs and, optionally, further buffer impurities such as betaine, Tetramethylammonium chloride, salts, buffering agents.
  • the at least one additional purification step B is not a core-bead flow through chromatography as defined in the context of step A.
  • the at least one additional purification step B can be carried out in with aqueous buffer systems which in particular means that step B can be carried out in the absence of organic solvents (e.g. ethanol, methanol, isopropanol, acetonitrile).
  • organic solvents e.g. ethanol, methanol, isopropanol, acetonitrile
  • the at least one additional purification step B can be carried out by directly subjecting the purified flowthrough of step A.
  • the at least one additional purification step B can be carried out using single use materials.
  • the at least one additional the purification step B is selected from ion exchange chromatography (in particular AEX), Hydrophobic interaction chromatography (HIC), Tangential flow filtration (TFF), Hydrophilic interaction liquid chromatography (HILIC), size exclusion chromatography (SEC), RP-HPLC, hydroxyapatite chromatography, filtration, precipitation, affinity purification, or any combination thereof.
  • ion exchange chromatography in particular AEX
  • HIC Hydrophobic interaction chromatography
  • THF Tangential flow filtration
  • HILIC Hydrophilic interaction liquid chromatography
  • SEC size exclusion chromatography
  • RP-HPLC hydroxyapatite chromatography
  • the at least one additional purification step B is selected from at least one ion exchange chromatography step, at least one hydrophobic interaction chromatography (HIC) step, or at least one tangential flow filtration (TFF) step, or any combinations thereof.
  • step B may comprise one or more ion exchange chromatography steps and one or more TFF steps.
  • step B may comprise one or more ion exchange chromatography steps and one or more TFF steps and one or more HIC steps.
  • step B may comprise one or more ion exchange chromatography steps and one or more HIC steps.
  • step B may comprise one or more TFF steps and one or more HIC steps.
  • the at least one additional purification step B is selected from ion exchange chromatography.
  • the DNA may be purified using at least one step of ion exchange chromatography (as step B).
  • a method of the invention can comprise one or more ion exchange chromatography steps.
  • the inventors have found that the implementation of at least one ion exchange chromatography step (e.g. an AEX step) enables a fast, scalable purification process for obtaining pure DNA with high yield and is particularly advantageous for removing dNTPs from the desired target DNA along with other potential impurities such as betaine, Tetramethylammonium chloride, salts, buffering agents.
  • the method of purifying DNA comprises at least one step of core-bead flowthrough chromatography (step A) and at least one ion exchange chromatography step (the at least one additional purification step B).
  • the method of purifying DNA comprises at least one step of core-bead flow through chromatography (step A) to provide a flow through that is then subjected to at least one ion exchange chromatography (the at least one additional purification step B).
  • the ion exchange chromatography is performed by contacting the DNA (contained in the flowthrough), preferably the in vitro amplified DNA, with an ion exchange material.
  • the ion exchange chromatography is performed by contacting the purified flow through obtained after step A comprising in vitro amplified DNA, preferably the purified flow through comprising PCR amplified DNA, with an ion exchange material.
  • the flow through obtained after step A has already been purified by core-bead flow through chromatography as defined herein, wherein in particular peptide or protein impurities such as DNA polymerase and DNA primers have been removed.
  • the flowthrough obtained after step A may still comprise at least one impurity, preferably dNTPs.
  • contacting the DNA, preferably the in vitro amplified DNA, with an ion exchange material is performed under conditions to allow binding of the target DNA to the ion exchange material and does not allow binding of impurities (e.g. dNTPs).
  • impurities e.g. dNTPs
  • the in vitro amplified DNA is contacted with the ion exchange material in the presence of a solution comprising a salt at a concentration that allows selective binding of the vitro amplified DNA of interest to the ion exchange material.
  • contacting the purified flow through obtained after step A with an ion exchange material is performed under conditions to allow binding of the in vitro amplified DNA (i.e. the target DNA) to the ion exchange material and does not allow binding of impurities (e.g. dNTPs).
  • impurities e.g. dNTPs
  • the impurities that do not bind to the ion exchange material comprise dNTPs and, optionally, PCR buffer components such as e.g. betaine, Tetramethylammonium chloride, salts, buffering agents
  • PCR buffer components such as e.g. betaine, Tetramethylammonium chloride, salts, buffering agents
  • the ion exchange chromatography is selected from an anion exchange chromatography (AEX).
  • AEX chromatography is a method of purification that leverages ionic interaction between positively charged material/sorbents and negatively charged molecules.
  • AEX sorbents typically include a charged functional group cross-linked to solid phase media.
  • AEX chromatography is typically characterized by loading a preparation to be purified onto the AEX material in a buffer comprising a salt/buffer with low ionic strength, i.e. at rather low salt concentrations. These conditions allow highly polar and/or charged molecules (e.g. DNA) to bind to the charged groups on the AEX material, while the components having lower polarity I hydrophilicity (e.g. certain impurities such as dNTPs) will not bind under such conditions.
  • highly polar and/or charged molecules e.g. DNA
  • the components having lower polarity I hydrophilicity e.g. certain impurities such as dNTPs
  • the molecule of interest (e.g., the target DNA) is then typically eluted from the column by successively increasing the concentration of the salt/buffer so that the increased number of ions compete with the binding of the molecules still bound to the AEX material.
  • a gradient increasing the ionic strength of the elution buffer is typically used for purifications via AEX chromatography.
  • strong and weak anion exchangers or exchange groups there are two categories of anion exchange media, “strong” and “weak” exchangers or exchange groups.
  • the classification of strong and weak anion exchangers refers to the ionization of the ability of the charged functional groups to ionize in response to a change in pH. Strong anion exchangers maintain the same charge density over a broad pH range (e.g. a pH range from 2 to 12, from 2 to 11 , from 3 to 9), while weak anion exchangers exhibit charge densities which vary with changes in pH. As a result, the binding capacity and selectivity of weak exchangers can be manipulated by the pH of the mobile phase.
  • Anion exchange sorbents or resins facilitate DNA capture due to the interaction with the negatively charged phosphate backbone of the DNA, providing an effective mode of separation.
  • the ion exchange chromatography is performed using a material that comprise a positively charged functional group linked to solid phase media.
  • the solid phase media is a strong anion exchanger (e.g., maintain a positive charge over a broad pH range, such as over pH 3 to pH 9), such as a quaternary amine.
  • a weak anion exchanger can be used, such as a polyethyleneimine, a diethylaminomethyl, a dimethylaminopropyl, or a polyallylamine.
  • the ion exchange chromatography is performed with a material functionalized with a weak ion exchange ligand (e.g. primary amine) or a strong ion exchange ligand (e.g. quaternary ammonium).
  • a weak ion exchange ligand e.g. primary amine
  • a strong ion exchange ligand e.g. quaternary ammonium
  • Suitable AEX materials that may be used in the context of the invention are for example available as an anion exchange membrane, an anion exchange resin, a three-dimensional microporous hydrogel structure, a packed bed of superporous beads, macroporous beads, a monolith, agarose beads, cross-linked agarose, silica beads, large pore gels, methacrylate-based beads, polystyrene-based beads, cellulose-based beads, dextran-based beads, bisacrylamide-based beads, polyvinylether- based beads, ceramic-based beads, polymer-based beads, Polyethersulfon membranes or cellulose-based membranes.
  • AEX materials examples include, among others, Sartobind Q®, Mustang Q®, Mustang E®, Poros XQ, Poros HQ, Nuvia Q, Capto Q, Bakerbond PolyQuat, DEAE Sepharose, NH2-750F, Q Sepharose, Giga Cap Q-650M, Fractogel EMD COO, NatriFlo HD-Q, and 3MTM EmphazeTM AEX Hybrid Purifier, CIMmultus SO3, CIMmultus QA, CIMmultus EDA, CIMmultus EV (all registered trademark names).
  • the ion exchange chromatography is performed with an ion exchange material selected from a bead, resin, monolith, or membrane.
  • the ion exchange chromatography is performed with an ion exchange membrane, preferably an AEX membrane.
  • the ion exchange chromatography is performed with a membrane adsorber, preferably a membrane adsorber functionalized with quaternary ammonium.
  • the membrane comprises cellulose or PES or a derivative thereof.
  • Particularly preferred are PES membrane adsorber materials.
  • PES membrane adsorber materials are Sartobind Q 15 and Sartobind Q nano.
  • the anion exchange membrane has an average pore size of about 3.0pm to 5.0pm.
  • AEX monolith e.g. methacrylate based Convective Interaction Media (CIM)) functionalized with a weak anion exchanger or a strong anion exchanger.
  • a methacrylate polymer e.g. methacrylate based Convective Interaction Media (CIM)
  • CIM Convective Interaction Media
  • the ion exchange chromatography comprises a step of loading the DNA, preferably loading the purified flow through of step A, onto the ion exchange chromatography material as defined herein, in particular the AEX chromatography material as defined herein.
  • Loading conditions for the AEX chromatography can be performed under denaturing conditions, partially denaturing conditions, or non-denaturing conditions.
  • loading of the DNA to the ion exchange chromatography is performed under conditions of low ionic strength, and subsequently the bound DNA is eluted by (gradually or stepwise) increasing salt concentration/ionic strength of the elution buffer.
  • loading of the DNA to the ion exchange chromatography is performed using a buffer system having a low ionic strength that preferably comprises a chelating agent (e.g. EDTA) and a buffer agent (e.g. Tris).
  • a chelating agent e.g. EDTA
  • a buffer agent e.g. Tris
  • the purified flow through obtained from the core-bead flow through chromatography step A can be directly used and loaded to the ion exchange chromatography of step B.
  • the purified flow through obtained from the core-bead flow through chromatography step A is diluted and/or re-buffered before loading to the ion exchange chromatography of step B.
  • a dilution step may e.g. be performed by a concurrent dilution step (e.g. to dilute the purified flow through of step A).
  • a dilution step re-buffering step may be performed using filtration orTFF.
  • the ion exchange chromatography step B is carried out in a “bind-elute mode”, in which the target DNA remains bound to the ion exchange material until eluted during an elution step.
  • the ion exchange chromatography comprises a step of eluting the bound DNA (in particular, the in vitro amplified target DNA) to obtain a pure preparation of DNA in particular, the in vitro amplified target DNA).
  • the eluting is performed using an eluent or salt buffer, preferably a high salt buffer.
  • the eluting is performed using a high salt buffer, preferably wherein the high salt buffer comprises at least 1 M, 1 ,5M, 2M, 3.5mM or 5mM salt.
  • the salt is selected from a chaotropic salt.
  • a chaotropic salt is a molecule in water solution that can disrupt the hydrogen bonding network between water molecules (i.e. exerts chaotropic activity). This has an effect on the stability of the native state of other molecules in the solution, mainly macromolecules (proteins, nucleic acids) by weakening the hydrophobic effect.
  • a chaotropic salt reduces the amount of order in the structure of a protein formed by water molecules, both in the bulk and the hydration shells around hydrophobic amino acids, and may cause its denaturation.
  • the chaotropic salt does not cause a denaturing of the DNA.
  • the salt is preferably the chaotropic salt and may be selected from ammonium chloride, potassium chloride, sodium chloride, magnesium sulphate, magnesium chloride, magnesium nitrate, guanidinium hydrochloride, or mixtures thereof.
  • the salt is NaCI.
  • the pure preparation of DNA obtained after step B may be present in a buffer comprising a chaotropic salt, such as NaCI, as defined herein.
  • a chaotropic salt such as NaCI
  • this pure preparation of DNA obtained after step B comprising the salt can directly be used for DNA immobilization or diluted to the desired salt concentration.
  • the ion exchange chromatography comprises one or more steps of washing off impurities such as dNTPs using a wash buffer.
  • the washing steps are typically performed after the DNA has been loaded to the ion exchange material and removes weakly bound impurities such as dNTPs.
  • the wash buffer is a salt buffer that preferably comprises less than or about 500mM salt, preferably wherein the salt is selected from NaCI.
  • the ion exchange chromatography is performed at a flow rate of at least 0.5 mL/min, preferably at least 1 mL/min, more preferably at least 3mL/min (e.g. between 2ml_/min and 15mL/min).
  • the flow rate is a linear flow rate.
  • the flow rate is controlled using a pump and, optionally, using a flow sensor.
  • the pump is part of a liquid chromatography system (e.g. FPLC) or part of an upstream device (e.g. a pump used for carrying out a PCR step).
  • a liquid chromatography system e.g. FPLC
  • an upstream device e.g. a pump used for carrying out a PCR step.
  • the ion exchange chromatography is performed at a pressure of less than 0.5 bar, e.g. about 0 bar.
  • the pressure is controlled using a pump and, optionally, using a pressure sensor.
  • the ion exchange chromatography step concentrates the DNA preferably by at least 1 .5-fold , 2-fold, 2.5-fold. Accordingly, the DNA concentration of the target DNA in the pure preparation obtained afterthe ion exchange chromatography step B may be increased by at least 1 .5-fold , 2-fold, 2.5-fold compared to the concentration of the target DNA in the purified flow through after step A.
  • the at least one additional purification step B is selected from hydrophobic interaction chromatography (HIC).
  • HIC hydrophobic interaction chromatography
  • Hydrophobic interaction chromatography is based on the hydrophobic interaction between hydrophobic moieties bound to a support material and hydrophobic regions of the molecule that binds to the matrix such as the target DNA. Binding is achieved at high salt concentrations and the molecule (the target DNA) is eluted from the matrix by decreasing the salt concentration and, optionally, the addition of organic solvents.
  • the DNA i.e. the target DNA
  • the DNA may be purified using at least one step of HIC (as step B).
  • a method of the invention can comprise one or more HIC steps.
  • the inventors have found that the implementation of at least one HIC step enables a fast, scalable purification process for obtaining pure DNA with high yield and is particularly advantageous for removing dNTPs from the desired target DNA along with other potential impurities such as betaine, Tetramethylammonium chloride, salts, buffering agents.
  • the method of purifying DNA comprises at least one step of core-bead flow through chromatography (step A) and at least one HIC step (the at least one additional purification step B).
  • the method of purifying DNA comprises at least one step of core-bead flow through chromatography (step A) to provide a flow through that is then subjected to at least one HIC step (the at least one additional purification step B).
  • the HIC is performed by contacting the DNA (contained in the flowthrough), preferably the in vitro amplified DNA, with a Hydrophobic interaction chromatography material. Accordingly, in preferred embodiments, the HIC is performed by contacting the purified flow through obtained after step A comprising in vitro amplified DNA, preferably the purified flow through comprising PCR amplified DNA, with a HIC material.
  • the flow through obtained after step A has already been purified by core-bead flow through chromatography as defined herein, wherein in particular peptide or protein impurities such as DNA polymerase and DNA primers have been removed.
  • the flowthrough obtained after step A may still comprise at least one impurity, preferably dNTPs.
  • contacting the purified flow through obtained after step A with a HIC material is performed under conditions to allow binding of the in vitro amplified DNA (i.e. the target DNA) to the HIC material and does not allow binding of impurities (e.g. dNTPs).
  • impurities e.g. dNTPs
  • the impurities that do not bind to the HIC material comprise dNTPs and, optionally, PCR buffer components such as e.g. betaine, Tetramethylammonium chloride, salts, buffering agents
  • the HIC is performed with a material functionalized with a weak hydrophobic ligand or a strong hydrophobic ligand.
  • a hydrophobic ligand may be selected from alkyl-, aryl-ligands, and combinations thereof.
  • hydrophobic ligands may be selected from butyl, hexyl, phenyl, octyl, or polypropylene glycol ligands.
  • the HIC media is selected from the group consisting of CaptoPhenyl, Phenyl SepharoseTM 6 Fast Flow with low or high substitution, Phenyl SepharoseTM High Performance, Octyl SepharoseTM High Performance, FractogelTM EMD Propyl, FractogelTM EMD Phenyl, Macro-PrepTM Methyl, Macro-PrepTM t-Butyl, WP Hll-Propyl (C3)TM, ToyopearlTM ether, ToyopearlTM phenyl, ToyopearlTM butyl, ToyoScreen PPG, ToyoScreen Phenyl, ToyoScreen Butyl, ToyoScreen Hexyl, HiScreen Butyl FF, HiScreen Octyl FF, Tosoh Hexyl, CIMmulus-OH, CIMmulus-C4-HLD.
  • the HIC is performed with a HIC material selected from a bead, resin, monolith, or membrane.
  • the HIC media is a column.
  • the method may be carried out using a monolith.
  • a monolith may be composed of a methacrylate polymer (e.g. methacrylate based Convective Interaction Media (CIM)) functionalized with a hydrophobic ligand as defined herein.
  • CIM Convective Interaction Media
  • the ion exchange chromatography comprises a step of loading the DNA, preferably loading the purified flowthrough of step A, onto the HIC material as defined herein, in particular the HIC chromatography material as defined herein.
  • Loading conditions for the HIC chromatography can be performed under denaturing conditions, partially denaturing conditions, or non-denaturing conditions.
  • loading of the DNAto the HIC is performed under conditions of high ionic strength, and subsequently the bound DNA is eluted by (gradually or stepwise) decreasing salt concentration/ionic strength of the elution buffer.
  • the purified flow through obtained from the core-bead flow through chromatography step A is re-buffered before loading to the HIC of step B.
  • a re-buffering may e.g. be performed by adding a salt (e.g. to increase the ionic strength of the purified flow through of step A).
  • a re-buffering step may be performed using filtration orTFF.
  • the loading or binding of the DNA is performed using a high salt buffer, preferably wherein the high salt buffer comprises at least 750mM, 1 M, 1 ,5M, 2M, 3.5M or 5M salt, preferably wherein the salt is selected from a chaotropic salt.
  • the HIC comprises a step of eluting the bound DNA (in particular, the in vitro amplified target DNA) to obtain a pure preparation of DNA in particular, the in vitro amplified target DNA.
  • the eluting is performed using an eluent or salt buffer, preferably a low salt buffer.
  • the eluting is performed using a low salt buffer, preferably wherein the low salt buffer comprises less than 5M, 3.5M, 2M, 1 ,5M, 1M, 750mM salt.
  • the salt is selected from a chaotropic salt, preferably NaCI.
  • the pure preparation of DNA obtained after step B may be present in a buffer comprising a chaotropic salt, such as NaCI, as defined herein.
  • a chaotropic salt such as NaCI
  • this pure preparation of DNA obtained after step B comprising the salt can directly be used for DNA immobilization or may be adjusted to the desired salt concentration.
  • the HIC comprises one or more steps of washing off impurities such as dNTPs using a wash buffer.
  • the washing steps are typically performed after the DNA has been loaded to the ion exchange material and removes weakly bound impurities such as dNTPs.
  • the HIC is performed at a flow rate of at least 0.5 mL/min, preferably at least 4ml_/min, more preferably at least 8ml_/min.
  • the flow rate is a linear flow rate.
  • the flow rate is controlled using a pump and, optionally, using a flow sensor.
  • the pump is part of a liquid chromatography system (e.g. FPLC) or part of an upstream device (e.g. a pump used for carrying out a PCR step).
  • a liquid chromatography system e.g. FPLC
  • an upstream device e.g. a pump used for carrying out a PCR step.
  • the HIC is performed at a pressure of less than 20bar.
  • the pressure is controlled using a pump and, optionally, using a pressure sensor.
  • the at least one additional purification step B is selected from tangential flow filtration.
  • Tangential Flow Filtration is a type of filtration.
  • TFF is different from dead-end filtration in which the feed is passed through a membrane or bed, the solids being trapped in the filter and the filtrate being released at the other end.
  • TFF gets its name because the majority of the feed flow travels tangentially across the surface of the filter, rather than into the filter.
  • the principal advantage of this is that the filter cake (which can blind the filter) is substantially washed away during the filtration process, increasing the length of time that a filter unit can be operational. It can be a continuous process, unlike batch-wise dead-end filtration.
  • This type of filtration is typically selected for feeds containing a high proportion of small particle size solids (where the permeate is of most value) because solid material can quickly block (blind) the filter surface with dead-end filtration.
  • Applied pressure causes one portion of the flow stream to pass through the membrane (filtrate/permeate) while the remainder (retentate) is recirculated back to the feed reservoir.
  • the primary applications for TFF are concentration, diafiltration (desalting and buffer/solvent exchange), and fractionation of large from small biomolecules.
  • TFF Membranes with different molecular weight cut-offs (MWCO) may be used for TFF.
  • MWCO molecular weight cut-offs
  • ultrafiltration membranes are preferably used for TFF.
  • the DNA i.e. the target DNA
  • the DNA may be purified using at least one step of TFF (as step B).
  • a method of the invention can comprise one or more TFF steps.
  • the inventors have found that the implementation of at least one TFF step enables a fast, scalable purification process for obtaining pure DNA with high yield and is particularly advantageous for removing dNTPs from the desired target DNA along with other potential impurities such as betaine, Tetramethylammonium chloride, salts, buffering agents.
  • the method of purifying DNA comprises at least one step of core-bead flowthrough chromatography (step A) and at least one TFF step (the at least one additional purification step B).
  • the method of purifying DNA comprises at least one step of core-bead flow through chromatography (step A) to provide a flow through that is then subjected to at least one TFF step (the at least one additional purification step B).
  • the flow through obtained after step A has already been purified by core-bead flow through chromatography as defined herein, wherein in particular peptide or protein impurities such as DNA polymerase and DNA primers have been removed.
  • the flowthrough obtained after step A may still comprise at least one impurity, preferably dNTPs.
  • the TFF is performed under conditions to allow retaining of the in vitro amplified DNA and removing impurities, preferably wherein the impurities comprise dNTPs and, optionally, PCR buffer components.
  • TFF is performed to concentrate and/or re-buffer and/or condition the purified flow through obtained after step A.
  • any desired buffer for the re-buffering may be chosen e.g. a buffer that is suitable for any downstream process (e.g. a buffer for DNA immobilization or a buffer for RNA in vitro transcription).
  • TFF may be carried out using any suitable filter membrane or system.
  • TFF may be carried out using a TFF hollow fiber module or a TFF membrane cassette.
  • the TFF is performed using a membrane cassette or a hollow fibre module. In particularly preferred embodiments, TFF is performed using a membrane cassette.
  • the molecular weight cut-off of the filter membrane may be selected depending on the size of the DNA, particularly the in vitro amplified DNA. The larger the target DNA, the higher the molecular weight cut-off of the membrane may be selected. In a preferred embodiment, the molecular weight cut-off of the filter membrane is ⁇ 500 kDa, preferably ⁇ 300 kDa and most preferably ⁇ 100 kDa.
  • the TFF is performed with a TFF material that has a MWCO of less than 500kDa, preferably of 300kDa or less than 300kDa, more preferably ⁇ 100kDa or ⁇ 50kDa.
  • the filter membrane of the TFF material may comprise any suitable filter material, e.g. polyethersulfone (PES), modified polyethersulfone (mPES), polysulfone (PS), modified polysulfone (mPS), ceramics, polypropylene (PP), cellulose, regenerated cellulose or a cellulose derivative e.g. cellulose acetate or combinations thereof.
  • PES polyethersulfone
  • mPES modified polyethersulfone
  • PS polysulfone
  • mPS modified polysulfone
  • ceramics e.g. polypropylene (PP), cellulose, regenerated cellulose or a cellulose derivative e.g. cellulose acetate or combinations thereof.
  • the TFF is performed with a TFF membrane cassette that preferably comprises a cellulose (or regenerated cellulose or a cellulose derivative) or PES membrane.
  • the TFF is performed with a TFF membrane cassette that preferably comprises a cellulose (or regenerated cellulose or a cellulose derivative) membrane
  • a cellulose-based membrane (cellulose, regenerated cellulose or a cellulose derivative) with a MWCO of ⁇ 1 OOkDa or ⁇ 300kDa.
  • the DNA membrane load of the TFF membrane (during to process of TFF) is about 0.1 to about 10 mg/cm 2 and preferably from about 0.5 to about 2 mg/cm 2 . In a preferred embodiment, the DNA membrane load of the TFF membrane is about 0.1 to 0.6 mg/cm2.
  • the feed flow rate in the at least one step of TFF in step B is 500 to 1 .500 l/h/m2, preferably 600 to 1 .200 l/h/m2, more preferably 700 to 1 .000 l/h/m2.
  • the at least one TFF step concentrates the DNA preferably by at least 1 .5-fold, 2- fold, 2.5-fold.
  • the method of purifying DNA comprises the steps A) purifying an impure preparation comprising in vitro amplified DNA by at least one step of core-bead flowthrough chromatography to provide a purified flowthrough; and B) purifying the flowthrough of step A by at least one additional purification step to obtain a pure preparation of DNA, in particular in vitro amplified DNA.
  • the pure preparation comprises a reduced amount of impurities, preferably comprises a reduced amount of proteins or enzymes (e.g. DNA polymerase, BSA, gelatine, antibodies for hot-start function), DNA primers, and dNTPs compared to the impure preparation or compared to the purified flow through obtained after step A.
  • the pure preparation comprises a reduced amount of dNTPs compared to the impure preparation or compared to the purified flow through obtained after step A.
  • the pure preparation is essentially free of proteins or enzymes (e.g. DNA polymerase, BSA, gelatine, antibodies for hot-start function), DNA primers, dNTPs, and PCR buffer component.
  • proteins or enzymes e.g. DNA polymerase, BSA, gelatine, antibodies for hot-start function
  • DNA primers e.g. DNA polymerase, BSA, gelatine, antibodies for hot-start function
  • dNTPs e.g. DNA polymerase, BSA, gelatine, antibodies for hot-start function
  • PCR buffer component e.g. DNA polymerase, BSA, gelatine, antibodies for hot-start function
  • the pure preparation is essentially free of dNTPs.
  • the pure preparation has a purity of more than 70%, 80%, 90%. Accordingly, the vitro amplified DNA contained in the pure preparation has a purity level of more than 70%, 80%, 90%. DNA purity may be determined using RP HPLC.
  • the vitro amplified DNA contained in the pure preparation has an integrity of more than 60%, 70%, 80%. Integrity of DNA may be determined using analytical RP HPLC agarose gel electrophoresis, or capillary gel electrophoresis.
  • the vitro amplified DNA contained in the pure preparation is suitable for use in a GXP compliant manufacturing of a medicament, preferably the manufacturing of an RNA-based medicament.
  • the purification method of the invention comprises the steps
  • step B purifying the flow through of step A by at least one step of ion exchange chromatography or TFF to obtaining a pure preparation of in vitro amplified DNA.
  • the purification method of the invention is preferably a method of purifying a PCR reaction product and comprises the steps
  • step B purifying the flow through of step A by at least one step of ion exchange chromatography or TFF to remove dNTPs thereby obtaining a pure preparation of in vitro amplified DNA.
  • the purification method of the invention is a method of purifying a PCR reaction product and comprises the steps A) purifying an impure preparation comprising PCR-amplified DNA by at least one step of core-bead flow through chromatography to remove DNA polymerase and DNA primers thereby providing a purified flow through; and
  • step B purifying the flow through of step A by at least one step of ion exchange chromatography, preferably AEX, to remove dNTPs thereby obtaining a pure preparation of in vitro amplified DNA.
  • the purification method of the invention (including step A and B) can be used for purifying a PCR-amplified templates for RNA in vitro transcription.
  • the purification method of the invention does not comprise a step of DNA precipitation.
  • the purification method of the invention does not comprise a purification method or precipitation step using an organic solvent.
  • the purification method of the invention allows for a purification of DNA (i.e. the target DNA) that has a size of at least 500 nucleotides.
  • the purification allows for a purification of DNA (i.e. the target DNA) that has a size ranging from at least about 500 nucleotides to 10000 nucleotides, preferably ranging from at least about 500 nucleotides to 7000 nucleotides, even more preferably ranging from at least about 1000 nucleotides to 5000 nucleotides.
  • the purified flow through of step A is directly subjected to the at least one additional purification step B.
  • the purification method of the invention is performed continuously or semi-continuously. In preferred embodiments, the purification method of the invention (including step A and B) can be carried out continuously. Suitably, the purification method of the invention (including step A and B) can be carried out in an automated manner.
  • the impure preparation is continuously subjected to the core-bead flow through chromatography of step A and continuously or as a batch subjected to the at least one additional purification step B.
  • the in vitro DNA amplification step (e.g. the PCR amplification) is fluidically connected to step A.
  • step A is fluidically connected to step B.
  • the vitro DNA amplification step preferably the PCR amplification step, is fluidically connected to step A, and step A is optionally fluidically connected to step B.
  • the fluidic connection is preferably facilitated by tubes, channels, microchannels, or microfluidic channels (depending on the desired scale of the method).
  • the purification method of the invention is scalable from DNA yields ranging from about 1 pg purified DNA to about 1000g purified DNA, preferably ranging from about 100pg purified DNA to about 100g purified DNA or from about 1 mg purified DNA to about 10g purified DNA.
  • the purification method of the invention is used in a microfluidics scale. 1
  • the purification method of the invention (including step A and B) can advantageously be performed under aqueous conditions, so methods of the invention do not require the use of organic solvents and are ideally performed without the use of organic solvents. Methods of the invention are therefore ideally performed without the use of acetonitrile, chloroform, phenol and/or methanol. Ideally, they can be performed without using any organic solvents.
  • the purification method of the first aspect can advantageously be performed under GMP conditions in the production process of medicaments, e.g. RNA-based medicaments.
  • the purification method of the invention can be performed using single-use materials (chromatography materials, tubes, optionally pumps) which is advantageous in the context of a pharmaceutical production as laborious cleaning procedures after completion of the purification can be avoided.
  • the present invention provides a method for producing DNA.
  • the method of producing DNA comprises at least one step of purifying DNA
  • target DNA The DNA intended to be produced and purified by the method of the invention is herein also referred to as “target DNA”.
  • the method of producing DNA preferably the method of producing in vitro amplified DNA, comprises the steps
  • the method of producing DNA preferably the method of producing in vitro amplified DNA, comprises the steps
  • the in vitro DNA amplification step comprises or consists of polymerase chain reaction (PCR) as defined herein, rolling circle amplification as defined herein, or isothermal amplification as defined herein.
  • PCR polymerase chain reaction
  • the in vitro DNA amplification step is PCR.
  • the PCR is performed using a suitable PCR master-mix.
  • the PCR master-mix does not interfere with the downstream purification step A as defined herein.
  • the PCR master-mix used for preparing the in vitro amplified DNA lacks compounds that have a negative effect on step A of the method defined in the context of the first aspect.
  • a typical PCR master-mix may comprise:
  • DNA Polymerase e.g. hot-start DNA Polymerase
  • a storage matrix for the DNA polymerase such as gelatine and/or BSA and/or glycerol
  • reaction buffer which typically comprises T ris
  • a DNA amplification template e.g. plasmid DNA
  • the polymerase chain reaction comprises a step of preparing a PCR master-mix by mixing a first solution comprising DNA primers (e.g. biotinylated DNA primers) and DNA amplification template with a second solution comprising PCR buffer components (e.g. DNA polymerase) using a mixing means.
  • the mixing is performed via pumps (e.g. a syringe pump, pulseless flow pump, peristaltic pump, microfluidics pump, or the like).
  • the mixing means is selected from a static mixer or a dynamic mixer.
  • a static mixer is used that may be selected from a T-piece or a Y-piece.
  • That process may be controlled by flow sensors. Moreover, the temperature of the first solution and second solution as well as the PCR master-mix may be controlled and/or regulated. Dynamic or static mixing can be achieved by a single pump controlled by a flow meter, or two separate pumps controlled by individual flow meters.
  • the step of preparing a PCR master-mix by mixing as outlined herein reduces side-products (e.g. unspecific binding of DNA primers to the DNA amplification template) and improves the stability of the PCR master-mix.
  • the step of preparing a PCR master-mix by mixing may be performed in a continuous manner, and the freshly produced PCR master-mix may be used in a continuous PCR process for preparing DNA.
  • the PCR buffer comprises a DNA polymerase, optionally a hot-start DNA polymerase.
  • the PCR buffer lacks antibodies or aptamers for mediating hot-start PCR.
  • antibodies are used to optimize the reaction. When such an antibody is bound to the DNA polymerase, the enzyme is rendered inactive. It provides an antibody-mediated hot-start that enhances the specificity and sensitivity of PCR.
  • antibodies for mediating hot-start PCR are not included, in particular in embodiments where the PCR master-mix is produced by mixing as defined above which reduces the formation of unwanted side-products. Not using an antibody has the advantage of reducing further impurities which is particularly important in the context of a pharmaceutical DNA production.
  • preparing the PCR master-mix as described herein allows a hot-start PCR without including an antibodies or aptamers for mediating hot-start PCR.
  • the prepared PCR master-mix is directly introduced into a PCR device, preferably a PCR device for continuous PCR.
  • a PCR device for continuous PCR.
  • continuous capillary PCR is preferred.
  • the PCR master-mix preparation is fluidically connected to the vitro DNA amplification step, preferably the PCR amplification step.
  • the fluidic connection is preferably facilitated by tubes, channels, microchannels, or microfluidic channels (depending on the desired scale of the method).
  • the PCR is performed using at least one biotinylated DNA primer so that the amplified DNA is biotinylated. This is particularly important if the produced PCR amplified DNA is used in immobilized form as a template for RNA in vitro transcription as further specified herein.
  • the DNA preparation obtained in step (i) is directly transferred to the purifying of step (ii) preferably via a tube, capillary, or microfluidic channel.
  • the transferring is performed under controlled flow rates and/or pressure.
  • pumps e.g. a syringe pump, pulseless flow pump, peristaltic pump, microfluidics pump, or the like
  • the transfer/pump is performed at controlled flow rates, wherein the flow rates are optionally controlled using a flow sensor.
  • the in vitro DNA amplification step (e.g. the PCR amplification) is fluidically connected to the purification method as defined in the context of the first aspect. Accordingly, the in vitro DNA amplification step (e.g. the PCR amplification) is fluidically connected to step A as defined herein, and step A is optionally fluidically connected to step B as defined herein.
  • the PCR master-mix preparation is fluidically connected to the vitro DNA amplification step, preferably the PCR amplification step, and the in vitro DNA amplification step (e.g. the PCR amplification) is fluidically connected to step A as defined herein, and step A is optionally fluidically connected to step B as defined herein.
  • the fluidic connection is preferably facilitated by tubes, channels, microchannels, or microfluidic channels (depending on the desired scale of the method).
  • the DNA i.e. the target DNA
  • the DNA is a DNA template for RNA in vitro transcription.
  • the DNA sequence may be transcribed into an RNA sequence (as further specified in the context of the third aspect). Therefore, the DNA template for RNA in vitro transcription may comprise certain sequence elements, depending on which type of RNA is to be produced.
  • the DNA template codes for (that is, serves as a template for) any type of therapeutic RNA, preferably an mRNA, a replicon RNA, or a circular RNA.
  • the DNA template for RNA in vitro transcription may comprise a promoter sequence for an RNA polymerase (e.g. T7 promoter, SP6 promoter), a Kozak sequence, UTR sequences (3’ UTR and/or 5’ UTR), a coding sequence (cds), a poly(A/T) sequence (e.g. comprising about 100 A/T nucleotides), and an optional histone- stern loop.
  • a promoter sequence for an RNA polymerase e.g. T7 promoter, SP6 promoter
  • a Kozak sequence e.g. T7 promoter, SP6 promoter
  • UTR sequences 3’ UTR and/or 5’ UTR
  • cds coding sequence
  • poly(A/T) sequence e.g. comprising about 100 A/T nucleotides
  • an optional histone- stern loop e.g. T7 promoter, SP6 promoter
  • a Kozak sequence e.g. T7 promoter, SP
  • the method for producing DNA produces more than 1 pg of DNA, more than 1 mg of DNA, more than 1g of DNA, more than 10g of DNA, more than 100g of DNA.
  • 1mg to 1000g of DNA preferably 1 mg to 100g.
  • the produced DNA has an DNA purity of at least 70%, preferably of at least 80%, more preferably of at least 90%, 95%, 96%, 97%, 98%, or 99%.
  • the produced DNA has an DNA integrity of at least 60%, at least 70%, preferably of at least 80%. DNA integrity may be determined using IP-RP-HPLC, agarose electrophoresis, or capillary gel electrophoresis.
  • the method of producing DNA of the invention comprises the steps
  • step B) purifying the flow through of step A by at least one step of ion exchange chromatography or TFF to obtain a pure preparation of in vitro amplified DNA, preferably wherein steps i), ii), and A) are performed in-line or continuously.
  • the method of producing DNA of the invention comprises the steps
  • step B) purifying the flow through of step A by at least one step of ion exchange chromatography, preferably AEX, to obtaining a pure preparation of in vitro amplified DNA, preferably wherein steps i), ii), and A) are performed inline or continuously.
  • steps i), ii), and A) are performed inline or continuously.
  • the method of producing DNA of the invention comprises the steps
  • step B) purifying the flow through of step A by at least one step of ion exchange chromatography, preferably AEX, to remove dNTPs to obtaining a pure preparation of in vitro amplified DNA, preferably wherein steps i), ii), and A) are performed in-line or continuously.
  • steps i), ii), and A) are performed in-line or continuously.
  • the method of producing DNA of the invention comprises the steps
  • step B) purifying the flow through of step A by at least one step of ion exchange chromatography, preferably AEX, to remove dNTPs to obtaining a pure preparation of in vitro amplified DNA, preferably wherein steps i), ii), and A) are performed in-line or continuously.
  • steps i), ii), and A) are performed in-line or continuously.
  • the present invention provides a method of producing RNA.
  • the method of producing RNA comprises at least one step of purifying and/or producing DNA.
  • the method of producing RNA comprises the steps purifying in vitro amplified DNA; and performing RNA in vitro transcription using the purified DNA as a template for RNA in vitro transcription.
  • the method of producing RNA comprises the steps purifying the in vitro amplified DNA as defined in the context of the first aspect; and performing RNA in vitro transcription using the purified DNA as a template for RNA in vitro transcription.
  • the method of producing RNA additionally comprises a step of generating a DNA preparation by in vitro DNA amplification method as defined herein.
  • the method of producing RNA comprises the steps generating a DNA preparation by an in vitro DNA amplification step; and purifying the in vitro amplified DNA; and performing RNA in vitro transcription using the purified DNA as a template for RNA in vitro transcription.
  • the method of producing RNA additionally comprises a step of generating a DNA preparation by an in vitro DNA amplification as defined in the second aspect.
  • the method of producing RNA comprises the steps generating a DNA preparation by an in vitro DNA amplification step as defined in the second aspect; and purifying the in vitro amplified DNA as defined in the first aspect; and performing RNA in vitro transcription using the purified DNA as a template for RNA in vitro transcription.
  • the method of producing RNA comprises step of immobilizing the in vitro amplified DNA on a solid support prior to the step of RNA in vitro transcription
  • the in vitro amplified DNA is a biotinylated PCR amplified DNA
  • the solid support is a streptavidin functionalized.
  • Suitable solid supports may be selected from a particle (e.g. magnetic particle), a column, ora membrane.
  • particle in that context has to be understood as solid supports that are suitable for the use in RNA in vitro transcription. Accordingly, the term “particle” as such is not limiting in scale, shape, or composition.
  • particles in the context of the invention have to be understood as free floating solid supports (e.g. free floating upon mixing, shaking etc.).
  • the solid support is a streptavidin functionalized magnetic particle.
  • the DNA immobilization step comprises a step of combining biotinylated DNA with a streptavidin functionalized solid support (e.g. magnetic particle) in an immobilization buffer.
  • the step may be performed in the reaction vessel that is used for the RNA production method.
  • DNA templates for RNA in vitro transcription immobilized on solid supports are particularly suitable in the context of an automated pharmaceutical RNA production as the immobilized template DNA can be re-used for several IVT cycles which increases the RNA yield of the RNA production process.
  • DNA may be removed easily (e.g., by filtration or magnetic forces) and, therefore, the DNA used for RNA synthesis does not contaminate the final RNA product.
  • a step of DNAse digestion is not needed as the DNA is immobilized and can easily be removed from the RNA product.
  • the RNA in vitro transcription is performed in the presence of an NTP mixture and an IVT buffer under conditions to allow transcribing of the DNA, in particular the immobilized DNA, into RNA.
  • the NTP mixture comprises adenine, cytosine, guanine and uracil.
  • the NTP mixture comprises modified nucleotides.
  • the modified nucleotide of the NTP mixture is a modified uracil nucleotide.
  • the NTP mixture comprises a modified nucleotide selected from pseudouridine (ip) and/or N1- methylpseudouridine (m1ip). Particularly preferred in the context of the invention is m1ip.
  • the NTPs mixture is optimized for the given RNA sequence to be produced (according to claims 1 to 35 of WD2015188933). Accordingly, for producing an RNA sequence with G:C:A:U of 1 :2:3:2, the respective sequence optimized NTP mixture comprises G:C:A:U in a molar ratio of 1 :2:3:2.
  • the IVT buffer comprises a buffer agent (e.g. Tris or HEPES), MgCb, an RNA polymerase.
  • a buffer agent e.g. Tris or HEPES
  • MgCb e.g. MgCb
  • RNA polymerase e.g. TgCb
  • RNA polymerases in the context of the invention may be selected from bacteriophage derived RNA polymerases, for example T7, T3, SP6, or Syn5 RNA polymerases. These RNA polymerases may be engineered to e.g. improve the quality of the synthetized RNA (e.g. reduced dsRNA content, reduced short abortive by-products, improved capping efficiency).
  • the IVT buffer may optionally comprise a cap analogue (preferably a cap1 analogue).
  • IVT buffer comprises a cap analogue, preferably a cap1 analogue.
  • cap analogue as used herein is intended to refer to a non-polymerizable di-nucleotide or tri-nucleotide that has cap functionality in that it facilitates translation or localization, and/or prevents degradation of an RNA molecule when incorporated at the 5’-end of the nucleic acid molecule.
  • Non-polymerizable means that the cap analogue will be incorporated only at the 5’-terminus because it does not have a 5’ triphosphate and therefore cannot be extended in the 3’- direction by a template-dependent polymerase, particularly, by template-dependent RNA polymerase.
  • the cap1 analogues m7G(5’)ppp(5’)(2’OMeA)pG or m7G(5’)ppp(5’)(2’OMeG)pG are contained in the NTP mixture of the IVT buffer.
  • a particularly preferred cap1 analogue in that context is m7G(5’)ppp(5’)(2’OMeA)pG.
  • the cap1 analogue is 3’OMe- m7G(5’)ppp(5’)(2’OMeA)pG.
  • a cap1 analogue as described in W02023007019 may be used.
  • the IVT buffer may comprise components to e.g., prevent or reduce RNA degradation (e.g., RNAse inhibitor), to prevent or reduce the formation of pyrophosphate (e.g., Pyrophosphatase), to maintain a preferred pH (e.g. buffering agents), to resolve secondary structures in the DNA and/or the RNA (e.g. betaine), to increase the performance of the RNA polymerase enzyme (spermidine), etc.
  • RNA degradation e.g., RNAse inhibitor
  • pyrophosphate e.g., Pyrophosphatase
  • a preferred pH e.g. buffering agents
  • RNA e.g. betaine
  • spermidine RNA polymerase enzyme
  • the method as outlined herein is suitable for producing any type of RNA.
  • the RNA is selected from a single stranded RNA or double stranded RNA, and/or a coding RNA or a non-coding RNA, and/or a linear RNA or a circular RNA.
  • the RNA may be a double-stranded non-coding RNA in circular form, or the RNA may be a single stranded non-coding RNA in linear form, or the RNA may be a double stranded coding RNA in linear form, etc.
  • the RNA is a single stranded coding RNA in linear or circular form.
  • the RNA is selected from viral RNA, retroviral RNA, replicon RNA, small interfering RNA (siRNA), antisense RNA, saRNA (small activating RNA), CRISPR RNA (small guide RNA, sgRNA), ribozymes, aptamers, riboswitches, immunostimulating RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), Piwi-interacting RNA (piRNA), selfreplicating RNA, circular RNA, or messenger RNA (mRNA).
  • siRNA small interfering RNA
  • antisense RNA small activating RNA
  • CRISPR RNA small guide RNA, sgRNA
  • ribozymes small guide RNA, sgRNA
  • aptamers riboswitches
  • immunostimulating RNA transfer RNA
  • tRNA transfer RNA
  • the RNA is a coding RNA.
  • the RNA comprises at least one coding sequence.
  • a coding RNA can be any type of RNA characterized in that said RNA comprises at least one coding sequence that is translated into at least one amino-acid sequence (e.g. upon administration to a cell).
  • the RNA is selected from an mRNA, a (coding) circular RNA, a (coding) selfreplicating RNA, a (coding) viral RNA, or a (coding) replicon RNA.
  • the RNA has a length ranging from about 500 to about 10000 nucleotides, ranging from about 500 to about 7000 nucleotides, ranging from about 1000 to about 5000 nucleotides.
  • the RNA is an mRNA. Accordingly, the method is a method for producing coding RNA, preferably mRNA. In preferred embodiments, the RNA is a therapeutic RNA.
  • RNA relates to an RNA providing a therapeutic modality.
  • therapeutic in that context has to be understood as “providing a therapeutic function” or as “being suitable for therapy or administration”.
  • a “therapeutic RNA” is typically produced using methods and compositions suitable for pharmaceutical production.
  • therapeutic in that context should not at all to be understood as being limited to a certain therapeutic modality.
  • therapeutic RNA does not include natural RNA extracts or RNA preparations (e.g., obtained from bacteria, or obtained from plants) that are not suitable for administration to a subject (e.g., animal, human).
  • the RNA sequence or the coding sequence has a G/C content of at least about 50%, 55%, or 60%.
  • the RNA or the coding sequence has a G/C content of at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%.
  • the method for producing RNA is a method for producing RNA that has an RNA sequence or the coding sequence with a G/C content of at least about 55%.
  • the produced RNA comprises at least one 3’UTR and/or at least one 5’UTR sequence.
  • Suitable UTR sequences and UTR combinations may be selected from published PCT patent application WO2019077001 , preferably selected from UTR sequences and UTR combinations as disclosed in claims 1 to 10 of WD2019077001 .
  • the 5’ UTR is selected or derived from HSD17B4, and the 3’ UTR is selected or derived from PSMB3.
  • the produced RNA comprises at least one histone stem loop sequence (hSL). Suitable hSL sequences that may be used within the present invention may be derived from formulae (I) or (II) of WD2012019780.
  • the produced RNA comprises at least one poly(A) sequence or at least two poly(A) sequences (see e.g., WO2016091391).
  • the poly(A) sequence represents the 3’ terminus of the RNA (see e.g., WD2022162027).
  • the poly(A) sequence has a length of about 50Ato about 150A. In some embodiments, the poly(A) sequence has a length of about 64A or 100A.
  • the method for producing RNA is a method for producing therapeutic coding RNA (e.g., therapeutic mRNA).
  • the RNA comprises at least one coding sequence that encodes at least one therapeutic peptide or protein.
  • therapeutic peptide or proteins are defined in the context of the first aspect.
  • the method of producing RNA comprises the steps of performing RNA in vitro transcription using the purified DNA as a template for RNA in vitro transcription, wherein the DNA is immobilized on a solid support (e.g. particle) and contained in a reaction vessel, followed by performing at least one IVT cycle by mixing/incubating the immobilized DNA with the IVT bufferto produce an RNA batch.
  • a solid support e.g. particle
  • the solid support is selected from magnetic particles on which the DNA template for RNA in vitro transcription is immobilized, preferably (super)paramagnetic particles.
  • magnetic DNA particles allow for a mixing of the DNA particles during the course of the IVT and/or a capturing of the DNA particles after the IVT cycle which is important for an automated RNA manufacturing process.
  • the mixing and/or capturing is preferably induced by magnetic force, e.g., by a magnet as further specified herein.
  • Preferred examples of particles that are suitable in the context of the invention are Dynabeads® such as Dynabeads® M-270, Dynabeads® M-280, Dynabeads® M-450, or Dynabeads® MyOne.
  • the RNA in vitro transcription step of the method is carried out in a bioreactor as described in WD2020002598, preferably as described in claims 1 to 59 of WD2020002598.
  • a bioreactor as described in WD2020002598, preferably as described in claims 1 to 59 of WD2020002598.
  • Particularly suitable bioreactors in the context of the invention are illustrated in Figures 1 to 11 of WD2020002598.
  • the produced RNA is capped in an enzymatic capping step (e.g. by using immobilized enzymes as described in WO2016193226).
  • the produced RNA is polyadenylated in an enzymatic polyadenylation step (e.g. by using immobilized enzymes as described in WD2016174271).
  • the method comprises a step of purifying the obtained RNA.
  • the DNA template for RNA in vitro transcription produced and purified according to the invention is highly pure, and as in preferred embodiments the DNA template is immobilized in the process of RNA transcription (therefore no DNAse treatment is required in these embodiments), the purification of the obtained RNA can be performed in an effective manner to obtain pure RNA.
  • the RNA purification comprises at least one step selected from RP-HPLC, AEX, SEC, HIC, hydroxyapatite chromatography, TFF, filtration, precipitation, core-bead flowthrough chromatography, oligo(dT) purification, cellulose-based purification, or any combination thereof.
  • the RNA purification comprises at least one step of oligo(dT) purification and/or RP-HPLC.
  • the method comprises a step of formulating the obtained RNA, preferably formulating the obtained purified RNA, into lipid-based carriers, preferably into LNPs.
  • LNPs may be formulated by mixing a lipid composition with an aqueous RNA composition via a T- piece or Y-piece mixing element.
  • LNPs may be formulated using microfluidic mixing. After LNP formulation, the LNPs may be purified or re-buffered using filtration and/or TFF.
  • a DNA template for IVT is used that is highly pure and that the DNA template for IVT is preferably purified according to the method of the first aspect to ensure that the downstream method steps are not impaired, e.g. by contaminations such as dNTPs, DNA polymerase enzymes, DNA primers.
  • the method for producing RNA produces more than 1 mg of RNA, more than 1g of RNA, more than 10g of RNA, more than 100g of RNA.
  • RNA preferably 1 mg to 1000g of RNA, preferably 1 mg to 100g.
  • the produced RNA has an RNA purity of at least 85%, preferably of at least 90%, more preferably of at least 95%, 96%, 97%, 98%, or 99%.
  • the produced RNA has an RNA integrity of at least 75%, preferably of at least 80%, more preferably of at least 85%. RNA integrity is determined using IP-RP-HPLC.
  • the step of performing RNA in vitro transcription using the purified DNA as a template for RNA in vitro transcription is flu idically connected to the upstream steps of purifying in vitro amplified DNA as defined herein and/or the upstream steps of producing DNA as defined herein.
  • the step of performing RNA in vitro transcription using the purified DNA as a template for RNA in vitro transcription may be flu idically connected to any downstream step as defined herein, including purifying and/or formulating the RNA.
  • the fluidic connection is preferably facilitated by tubes, channels, microchannels, or microfluidic channels (depending on the desired scale of the method).
  • the present invention provides a system for purifying DNA, in particular a system for purifying an impure preparation comprising DNA (e.g. for purifying a PCR reaction product).
  • the system for purifying DNA comprises at least one core-bead flow through chromatography unit.
  • the device for purifying DNA comprises at least one downstream purification unit.
  • the device for purifying DNA comprises at least one core-bead flow through chromatography unit and at least one downstream purification unit.
  • the system for purifying DNA preferably in vitro amplified DNA, is configured to carry out a method for purifying DNA of the first aspect.
  • the system for purifying DNA preferably in vitro amplified DNA, is configured to perform a method of the first aspect and comprises at least one core-bead flow through chromatography unit and at least one downstream purification unit.
  • the system for purifying DNA is a device for purifying in vitro amplified DNA (contained in an impure preparation), in particular PCR amplified DNA (contained in an impure preparation). More preferably, the system for purifying DNA is for purifying a PCR reaction product.
  • the at least one core-bead flow through chromatography unit comprises a core-bead flow through chromatography material as defined in the context of the first aspect, in particular in form of a column.
  • the core-bead flow through chromatography material are beads that have a molecular weight cut-off of below 700kDa, 600kDa, 500kDa, preferably 400kDa.
  • the core-bead flow through chromatography is configured for single-use.
  • the downstream purification unit comprises at least one ion exchange chromatography unit, at least one HIC unit, or at least one TFF unit, or any combinations thereof. In preferred embodiments, the downstream purification unit comprises at least one ion exchange chromatography unit and/or at least one TFF unit. In particularly preferred embodiments, the downstream purification unit comprises at least one ion exchange chromatography unit.
  • the ion exchange chromatography unit comprises ion exchange chromatography material, preferably AEX material, as defined in the context of the first aspect, in particular an anion exchange membrane.
  • the anion exchange membrane is preferably selected from a membrane adsorber functionalized with quaternary ammonium.
  • the ion exchange chromatography element is configured for single-use
  • the HIC unit comprises HIC material as defined in the context of the first aspect.
  • the TFF unit comprises a TFF as defined in the context of the first aspect, preferably a TFF membrane cassette that preferably comprises a cellulose or PES membrane.
  • the TFF material that has a MWCO of less than 500kDa, preferably 10OkDa or 300kDa.
  • the system for purifying DNA additionally comprises a pump fortransporting the impure preparation comprising DNA, preferably in vitro amplified DNA, into the core-bead flow through chromatography unit and, optionally, a pump fortransporting the purified flow through of the core-bead flow through chromatography unit into the downstream purification unit, preferably the ion exchange chromatography unit and/or the TFF unit.
  • pumps may be selected from a syringe pump, pulseless flow pump, peristaltic pump, a microfluidics pump, orthe like.
  • the downstream purification unit preferably the ion exchange chromatography unit and/or the TFF unit, are connected to a unit that provides media and/orwash buffers (e.g. in bags or containers).
  • the media and/orwash buffers may be transported via a pump into the DNA purification system.
  • the system for purifying DNA additionally comprises a pressure sensor upstream of the core-bead flowthrough chromatography unit and/or upstream of the downstream purification unit, e.g. upstream of the ion exchange chromatography unit.
  • the system for purifying DNA additionally comprises at least one further sensor selected from a conductivity sensor, an UV sensor, a flow sensor.
  • the system for purifying DNA comprises at least one conductivity sensor downstream of the downstream purification unit.
  • the system for purifying DNA additionally comprises at least one inlet element for connecting the system for purifying DNA with an optional upstream DNA amplification unit or for providing the impure preparation.
  • the system for purifying DNA additionally comprises at least one exit element for connecting the system for purifying DNA with an optional downstream unit or for obtaining the pure preparation.
  • the core-bead flowthrough chromatography unit and the at least one downstream purification unit are flu id ically connected to each other.
  • the fluidic connection is preferably facilitated by tubes, channels, microchannels, or microfluidic channels (depending on the desired scale of the method).
  • the components of the system for purifying DNA as defined herein may be integrated into a closed device, a microfluidics chip, microfluidics system, or microfluidics cartridge. Accordingly, in embodiments, the system for purifying DNA is a device, a microfluidics chip, a microfluidics system, or a microfluidics cartridge.
  • the system for purifying in vitro amplified DNA as defined herein is a single use system. Accordingly, all fluidic channels, purification units, or bags/containers that are in direct contact with the DNA preparation may be discarded after a purification run to avoid cleaning procedures. In embodiments, where the system is integrated into microfluidics system, or a microfluidics cartridge, the whole microfluidics system, or a microfluidics cartridge may be discarded after a purification run.
  • the system for purifying DNA may be integrated into a DNA or RNA manufacturing device, e.g. a portable DNA or RNA manufacturing device or DNA or RNA manufacturing device in a facility.
  • a DNA or RNA manufacturing device e.g. a portable DNA or RNA manufacturing device or DNA or RNA manufacturing device in a facility.
  • Figure 1 shows schematically and exemplarily a system for purifying DNA according to the invention.
  • the system for purifying DNA (1) may comprise a core-bead flow through chromatography unit (2) and a downstream purification unit (3).
  • the downstream purification unit (3) may be selected from a TFF, HIC, or an ion exchange chromatography unit, preferably an AEX unit.
  • the system comprises pumps (4a, 4b), in particular a pump for a core-bead flow through chromatography unit (4a) and a pump for a downstream purification unit (4b).
  • the pump for a core-bead flow through chromatography unit (4a) may be contained in an upstream DNA amplification unit (e.g. a PCR unit) or may be a separate pump e.g. contained in an FPLC system.
  • the pump for a downstream purification unit (4b) may provide wash and/or elution buffers to the downstream purification unit (3).
  • the wash and/or elution buffers may be contained in respective containers or bags (8).
  • valves may be included to control the fluid flow of the system (7).
  • the different components of the system for purifying DNA (1) may be connected via fluidic connections (9) that may be, depending on the scale, tubes, channels, microchannels, or microfluidic channels.
  • the system for purifying DNA (1) may additionally comprise one or more pressure sensors (5), and one or more conductivity sensor I UV sensors (6).
  • An inlet element (10) may provide the impure preparation (in embodiments where (4a) is a pump or a pump of an FPLC system) and/or may serve as a connection element for connecting the system for purifying DNA (1) to an upstream unit, e.g. a DNA amplification unit (13).
  • the pure preparation containing DNA may be harvested via an exit element (11) and/or the exit element (11) may serve as a connection element for connecting the system for purifying DNA (1) to a downstream unit (e.g. a DNA trimming unit, a DNA immobilization unit, an RNA in vitro transcription unit (21)).
  • the system may allow for continuous purification of an impure preparation of DNA, e.g. a PCR reaction product.
  • system for purifying DNA is a system as depicted in Figure 1 .
  • the present invention provides a system for producing DNA.
  • the system for producing in vitro amplified DNA comprises at least one in vitro DNA amplification unit.
  • the system for producing in vitro amplified DNA comprises at least one DNA purification system as defined in the context of the fourth aspect.
  • the system for producing in vitro amplified DNA is configured to perform a method as defined in the context of the second aspect.
  • the system for producing in vitro amplified DNA is configured to perform a method as defined in the context of the second aspect and comprises at least one vitro DNA amplification unit as further defined below and at least one DNA purification system as defined in the context of the fourth aspect.
  • the in vitro DNA amplification unit is configured to perform polymerase chain reaction (PCR), rolling circle amplification, or isothermal amplification.
  • PCR polymerase chain reaction
  • the in vitro DNA amplification unit can be a microchannel, a tube, a vial, a container, or a bioreactor.
  • the in vitro DNA amplification unit can be used for pharmaceutical production (e.g. GMP compliant).
  • the in vitro DNA amplification unit can be composed of materials selected from glass, metal, ceramics, or a polymer (e.g. plastic material).
  • the in vitro DNA amplification unit can have a volume (e.g. the volume in which the DNA is contained) of 1 pL to 1000L, preferably 1 mL to 10L, more preferably 10mL to 1 L, even more preferably 10mL to 500mL.
  • the in vitro DNA amplification unit is a PCR device, preferably a continuous PCR device.
  • the in vitro DNA amplification unit is a PCR device as described in WD2022112498, in particular as described in claims 1 to 28, or as illustrated in Figures 1 to 11 of WD2022112498.
  • the in vitro DNA amplification unit is a PCR device configured to perform capillary PCR.
  • the in vitro DNA amplification unit is a PCR device configured to perform continuous PCR.
  • Capillary PCR can be understood as PCR done in a tube, a pipe, or a capillary.
  • the tube or capillary may have a very small diameter, which is in a range of about 0.5mm to about 1 ,5mm, preferably about 0.75mm to about 1 ,25mm.
  • the PCR master mix can be filled into the tube, a pipe, or a capillary, e.g. using a pump, and thereby be guided through the PCR device. While the PCR reaction is guided through the compartments, preferably continuously, the DNA product is generated.
  • the system for producing in vitro amplified DNA additionally comprises a mixing unit for preparing the PCR master mix.
  • the mixing unit comprises a mixing element (e.g. a T- piece, a Y-piece). It may also comprise a pump unit (e.g. a syringe pump, pulseless flow pump, peristaltic pump, a microfluidics pump, orthe like).
  • the mixing unit comprises a first container or bag for holding DNA primers and/or the DNA amplification template, a second container or bag for holding PCR buffer components (e.g. including DNA polymerase), and at least one pump for combining the DNA primers with the PCR buffer components via a mixing means, preferably a T-piece or a Y-piece mixing means.
  • the mixing unit comprises a pump for the first container or bag and a pump for the second container or bag, each pump optionally monitored by individual flow controllers.
  • the mixing unit comprises at least one pump and at least one flow controller to provide PCR master mix into the PCR device.
  • the system for producing in vitro amplified DNA additionally comprises, downstream of the DNA purification system, a DNA immobilization unit and/or a DNA trimming unit.
  • the DNA trimming unit comprises DNA restriction endonucleases, optionally immobilized DNA restriction endonucleases, to trim the 3’ end or 5’ end of the obtained DNA.
  • a DNA trimming may be required when DNA templates for RNA in vitro transcription are produced that should ideally contain a 3’ terminal poly(A) stretch.
  • the DNA trimming unit may comprise a restriction endonuclease (e.g., type Ils) to remove excessive nucleotides from the 3’ terminus of the DNA to obtain a 3’ terminal poly(A) end.
  • the system for producing in vitro amplified DNA additionally comprises, upstream of the in vitro DNA amplification unit, a DNA de-novo synthesis unit.
  • the in vitro DNA amplification unit, the at least one DNA purification system, and the optional DNA immobilization unit and/or DNA trimming unit are flu id ically connected to each other, preferably via tubes, capillaries, or microfluidic channels.
  • said mixing unit is fluidically connected to the in vitro DNA amplification unit. The fluidic connection is preferably facilitated by tubes, channels, microchannels, or microfluidic channels (depending on the scale).
  • Figure 2 shows schematically and exemplarily a system for producing DNA according to the invention.
  • the system for producing DNA (12) may comprise a DNA amplification unit (13) which is preferably a PCR device.
  • the system may also comprise a mixing unit for preparing PCR master mix (15a) that may comprise a container or bag for holding PCR buffer components (17) and a container or bag for holding DNA primers and DNA amplification template (18), pumps (14b) and flow sensors (5) and a mixing element (16), preferably a T-piece or a Y-piece.
  • the mixing unit for preparing PCR master mix (15a) may be configured to produce, preferably in a continuous manner, PCR master mix that may be introduced into the DNA amplification unit (13) optionally via a pump (14a) through fluidic connection (9).
  • the system may comprise a unit for holding PCR master mix (15b) that may comprise a container or bag for holding PCR master mix (19) and, optionally a pump (14c) and flow sensors (5).
  • the unit for holding PCR master mix (15b) may introduce the PCR master mix into the DNA amplification unit (13), optionally via a pump (14a) through fluidic connection (9).
  • the system for producing DNA (12) may also comprise a system for purifying the in DNA as defined herein, in particular as depicted in Figure 1 .
  • the system may comprise a core-bead flow through chromatography unit (2) and a downstream purification unit (3).
  • the different components of the system for producing DNA (12) may be connected via fluidic connections (9) that may be, depending on the scale, tubes, channels, microchannels, or microfluidic channels.
  • the system for producing DNA (12) may additionally comprise one or more pressure sensors (5), and one or more conductivity sensor / UV sensors (6).
  • the pure preparation containing DNA may be harvested via an exit element (11) and/or may serve as a connection element for connecting the system for purifying DNA (1) to a downstream unit (e.g. a DNA trimming unit, a DNA immobilization unit, an RNA in vitro transcription unit (21)).
  • the system may allow for continuous production and purification of a PCR product.
  • the system for producing DNA is a system as depicted in Figure 2. 6.
  • An RNA manufacturing system :
  • the present invention provides an RNA manufacturing system.
  • the RNA manufacturing system comprises at least one RNA in vitro transcription unit. In preferred embodiments, the RNA manufacturing system comprises at least one DNA purification system as defined in the context of the fourth aspect. In preferred embodiments, the RNA manufacturing system comprises at least one DNA production system as defined in the context of the fifth aspect. In preferred embodiments, the RNA manufacturing system is configured to perform a method as defined in the context of the third aspect.
  • the RNA production system is configured to perform a method as defined in the context of the third aspect and comprises at least one RNA in vitro transcription unit and at least one DNA purification system as defined in the context of the fourth aspect or, alternatively, at least one DNA production system as defined in the context of the fifth aspect.
  • the RNA in vitro transcription unit comprises a bioreactor for RNA in vitro transcription that preferably comprises a reaction vessel configured to hold a DNA template for RNA in vitro transcription immobilized on a solid support (e.g. a column or a bead).
  • a bioreactor for RNA in vitro transcription that preferably comprises a reaction vessel configured to hold a DNA template for RNA in vitro transcription immobilized on a solid support (e.g. a column or a bead).
  • a reaction vessel can be a microchannel, a tube, a vial, a container, or a bioreactor.
  • the reaction vessel is a reaction vessel that can be used for pharmaceutical production (e.g. GMP compliant).
  • the reaction vessel can be composed of materials selected from glass, metal, ceramics, or a polymer (e.g. plastic material).
  • the reaction vessel can have a volume (e.g. the volume in which the RNA in vitro transcription composition is contained) of 1pLto WOOL, preferably 1mLto WL, more preferably WmL to 1L, even more preferably 10mL to 500mL.
  • the RNA manufacturing device comprises a bioreactor as described in WD2020002598, preferably as described in claims 1 to 59 of WD2020002598. Particularly suitable bioreactors in the context of the invention are illustrated in Figures 1 to 11 of WD2020002598.
  • the RNA production system additionally comprises a DNA immobilization unit as separate entity.
  • DNA immobilization is carried out in the RNA in vitro transcription unit.
  • the RNA production system additionally comprises at least one further unit selected from an RNA purification unit, an RNA formulation unit, a fill-and-fin ish unit, a media supply unit.
  • the RNA manufacturing device comprises at least one RNA purification unit, preferably configured to perform purification of RNA using RP-HPLC, AEX, SEC, HIC, hydroxyapatite chromatography, TFF, filtration, precipitation, core-bead flowthrough chromatography, oligo(dT) purification, cellulose-based purification, or any combination thereof.
  • the RNA purification unit comprises an oligo(dT) purification and/or a RP-HPLC element.
  • the RNA manufacturing device additionally comprises a formulation module, preferably an LNP formulation module and/or a fill and finish module suitable for aseptic filling of an RNA medicament.
  • a formulation module preferably an LNP formulation module and/or a fill and finish module suitable for aseptic filling of an RNA medicament.
  • the bioreactor for RNA in vitro transcription and further optional modules are integrated into a GMP manufacturing device, module, or system.
  • the GMP manufacturing device, module, or system is a device, module, or system as described in WD2022049093, in particular as described in claims 1 to 72 of WD2022049093, or as illustrated in Figures 1 to 6 of WD2022049093.
  • the in vitro DNA amplification unit, the at least one DNA purification system, and the RNA in vitro transcription unit are fluidically connected to each other, preferably via tubes, capillaries, or microfluidics channels.
  • said mixing unit is fluidically connected to the in vitro DNA amplification unit.
  • the fluidic connection is preferably facilitated by tubes, channels, microchannels, or microfluidic channels (depending on the desired scale).
  • the components of the RNA manufacturing system as defined herein may be integrated into a device, a microfluidics chip, microfluidics system, or microfluidics cartridge. Accordingly, in embodiments, the RNA manufacturing system is a device, a microfluidics chip, a microfluidics system, ora microfluidics cartridge.
  • the system for producing DNA as defined herein is a single use system. Accordingly, all fluidic channels, purification units, or bags/containers that are in direct contact with the DNA preparation may be discarded after a production run to avoid cleaning procedures. In embodiments, where the system is integrated into microfluidics system, or a microfluidics cartridge, the whole microfluidics system, or a microfluidics cartridge may be discarded after a DNA production run.
  • the RNA manufacturing system as defined herein is a single use system. Accordingly, all fluidic channels, purification units, or bags/containers that are in direct contact with the DNA or RNA preparation may be discarded after a manufacturing run to avoid cleaning procedures. In embodiments, where the system is integrated into microfluidics system, or a microfluidics cartridge, the whole microfluidics system, ora microfluidics cartridge may be discarded after a purification run.
  • the RNA manufacturing system as defined herein may be integrated into an RNA manufacturing device, e.g. a portable RNA manufacturing device or RNA manufacturing device in a facility.
  • Figure 3 shows schematically and exemplarily an RNA manufacturing system according to the invention.
  • the RNA manufacturing system (20) may comprise an RNA in vitro transcription unit (21), an optional separate DNA immobilization unit, and a media supply unit (22) for supplying components of the IVT master mix or components for DNA immobilization.
  • the media supply unit (22) comprises a pump for providing IVT master mix or DNA immobilization components (23) and containers or bags for holding for holding IVT or DNA immobilization components (24).
  • DNA immobilization may take place in the RNA in vitro transcription unit (21) or in a separate DNA immobilization unit.
  • the in vitro transcribed RNA may be purified in an RNA purification unit (e.g. comprising an oligo d(T) purification).
  • the obtained RNA may be harvested via an exit element for RNA (25) and/or the system may be connected via exit element (25) to a formulation unit.
  • the system for RNA manufacturing may preferably comprise the components of the system for DNA purification (see Figure 1) and/or the components of the system for DNA production (see Figure 2).
  • the different components of the RNA manufacturing system (20) may be connected via fluidic connections (9) that may be, depending on the scale, tubes, channels, microchannels, or microfluidic channels.
  • system for producing DNA is a system as depicted in Figure 3.
  • Figure 1 shows an exemplary system for purifying DNA (1 ) according to the fourth aspect.
  • Figure 2 shows an exemplary system for producing DNA (12) according to the fifth aspect.
  • Figure 3 shows exemplary RNA manufacturing system (20) according to the sixth aspect.
  • Figure 4 shows that core-bead flow through chromatography material with MWCO of 400 is suitable for purifying DNA.
  • Figure 4A shows that all DNA fragments of a DNA ladder (0.5kbp to 10kbp) were present in the flow through fractions;
  • Figure 4B shows that DNA fragments of a DNA ladder (25bp to 500bp) shorter than 150bp were strongly reduced in the flow-through fractions;
  • Figure 4C shows that DNA fragments of a DNA ladder (25bp to 500bp) shorter than 150bp could be washed off by NaOH from the core-bead flow through chromatography beads. Further details are provided in Example 1 and Example 2.
  • Figure 5 shows that core-bead flow through chromatography material with MWCO of 400 is suitable for removing DNA primers but not effective in removing dNTPs.
  • Figure 5A shows that only the NaOH fractions contained DNA primers washed off from the beads. DNA primers were absent in the flow through or the TE wash fractions.
  • Figure 5B shows that dNTPs were present in the flow through and TE fractions, and a small proportion of dNTPs could be washed off from the beads. Further details are provided in Example 3 and Example 4.
  • Figure 6 shows that in-line purification of a PCR reaction product using core-bead flow through chromatography yields a pre-purified flowthrough containing high DNA concentrations (bars) and DNA with excellent integrity (dots). Further details are provided in Example 5.
  • Figure 7 shows that ion exchange chromatography was capable of removing dNTPs from a pre-purified flow-through.
  • Figure 7A shows that the ion exchange chromatography material did not bind dNTPs.
  • Figure 7B shows that the ion exchange chromatography material effectively captured the target DNA, and that the target DNA could be eluted from the ion exchange chromatography material. Further details are provided in Example 6.
  • Figure 8 shows that an optimized ion exchange chromatography process, indicating that NTPs and impurities are removed, and that purified DNA can be eluted using a high salt elution buffer (comprising about 5mM NaCI). Further details are provided in Example 7.
  • the goal of the present example was to evaluate whether core-bead flow through chromatography is suitable for the purification of DNA, and, in particular, which molecular weight cut-off is most appropriate for allowing flow- through purification of larger DNA fragments (e.g. DNA fragments having the typical size of PCR products).
  • a DNA size ladder was applied to two different core-bead flowthrough chromatography materials with different molecular weight cut-off (MWCO of 400 and 700). Accordingly, the DNA size ladder was applied to 4.7mL HiScreen columns containing CaptoCore 400 or CaptoCore 700 resin (Cytiva).
  • the applied DNA size ladder contained DNA fragments in range from 0.5kbp to 10kbp (1 kb Ladder N322L from New England Biolabs). 160pg DNA size ladder was loaded at 1 mL/min on a core-bead flowthrough chromatography columns equilibrated in 1xTE buffer (10mM Tris, 1mM EDTA pH 8.0). Loading was performed at 1 mL/min and the column was washed initially with 40mL of 1xTE buffer and subsequently with 40mL of 0.1 M NaOH. Fractions of 1 mL volume were collected for both washing steps. DNA fragments in the fractions were analyzed using a Fragment Analyzer 5300 System (Agilent).
  • core-bead flow through chromatography material with MWCO of 700 is not suitable for the purification of linear DNA fragments (e.g. in vitro amplified DNA such as PCR amplified DNA) in a flow-through mode.
  • Core-bead flow through chromatography material with MWCO of 400 seems to be suitable for purification of linear DNA fragments (e.g. in vitro amplified DNA such as PCR amplified DNA) in a flow-through mode.
  • Example 2 Core-bead flow through chromatoqraphy with MWCO 400 is removes short DNA fragments
  • the goal of the present example was to evaluate whether core-bead flow through chromatography with MWCO of 400 is suitable forthe removal of short DNA fragments from the flow-through. Therefore, a small size DNA ladder was applied, and the capability of binding small DNA fragments was tested.
  • a DNA size ladder was applied to a core-bead flowthrough chromatography with MWCO of 400. Accordingly, the DNA size ladder was applied to 4.7mL HiScreen columns containing CaptoCore 400 resin (Cytiva). The DNA size ladder contained fragments in range from 25bp to 500bp (SM1191 Low Range DNA Ladder; Thermo Scientific).
  • DNA fragments could be washed off by NaOH from the core-bead flow through chromatography beads.
  • 150bp DNA fragments were partially or completely retained by the resin and could be recovered upon the 0.1 M NaOH washing steps.
  • DNA fragments having sizes of25bp, 50bp, 75bp could be completely retained by the CaptoCore 400 resin.
  • core-bead flow through chromatography material with MWCO of 400 is suitable for the purification of larger linear DNA fragments (e.g. in vitro amplified DNA such as PCR amplified DNA) in a flow- through mode.
  • DNA fragments having a length of25bp, 50bp, and 75bp can be completely removed from the flowthrough as these shorter DNA fragments may enter the pores of the core-bead flowthrough chromatography beads.
  • the data shows that core-bead flow through chromatography with MWCO of 400 is suitable for the removal of DNA primers (typically having a length of below 150bp) from a preparation containing larger linear DNA fragments (e.g. target DNA).
  • Example 3 Core-bead flow through chromatography with MWCO of 400 is suitable for DNA primer removal
  • the goal of the present example was to evaluate whether core-bead flow through chromatography with MWCO of 400 is suitable forthe removal of DNA primers from the flow-through. Therefore, DNA primers were applied, and the capability of binding (retaining) these DNA primers was tested.
  • the DNA primers were applied to 4.7mL HiScreen columns containing CaptoCore 400 resin (Cytiva). 120nmol of a DNA primer pair (55nt and 32nt in length; SEQ ID NO: 1 and 2) in IxKapaHifi buffer (Kapa Biosystems) at a concentration of 6pM each was loaded at 1 mL/min on the core-bead flow through chromatography columns equilibrated in 1xTE buffer (10mM Tris, 1mM EDTA pH 8.0). Loading was performed at 1 mL/min and the column was washed initially with 15mL of 1xTE buffer and subsequently with 15mL of 0.1 M NaOH (to wash off bound DNA primers). Fractions of 1 mL volume were collected for both washing steps. The DNA primer concentration in the respective fractions was monitored by NanodropOne ( Figure 5A).
  • Example 4 Core-bead flow through chromatoqraphy does not effectively remove dNTPs
  • dNTPs are used in in vitro DNA amplification methods such as PCR or RCA. Therefore, dNTPs were applied, and the capability of binding (retaining) these dNTPs was tested. dNTPs were applied to 4.7mL HiScreen columns containing CaptoCore 400 resin (Cytiva).
  • dNTPs could be detected in flowthrough and 1xTE washing step. A smaller proportion of dNTPs could be recovered in 0.1 M NaOH elution fraction 7-10. Accordingly, the data surprisingly shows that core-bead flow through chromatography does not effectively remove dNTPs from the flow-through even though dNTPs should be small enough to enter the pores of the core-bead flow through chromatography material.
  • a removal of dNTPs is desired (e.g. for the purification of a PCR reaction product) an additional purification step capable of removing dNTPs may be required.
  • Example 5 Core-bead flow through chromatography with MWCO of 400 is suitable for protein removal
  • the goal of the present example was to evaluate whether core-bead flow through chromatography with MWCO of 400 is suitable for the removal of proteins such as DNA polymerase from the flow-through.
  • DNA polymerases are used in in vitro DNA amplification methods such as PCR or RCA. Therefore, DNA polymerase was applied, and the capability of binding (retaining) the DNA polymerase was tested.
  • DNA polymerase was applied to 4.7mL HiScreen columns containing CaptoCore 400 resin (Cytiva).
  • each fraction was transferred to a PCR mix containing 0.3pM of forward and reverse primer (SEQ ID NO: 1 and 2), 2ng/pL of a pDNA amplification template, 0.4mM each dNTP, and Sybr Green I dye in IxKapaHifi buffer.
  • Real-time PCR performed with the cycling program in Table 1 .
  • DNA polymerase activity could only be detected for the loaded sample and no activity could be detected in the flowthrough and 1xTE washing fractions. Therefore, the complete DNA polymerase protein fraction was bound in the core-bead flowthrough chromatography beads and effectively removed from the flow-through.
  • the data shows that core-bead flow through chromatography is suitable for removing proteins such as DNA polymerase.
  • the goal of the present example was to evaluate whether core-bead flow through chromatography with MWCO of 400 is suitable forthe purification, in particular the continuous purification, of a PCR reaction product to obtain a purified target DNA.
  • PCR Master Mix 200mL of a PCR Master Mix was prepared based on 0.02U/pL KapaHifi polymerase, 0.3pM forward and reverse primer (SEQ ID NO: 1 and 2), 0.02ng/pL pDNA amplification template, and 0.4mM each dNTP in IxKapaHifi buffer.
  • PCR was performed in a continuous PCR device at a flow rate of 1 mL/min to amplify the target DNA.
  • the denaturation temperature was set to 98°C and the annealing and elongation temperature to 63.5°C.
  • the amplified target DNA was a DNA template for RNA in vitro transcription encoding a viral antigen (SEQ ID NO: 3; 1938nt in length).
  • the obtained PCR reaction product comprising the target DNA (SEQ ID NO: 3) was continuously loaded on a downstream core-bead flow through chromatography resin with a MWCO of 400.
  • a pressure sensor was connected between the continuous PCR device and a 4.7mL HiScreen column with CaptoCore 400 resin. The pressure sensor ensured that the system pressure was below the pressure limit of the used column.
  • the linear DNA integrity was determined using a 5300 Fragment Analyzer system with the DNF-930 kit ( Figure 6).
  • DNA primer in the 0.1 M NaOH washing step were resolved using a 5300 Fragment Analyzer system with the DNF-905 kit (Agilent).
  • polymerase activity as monitored by monitored by real time PCR as described in Example 5.
  • the obtained purified PCR product was essentially free of DNA polymerase and DNA primers.
  • the in-line purification using core-bead flow through chromatography was characterized by high DNA yield and DNA integrity.
  • the data shows that core-bead flow through chromatography is particularly suitable for a continuous in-line purification of PCR reaction products.
  • Example 7 Ion exchange chromatography effectively removes dNTPs and buffer components
  • core-bead flow through chromatography is particularly suitable for a flow- through purification of PCR reaction products as impurities such as DNA primers and DNA polymerase bind to the core-bead flowthrough material and the desired target DNA (the PCR amplified DNA) does not bind to the core-bead flowthrough material. Therefore, the desired target DNA can be obtained in purified form in the flow through.
  • dNTPs that are typically present in in PCR reaction products can not effectively be purified using core-bead flowthrough chromatography.
  • the goal of the present example was to evaluate ion exchange chromatography as an additional purification method to remove dNTPs from a purified flowthrough obtained from core-bead flowthrough chromatography.
  • a purification step using an anion exchange chromatography material (Sartobind Q nano membrane absorber; Sartorius) was implemented by using a syringe pump.
  • the Sartobind Q nano membrane absorber was washed with 90mL 1 M NaOH followed by washing with 30mL of 1xTE + 1 M NaCI.
  • the membrane absorber was equilibrated with 30mL of 1xTE buffer. 94mL of the pre-purified flow through pool of Example 6 was loaded onto the membrane absorber at a flow rate of 3mL/min and fractions of 6mL were collected.
  • the flow through did not contain a dsDNA concentration abovel ng/pL as determined by Qubit BR dsDNA assay, indicating that the target DNA bound to the membrane absorber.
  • a high absorbance at 260nm indicated the presence of a high concentration of dNTP in the flowthrough fractions ( Figure 7A), indicating that the dNTPs did not bind to the membrane absorber.
  • the Sartobind Q nano purification step was further optimized using a AktaPure FPLC system (see Figure 8). Increasing the NaCI concentration in the elution step to about 5M led to a sharpened elution profile. A step yield of 83% was obtained for the Sartobind Q nano purification step.
  • the additional purification of the pre-purified flow through using ion exchange chromatography (here: anion exchange membrane) let to an effective removal of dNTPs and buffer components.
  • anion exchange membrane ion exchange membrane
  • the target DNA effectively bound to the anion exchange membrane, whereas contaminations such as dNTPs and buffer components did not bind and could therefore be removed.
  • the combination of core-bead flowthrough chromatography and ion exchange chromatography let to an effective purification of an in vitro amplified DNA preparation (PCR reaction product).
  • the obtained purified target DNA may be used fordownstream applications such as RNA in vitro transcription.
  • the high purity of the in vitro amplified DNA also allows the use of said pure DNA in the manufacturing of a medicament (e.g. an RNA- based medicament).

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Abstract

La présente invention concerne, entre autres, un procédé de purification d'ADN amplifié in vitro, en particulier d'ADN amplifié par PCR, comprenant au moins une étape d'écoulement noyau-bille par chromatographie et au moins une étape de purification supplémentaire, par exemple une chromatographie par échange d'ions ou une filtration tangentielle. De plus, l'invention concerne des procédés de production d'ADN et des procédés de production d'ARN par transcription in vitro d'ARN à l'aide de l'ADN amplifié in vitro purifié en tant que matrice. D'autres aspects concernent un système de purification d'ADN amplifié in vitro, un système de production d'ADN amplifié in vitro, et un système de production d'ARN.
PCT/EP2023/073229 2023-08-24 2023-08-24 Procédé de purification d'adn amplifié in vitro WO2025040263A1 (fr)

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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001081566A2 (fr) * 2000-04-21 2001-11-01 Transgenomic, Inc. Appareil et procede de separation et de purification de polynucleotides
US20130237699A1 (en) * 2010-11-09 2013-09-12 Qiagen Gmbh Method and device for isolating and purifying double-stranded nucleic acids
WO2016091391A1 (fr) 2014-12-12 2016-06-16 Curevac Ag Molécules d'acides nucléiques artificielles destinées à améliorer l'expression de protéines
WO2016174271A1 (fr) 2015-04-30 2016-11-03 Curevac Ag Poly(n)polymérase immobilisée
WO2016193226A1 (fr) 2015-05-29 2016-12-08 Curevac Ag Procédé d'ajout de structures de coiffe à un arn au moyen d'enzymes immobilisées
WO2019077001A1 (fr) 2017-10-19 2019-04-25 Curevac Ag Nouvelles molécules d'acide nucléique artificielles
WO2020002598A1 (fr) 2018-06-28 2020-01-02 Curevac Ag Bioréacteur pour transcription in vitro d'arn
WO2022112498A1 (fr) 2020-11-27 2022-06-02 CureVac RNA Printer GmbH Dispositif de préparation d'un produit d'adn au moyen d'une réaction en chaîne par polymérase capillaire
WO2022162027A2 (fr) 2021-01-27 2022-08-04 Curevac Ag Procédé de réduction des propriétés immunostimulatrices d'arn transcrit in vitro

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001081566A2 (fr) * 2000-04-21 2001-11-01 Transgenomic, Inc. Appareil et procede de separation et de purification de polynucleotides
US20130237699A1 (en) * 2010-11-09 2013-09-12 Qiagen Gmbh Method and device for isolating and purifying double-stranded nucleic acids
WO2016091391A1 (fr) 2014-12-12 2016-06-16 Curevac Ag Molécules d'acides nucléiques artificielles destinées à améliorer l'expression de protéines
WO2016174271A1 (fr) 2015-04-30 2016-11-03 Curevac Ag Poly(n)polymérase immobilisée
WO2016193226A1 (fr) 2015-05-29 2016-12-08 Curevac Ag Procédé d'ajout de structures de coiffe à un arn au moyen d'enzymes immobilisées
WO2019077001A1 (fr) 2017-10-19 2019-04-25 Curevac Ag Nouvelles molécules d'acide nucléique artificielles
WO2020002598A1 (fr) 2018-06-28 2020-01-02 Curevac Ag Bioréacteur pour transcription in vitro d'arn
WO2022112498A1 (fr) 2020-11-27 2022-06-02 CureVac RNA Printer GmbH Dispositif de préparation d'un produit d'adn au moyen d'une réaction en chaîne par polymérase capillaire
WO2022162027A2 (fr) 2021-01-27 2022-08-04 Curevac Ag Procédé de réduction des propriétés immunostimulatrices d'arn transcrit in vitro

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