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• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical

Definitions

• the present invention relates to methods for the removal of double- and/or multistranded nucleic acid impurities from an RNA preparation, comprising the steps of incubating the RNA preparation at a pH in the range of pH 1 to pH 5, and subjecting the RNA preparation to purification to remove fragments produced by the dissociation of the double- and/or multi-stranded nucleic acid impurities.
• RNAse III A third approach for removal of dsRNA is enzymatic degradation using RNAse III.
• RNAse III is non-specific, i.e., it digests dsRNA as well as ssRNA.
• enzymes in the production process is undesirable, so this approach is not feasible for commercial-scale manufacturing of RNA.
• step (a) of the method of the present invention is performed as part of an affinity chromatography step, in which the RNA preparation is loaded onto the affinity chromatography medium at a pH in the range of pH 6 to pH 8, e.g., at a pH of about 7, and subsequently the pH is lowered to a pH in the range of pH 1 to pH 5, thus fulfilling step (a) of the method of the present invention.
• Suitable affinity ligands in this respect are not particularly limited and are known in the art. They include nucleic acids that are complementary to the target RNA, e.g., oligo-dT.
• steps (a) and (b) are separate steps that are performed subsequently in the order of first performing step (a), and then performing step (b).
• steps (a) and (b) are separate steps that are performed subsequently in this indicated order.
• step (b) can be performed at a pH that is the same as the pH used in step (a), or the pH can be increased prior to step (b).
• steps (a) and (b) can be performed concurrently, i.e., the incubation of the RNA preparation at a pH in the range of pH 1 to pH 5 according to the step (a) can be a part of the purification process to remove fragments produced by the dissociation of the double- and/or multi-stranded nucleic acid impurities in step (b).
• step (a) of the methods of the present invention can be performed while the RNA preparation is bound to a chromatographic medium, and step (b) of the methods of the present invention is effected using the chromatographic medium.
• precipitation/extraction techniques that is/are performed at a pH in the range of pH 1 to pH 5, or at a pH in the range of more than pH 5 to pH 7.
• these purification techniques can be performed at a pH in the range of pH 1 to pH 5, which range expressly includes the boundary values pH 1 and pH 5, or at a pH in a preferred range as defined above for step (a) of the method of the present invention.
• these purification techniques are performed at the same pH as step (a) of the method of the present invention.
• guanosine requires N1 protonation (pH > 1.6)
• adenosine requires unprotonated N1 (pH > 3.5)
• cytosine requires unprotonated N3 (pH > 4.2)
• uridine requires protonated N3 (pH ⁇ 9.2).
• RNA-RNA or RNA-DNA At pH ranges standardly used for RNA production, purification, and storage (i.e., pH 5 to 9), all four nucleosides are thus also suitably protonated for base-pairing with affinity ligands immobilized to chromatographic supports, e.g., poly- deoxythymidinic acid (Oligo dT).
• affinity ligands immobilized to chromatographic supports e.g., poly- deoxythymidinic acid (Oligo dT).
• a decrease in pH in accordance with the present invention disrupts the base-pairing with affinity ligand, thus releasing the target RNA molecule, while simultaneously denaturing multi- and/or double-stranded interactions (RNA-RNA or RNA-DNA).
• RNA duplexes ⁇ 20 mers
• protonation of hydrogen bonding acceptors disrupts base-pairing and leads to denaturation of RNA duplexes, measured as a decrease in the melting temperature of RNA duplexes with decreasing pH.
• low pH leads to denaturation (or at least destabilization) of nucleic acids due to protonation of G-C base pairs and resultant base pair formation.
• protonation stabilizes non-canonical interactions, e.g. A-C, and C-C in DNA duplexes. Denaturation of double-stranded DNA was shown by incubation in acidic conditions.
• RNA duplexes are not representative of therapeutically relevant RNA such as mRNA
• dsDNA is not representative of ssRNA contaminated with dsRNA, ssRNA, ssDNA or other double- and/or multi-stranded nucleic acid impurities.
• the present invention concerns the denaturation of double- and/or multi-stranded nucleic acid impurities present in RNA preparations to singlestranded form through low pH-driven strand separation, leading to disruption of canonical base-pairing.
• Double- and/or multi-stranded nucleic acid impurities are disassociated from the RNA by incubation of the RNA preparation at ambient to mildly elevated temperatures at a pH range of pH 1 to pH 5, preferably pH 2 to pH 4. This pH range is sufficient for protonation of bases involved in base-pairing, rendering them unable to support canonical base-pairing.
• the phosphate backbone remains unprotonated, and RNA thus retains an overall negative charge.
• the present invention can exploit the unusual pH range used for disruption of dsRNA and RNA-DNA double- and/or multi-stranded structures and for binding of RNA to be prepared to chromatographic media (porous particles, nanofibers, membranes, monoliths) used in ion exchange (cation and anion exchange), size exclusion, reversed phase, hydrophobic interaction, or multi-modal chromatographic supports.
• chromatographic media porous particles, nanofibers, membranes, monoliths
• ion exchange cation and anion exchange
• size exclusion size exclusion
• reversed phase hydrophobic interaction
• hydrophobic interaction hydrophobic interaction
• multi-modal chromatographic supports e.g., tangential-flow filtration devices.
• FIG. 14 mRNA (eGFP, 995 nt) was diluted in mobile phase A (50 mM Na-phosphate, 0.5 M NaCI, pH 7.5) and loaded onto Poros Oligo (dT)25 column (1 mL) at 1 mL/min. Column was washed with 9 mL of mobile phase B (50 mM Na-phosphate, pH 7.5), then eluted with a step gradient to mobile phase C (100 mM Glycine, pH 3). Elution fractions were analyzed by J2 dot-blot to confirm denaturation of dsRNA.
• mobile phase A 50 mM Na-phosphate, 0.5 M NaCI, pH 7.5
• dT Poros Oligo
• FIG. 15 mRNA (eGFP, 995 nt) was diluted in mobile phase A (50 mM Na-phosphate, 0.5 M NaCI, pH 7.5) and loaded onto Poros Oligo dT column (1 mL) at 1 mL/min. Column was washed with 9 mL of mobile phase B (50 mM Na-phosphate, pH 7.5), then eluted with a step gradient to mobile phase C (100 mM Glycine, pH 3). Resulting pH 3 eluate was then diluted in mobile phase A (10 mM Glycine, pH 3) and loaded onto CIM C4 HLD column at 1 mL/min.
• mobile phase A 50 mM Na-phosphate, 0.5 M NaCI, pH 7.5
• Poros Oligo dT column 1 mL/min.
• mRNA encoding for eGFP was incubated in 75 mM glycine, pH 3.0 at ambient temperature for 20 min. Sample was loaded onto a CIM C4 HLD column and eluted with a step gradient to 10 mM Citrate pH 5.1 ( Figure 11 , i). The starting material (mRNA untreated) and elution fraction were analyzed by AGE and dot-blot with J2 antibody ( Figure 11 , iii).

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• 2022
  • 2022-04-07 EP EP22167222.3A patent/EP4257685A1/en active Pending
• 2023
  • 2023-04-06 WO PCT/EP2023/059215 patent/WO2023194562A1/en not_active Ceased
  • 2023-04-06 US US18/850,226 patent/US20250207121A1/en active Pending
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