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WO2018141371A1 - Purification et/ou formulation d'arn - Google Patents

Purification et/ou formulation d'arn Download PDF

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Publication number
WO2018141371A1
WO2018141371A1 PCT/EP2017/052074 EP2017052074W WO2018141371A1 WO 2018141371 A1 WO2018141371 A1 WO 2018141371A1 EP 2017052074 W EP2017052074 W EP 2017052074W WO 2018141371 A1 WO2018141371 A1 WO 2018141371A1
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WO
WIPO (PCT)
Prior art keywords
rna
sample
lyophilization
solvent
butanol
Prior art date
Application number
PCT/EP2017/052074
Other languages
English (en)
Inventor
Kiriaki RAPTOPOULOU
Fabian Johannes EBER
Aniela WOCHNER
Janis Nicole KIEBEL
Original Assignee
Curevac Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Curevac Ag filed Critical Curevac Ag
Priority to PCT/EP2017/052074 priority Critical patent/WO2018141371A1/fr
Publication of WO2018141371A1 publication Critical patent/WO2018141371A1/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

Definitions

  • immunostimulatory RNA molecules e.g., WO2009095226
  • other noncoding RNAs e.g., microRNAs or RNAs suitable for genome editing
  • CRISPR/Cas9 guide RNAs CRISPR/Cas9 guide RNAs
  • Short RNA molecules can be synthesized by chemical methods, whereas long RNAs are typically produced by in vitro transcription using suitable DNA templates with a promoter and RNA polymerases, for example bacteriophage SP6, T3 or T7 RNA polymerases which are able to bind to said promoter.
  • RNA polymerases for example bacteriophage SP6, T3 or T7 RNA polymerases which are able to bind to said promoter.
  • the non-purified sample typically contains the desired RNA species alongside various contaminants, such as undesired RNA species (abortive sequences), proteins, spermidine, DNA or fragments thereof, pyrophosphates, free nucleotides, endotoxins, detergents, and organic solvents.
  • RNA-containing HPLC fractions contain an organic solvent, mostly acetonitrile, and triethylammonium acetate (TEAA), which have to be removed before the produced RNA can be applied, or before the RNA is subjected to further formulation steps.
  • organic solvent mostly acetonitrile, and triethylammonium acetate (TEAA), which have to be removed before the produced RNA can be applied, or before the RNA is subjected to further formulation steps.
  • TEAA triethylammonium acetate
  • the method is performed in a multi-well plate.
  • a multi-well plate typically used in laboratory setting is particularly preferred, such as e.g. a 12- well plate, a 24-well plate, a 48-well plate or a 96-well plate. It can furthermore be particularly preferred that the multi-well plate is a glass plate or a glass-coated plate.
  • the method is performed in a high throughput or robot-assisted setting.
  • the RNA subject to the inventive method is in vitro transcribed RNA.
  • Said in vitro transcribed RNA has furthermore been subject to an HPLC purification step in order to remove contaminants from the RNA production, where the RNA may preferably be eluted in the HPLC purification with a mixture of triethylammonium acetate and an organic solvent, such as e.g. acetonitrile.
  • said in vitro transcribed R A has been purified via HPLC using the PureMessenger® system.
  • the method may comprise a step of adjusting the RNA concentration in the sample to about 0.2 to about 2.0 g/1 prior to carrying out the lyophilizing step.
  • the lyophilizing step comprises steps of freezing, primary drying, and secondary drying.
  • the temperature of the freezing step is in the range of from about -60 to about -40°C, preferably about -50°C, more preferably -48°C. It is further preferred that the sample is cooled to said temperature at a rate of about -0.5°C / minute. Furthermore, the freezing step takes preferably between about 3 to about 6 hours, preferably between about 4 to about 5 hours, and more preferably about 4.5 hours.
  • the lyophilization step is repeated twice.
  • the RNA is dissolved in between the steps, preferably in ddH 2 0.
  • three lyophilization cycles are carried out in total, wherein the R A is dissolved in between the steps in ddH 2 0.
  • the sample is substantially free from triethylammonium acetate after the lyophilization step.
  • a co-solvent is added to the sample prior to the (first) lyophilization step.
  • Said co-solvent may be selected from the group consisting of t- Butanol, 2-Butanol, Ethanol, n-Propanol, n-Butanol, Isopropanol, Ethyl acetate, Dimethyl carbonate, Dichloromethane, Methyl ethyl ketone, Methyl isobutyl ketone, Acetone, 1- pentanol, Methyl acetate, Methanol, Carbon tetrachloride, Dimethyl sulfoxide,
  • the t-Butanol is added such that the resulting t-Butanol concentration in the sample is between about 5 and about 50% (f.c). Even more preferably, the t-Butanol is added such that the resulting t-Butanol concentration in the sample is about 20% (f.c).
  • the freeze point of the sample after t-Butanol addition is in the range of from about -50 to about -10°C.
  • the freeze point of the sample after the addition of t-Butanol is in the range of from about -30 to about -20°C.
  • the lyophilization step is repeated once if a co-solvent is used.
  • the co-solvent is only added once, namely prior to the first lyophilization step (and not prior to the second lyophilization step; for the second
  • lyophilization step ddH 2 0 is preferably used).
  • two lyophilization steps are preferably carried out if a co-solvent is present.
  • the sample is substantially free from the co-solvent after the lyophilization step.
  • the sample additionally comprises an organic solvent
  • the method additionally comprises the step of rotary vacuum concentration and/or evaporation using nitrogen, argon or carbon dioxide prior to the lyophilization step. Said concentration and/or evaporation is carried out in order to remove the organic solvent from the sample and to purify said sample from the organic solvent, respectively.
  • the rotary vacuum concentration is performed at a temperature ranging from about 10 to about 40°C, more preferably from about 10 to about 30°C and most preferably at a temperature of about 30°C, with 28°C being most preferred. It can be preferred that the rotary vacuum concentration takes between about 2 to about 10 hours, preferably about 2 to about 6 hours, more preferably about 4 hours.
  • a salt may be added to the sample, wherein said salt is preferably NaCl. NaCl may be added to a final concentration of about 10 to 2 mmol/g R A, preferably about 8 to 4 mmol/g RNA and most preferably about 6 mmol/g RNA.
  • said organic solvent is selected from the group consisting of acetonitrile, methanol, ethanol, 1-propanol, 2-propanol, ethyl acetate, tetrahydrofuran, acetone and a mixture of any of the foregoing. Most preferably, said organic solvent is acetonitrile.
  • the sample is substantially free from the organic solvent after the rotary vacuum concentration and/or evaporation.
  • the method comprises the step of adding a lyoprotectant, wherein said lyoprotectant is added prior to the final lyophilization cycle.
  • Said lyoprotectant may be selected from the group consisting of sucrose, fructose, glucose, mannose, trehalose, mannitol, polyvinylpyrrolidone, and Ficoll 70.
  • the method comprises the step of adding a complexing agent, wherein said complexing agent is added prior to the final lyophilization cycle.
  • Said complexing agent is preferably a cationic or polycationic compound, wherein said cationic or polycationic compound is preferably a cationic or polycationic peptide or protein.
  • the present invention is directed to R A purified and/or formulated according to the method described and claimed herein.
  • the present invention is directed to the RNA of the second aspect for use in therapy.
  • the present invention is directed to the use of a method according to the first aspect for the small-scale production of multiple RNAs in parallel.
  • the present invention is directed to the use of a method according to the first aspect for the screening of RNAs in parallel.
  • Figure 1 shows the effect of the number of lyophilization cycles on TEAA removal by single-solvent lyophilization from RNA samples in deep well plates. For experimental details, see Example 2.
  • Figure 2 shows the integrity of RNA in 6 different samples before (black bars) and after
  • Figure 3 shows the RNA yield in 6 different vials, for each vial before (left column), after one (second column), after two (third column) and after three (right column) lyophilization cycles.
  • Figure 3 shows the RNA yield in 6 different vials, for each vial before (left column), after one (second column), after two (third column) and after three (right column) lyophilization cycles.
  • Figure 4 shows the impact of TEAA concentration and content of t-BuOH on the freeze point of the sample. For experimental details, see Example 5.2.
  • Figure 5 shows the duration of single-solvent and co-solvent lyophilization.
  • Example 6.5 shows the RNA solubility after single solvent (right side) and co-solvent (left side) lyophilization combined with heat dissolution at 45-50 °C.
  • RNA is the usual abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e. a polymer consisting of nucleotide monomers.
  • nucleotides are usually adenosine monophosphate (AMP), uridine monophosphate (UMP), guanosine monophosphate (GMP) and cytidine monophosphate (CMP) monomers or analogues thereof (particularly as defined herein below), 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 order of the monomers i.e. the order of the bases linked to the sugar/phosphate-backbone, is called the RNA sequence.
  • RNA may be obtainable by transcription of a DNA sequence, e.g., inside a cell.
  • transcription is typically performed inside the nucleus or the mitochondria.
  • transcription of DNA usually results in the so-called premature RNA (also called pre-mRNA, precursor mRNA or heterogeneous nuclear RNA), which has to be processed into so-called messenger RNA, usually abbreviated as mRNA.
  • Processing of the premature RNA e.g. in eukaryotic organisms, comprises a variety of different posttranscriptional modifications such as splicing, 5 '-capping, polyadenylation, export from the nucleus or the mitochondria and the like. The sum of these processes is also called maturation of RNA.
  • the mature messenger RNA usually provides the nucleotide sequence that may be translated into an amino acid sequence of a particular peptide or protein.
  • a mature mRNA comprises a 5 '-cap, optionally a 5 'UTR, an open reading frame, optionally a 3 'UTR and a poly(A) tail.
  • messenger RNA several non-coding types of RNA exist which may be involved in regulation of transcription and/or translation, and immunostimulation.
  • RNA further encompasses any type of single stranded (ssRNA) or double stranded RNA (dsRNA) molecule known in the art, such as viral RNA, retroviral RNA and replicon RNA, small interfering RNA (siRNA), antisense RNA (asRNA), circular RNA (circRNA), CRISPR/Cas9 guide RNA, ribozymes, aptamers, riboswitches, immunostimulating RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA).
  • ssRNA single stranded
  • dsRNA double stranded RNA
  • viral RNA viral RNA
  • retroviral RNA and replicon RNA small interfering RNA
  • siRNA small interfering RNA
  • asRNA antisense
  • HPLC HPLC (abbreviation for "High Performance (High Pressure) Liquid
  • HPLC Chromatography
  • An HPLC apparatus consists in the simplest case of a pump with eluent reservoir containing the mobile phase, a sample application system, a separation column containing the stationary phase, and the detector.
  • a fraction collector may also be provided, with which the individual fractions may be separately collected after separation and are thus available for further applications.
  • RNA can be purified from various contaminations from previous manufacturing steps. These include buffer contaminations, protein impurities (Escherichia coli proteins, Restriction enzymes, T7-RNA-Polymerase, RNase-Inhibitor, DNase I, and BSA), impurities from RNA- RNA hybrids, from DNA-RNA hybrids or their fragments, from pDNA contaminations and bacterial genomic DNA contaminations, and solvent contaminations (Acetonitrile,
  • Reversed phase chromatography is preferably used when purifying RNA.
  • Reversed phase HPLC consists of a non-polar stationary phase and a moderately polar mobile phase. The retention time is therefore longer for molecules, which are more non-polar in nature, allowing polar molecules to elute more readily. Retention time is increased by the addition of polar solvent to the mobile phase and decreased by the addition of more hydrophobic solvent.
  • Reversed phase HPLC may comprise the use of a porous reserved phase as stationary phase, which may be provided with a particle size of 8.0 ⁇ to 50 ⁇ , in particular 8.0 to 30 ⁇ , still more preferably about 30 ⁇ .
  • the reversed phase material may be present in the form of small spheres.
  • the reversed phase may be porous and may have specific particle sizes.
  • the reversed phase has a pore size of 1000 A to 5000 A, in particular a pore size of 1000 A to 4000 A, more preferably 1500 A to 4000 A, 2000 A to 4000 A or 2500 A to 4000 A.
  • Particularly preferred pore sizes for the reversed phases are 1000 A to 2000 A, more preferably 1000 A to 1500 A and most preferably 1000 A to 1200 A or 3500-4500 A. Most preferred is a pore size of 4000 A.
  • a pore size of 1000 A to 5000 A in particular a pore size of 1000 A to 4000 A, more preferably 1500 A to 4000 A, 2000 A to 4000 A or 2500 A to 4000 A may be suitable to separate a RNA from other components of a mixture, the RNA having a size as mentioned above of up to about 15000 nucleotides (as single stranded RNA molecule) or base pairs (as double stranded RNA molecule), in particular 100 to 10000, more preferably 500 to 10000 nucleotides or base pairs, even more preferably 800 to 5000 nucleotides or base pairs and even more preferably 800 to 2000 nucleotides or base pairs.
  • a pore size for the reversed phase of about 2000 A to about 5000 A, more preferably of about 2500 to about 4000, most preferably of about 3500 to about 4500 A may thus be used to separate larger RNA molecules, e.g. RNA molecules of 100 to 10000, more preferably 500 to 10000 nucleotides or base pairs, even more preferably 800 to 5000 nucleotides or base pairs and even more preferably 800 to 2000 nucleotides or base pairs.
  • a pore size for the reversed phases of about 1000 A to about 2500 A, more preferably of about 1000 A to about 2000 A, and most preferably of about 1000 A to 1200 A may be used to separate smaller RNA molecules, e.g.
  • RNA molecules of about 30- 1000, 50-1000 or 100-1000 or 20-200, 20-100, 20-50 or 20-30 nucleotides may also be separated in this way.
  • any material known to be used as reverse phase stationary phase in particular any polymeric material may be used, if that material can be provided in porous form.
  • the stationary phase may be composed of organic and/or inorganic material.
  • polymers examples include (non-alkylated) polystyrenes, (non-alkylated) polystyrenedivinylbenzenes, silica gel, silica gel modified with non-polar residues, particularly silica gel modified with alkyl containing residues, more preferably with butyl-, octyl and/or octadecyl containing residues, silica gel modified with phenylic residues, polymethacrylates, etc. or other materials suitable e.g. for gel chromatography or other chromatographic methods as mentioned above, such as dextran, including e.g. Sephadex® and Sephacryl® materials, agarose, dextran/agarose mixtures, polyacrylamide, etc.
  • dextran including e.g. Sephadex® and Sephacryl® materials, agarose, dextran/agarose mixtures, polyacrylamide, etc.
  • the material for the reversed phase is a porous polystyrene polymer, a (non- alkylated) (porous) polystyrenedivinylbenzene polymer, porous silica gel, porous silica gel modified with non-polar residues, particularly porous silica gel modified with alkyl containing residues, more preferably with butyl-, octyl and/or octadecyl containing residues, porous silica gel modified with phenylic residues, porous polymethacrylates, wherein in particular a porous polystyrene polymer or a non-alkylated (porous)
  • polystyrenedivinylbenzene may be used. Stationary phases with polystyrenedivinylbenzene are known per se and may be used.
  • a non-alkylated porous polystyrenedivinylbenzene is one which, without being limited thereto, may have in particular a particle size of 8.0 ⁇ 1.5 ⁇ , in particular 8.0 ⁇ 0.5 ⁇ , and a pore size of 1000- 1500 A, in particular 1000-1200 A or 3500-4500 A and most preferably a particle size of 4000 A.
  • This stationary phase described in greater detail above is conventionally located in a column.
  • V2A steel is conventionally used as the material for the column, but other materials may also be used for the column provided they are suitable for the conditions prevailing during HPLC.
  • the column is straight. It is favorable for the HPLC column to have a length of 5 cm to 100 cm and a diameter of 4 mm to 50 cm.
  • Columns may in particular have the following dimensions: 25 cm long and 20 mm in diameter or 25 cm long and 50 mm in diameter, or 25 cm long and 10 cm in diameter or any other dimension with regard to length and diameter, which is suitable for preparative recovery of RNA, even lengths of several metres and also larger diameters being feasible in the case of upscaling.
  • the dimensions are here geared towards what is technically possible with liquid chromatography.
  • the mobile phase depends on the type of separation desired. This means that the mobile phase established for a specific separation, as may be known for example from the prior art, cannot be straightforwardly applied to a different separation problem with a sufficient prospect of success.
  • the ideal elution conditions, in particular the mobile phase used have to be determined by empirical testing.
  • a mixture of an aqueous solvent and an organic solvent is used as the mobile phase for eluting the RNA. It is favorable for a buffer to be used as the aqueous solvent which has in particular a pH of 6.0-8.0, for example of about 7, for example.
  • the buffer is triethylammonium acetate (TEAA), particularly preferably a 0.02 M to 0.5 M, in particular 0.08 M to 0.12 M, very particularly an about 0.1 M TEAA buffer, which also acts as a counterion to the RNA in the ion pair method.
  • TEAA triethylammonium acetate
  • the organic solvent which is used in the mobile phase comprises acetonitrile, methanol, ethanol, 1-propanol, 2-propanol, ethyl acetate, tetrahydrofuran and acetone or a mixture thereof, very particularly preferably acetonitrile.
  • the mobile phase is a mixture of 0.1 M triethylammonium acetate, pH 7, and acetonitrile.
  • the mobile phase it has proven favorable for the mobile phase to contain 5.0 vol.% to 25.0 vol.% organic solvent, relative to the mobile phase, and for this to be made up to 100 vol.% with the aqueous solvent.
  • the proportion of organic solvent is increased, in particular by at least 10%, more preferably by at least 50% and most preferably by at least 100%, optionally by at least 200%, relative to the initial vol.% in the mobile phase.
  • the proportion of organic solvent in the mobile phase amounts in the course of HPLC separation to 3 to 9, preferably 4 to 7.5, in particular 5.0 vol.%, in each case relative to the mobile phase.
  • the proportion of organic solvent in the mobile phase is increased in the course of HPLC separation from 3 to 9, in particular 5.0 vol.% to up to 20.0 vol.%, in each case relative to the mobile phase. Still more preferably, the method is performed in such a way that the proportion of organic solvent in the mobile phase is increased in the course of HPLC separation from 6.5 to 8.5, in particular 7.5 vol.%, to up to 17.5 vol.%, in each case relative to the mobile phase.
  • the mobile phase may contain 7.5 vol.% to 17.5 vol.%) organic solvent, relative to the mobile phase, and for this to be made up to 100 vol.%) with the aqueous buffered solvent.
  • the elution may proceed isocratically or by means of gradient separation.
  • isocratic separation elution of the RNA proceeds with a single eluent or a constant mixture of a plurality of eluents, wherein the solvents described above in detail may be used as eluent.
  • the composition of the eluent is varied by means of a gradient program.
  • the equipment necessary for gradient separation is known to a person skilled in the art.
  • Gradient elution may here proceed either on the low pressure side by mixing chambers or on the high pressure side by further pumps.
  • the proportion of organic solvent is increased relative to the aqueous solvent during gradient separation.
  • the above-described agents may here be used as the aqueous solvent and the likewise above-described agents may be used as the organic solvent.
  • the proportion of organic solvent in the mobile phase may be increased in the course of HPLC separation from 5.0 vol.% to 20.0 vol.%, in each case relative to the mobile phase.
  • the proportion of organic solvent in the mobile phase may be increased in the course of HPLC separation from 7.5 vol.% to 17.5 vol.%, in particular 9.5 to 14.5 vol.%, in each case relative to the mobile phase.
  • the flow rate of the eluent is so selected that good separation of the RNA from the other constituents contained in the sample to be investigated takes place.
  • the eluent flow rate may amount to from 1 ml/min to several litres per minute (in the case of upscaling), in particular about 1 to 1000 ml/min, more preferably 5 ml to 500 ml/min, even more preferably more than 100 ml/min, depending on the type and scope of the upscaling.
  • This flow rate may be established and regulated by the pump.
  • Detection proceeds favorably with a UV detector at 254 nm, wherein a reference measurement may be made at 600 nm. However, any other detection method may be used.
  • the HPLC is performed as described in WO2008077592 in order to obtain the HPLC sample comprising the RNA.
  • sample comprising RNA and triethylammonium acetate (and optionally acetonitrile):
  • sample describes a sample or fraction obtained after purifying RNA via HPLC as set out above. An HPLC sample will therefore be essentially free of the
  • RNA contaminants removed by HPLC e.g. small RNA contaminants (e.g. abortive RNA sequences), proteins, residual nucleotides and the residuals of the DNA template after its hydrolysis.
  • the HPLC sample will comprise RNA molecules, as well triethylammonium acetate (and optionally also acetonitrile) as remnants / contaminants from the mobile phase described above.
  • the focus of the present invention is on the removal of the contaminant triethylammonium acetate since this is generally more difficult to achieve compared to the removal of the acetonitrile.
  • Purification/purifying The terms "purification”, “purified” or “purifying” as used herein mean that RNA is separated and/or isolated from unwanted contaminants.
  • HPLC is used to purify RNA from by-products and components of the RNA production (e.g. chemical synthesis, RNA in vitro transcription).
  • the RNA is separated and/or isolated by HPLC from the by-products and the components of the RNA in vitro transcription reaction present in the sample after the RNA in vitro transcription reaction is complete.
  • the purified RNA sample has a higher purity than the RNA-containing sample after the production and prior to purification.
  • the HPLC sample can then further be purified by a method as claimed herein by removing triethylammonium acetate (and optionally also the acetonitrile), which was introduced during the HPLC purification.
  • the purified HPLC sample has a higher RNA purity than the HPLC sample prior to purification, i.e. the amount of TEAA (and optionally also of the acetonitrile) is substantially lower, if not completely absent.
  • the content of acetonitrile will be lower than 25%, more preferably lower than 20%, lower than 15%, lower than 10%, lower than 5%, lower than 2.5%, lower than 1%.
  • the purified HPLC sample will be substantially free of acetonitrile.
  • the content of TEAA will be lower than 5 g per g RNA, more preferably lower than 4 g per g RNA, lower than 3 g per g RNA, lower than 2 g per g RNA, and even more preferably lower than 1 g per g RNA.
  • the purified HPLC sample will be substantially free of acetonitrile.
  • substantially free of is used to describe a sample from which virtually all of a specific substance has been removed. Specifically, a sample is designated to be substantially free of acetonitrile at a concentration of ⁇ about 1000 ppm (preferably at a concentration of ⁇ about 400 ppm), substantially free of TEAA at a concentration of ⁇ about 1.0 g per g RNA (preferably at a concentration of ⁇ about 0.5 g per g RNA), and substantially free of t-Butanol at a concentration of ⁇ about 0.10 g per g RNA (preferably at a
  • Formulation/ formulating describes the process in which different components, including an active component, are combined to produce a final medicinal or therapeutic product.
  • formulation is often used synonymous with a dosage form in the meaning of the dosage form ready for use.
  • formulation can also refer to the state of each component when these components are combined to result in a medicinal or therapeutic product.
  • RNA as the active component is formulated in a specific way prior to combining the RNA with the further components of the dosage form to be applied.
  • Lyophilization is often a preferred formulation for therapeutic components, such as nucleic acids and in particular RNAs, because the long-term stability of many materials increases in the lyophilized state.
  • RNA in particular providing the RNA in a lyophilized form prepared according to the inventive method as one component of a dosage form.
  • the lyophilized RNA formulated according to the present invention may then be dissolved in ddH 2 0 or buffer or the like during this preparation process.
  • Lyoprotectants and/or complexing agents may be added prior to the final lyophilization when generating formulated RNA, which may accordingly be provided as complexed RNA and/or as RNA additionally comprising a lyoprotectant (e.g. trehalose).
  • a lyoprotectant e.g. trehalose
  • Rotary vacuum concentration is based on lowering the pressure above a bulk liquid, which in turn lowers the boiling points of the component liquids in it.
  • the component liquids of interest in applications of rotary evaporation are solvents that one desires to remove from a sample after an extraction, such as following a natural product isolation or a step in an organic synthesis. Liquid solvents can be removed without excessive heating of what are often complex and sensitive solvent-solute
  • Rotary evaporation is most often and conveniently applied to separate "low boiling" solvents such a n-hexane or ethyl acetate from compounds which are solid at room temperature and pressure.
  • "low boiling” solvents such as n-hexane or ethyl acetate
  • careful application also allows removal of a solvent from a sample containing a liquid compound if there is minimal co-evaporation (azeotropic behavior), and a sufficient difference in boiling points at the chosen temperature and reduced pressure.
  • Rotary vacuum evaporation using centrifuges in particular enables high throughput settings, i.e. the processing of many samples in parallel, for example in multi well plates.
  • a liquid sample subjected to RVC may be referred to as "concentrated" sample since the sample is more concentrated after RVC then prior to this step.
  • Lyophilization/lyophilizing describe a dehydration process typically used to preserve a perishable material or make the material more convenient for transport. It is also referred to as freeze-drying or cryodessication.
  • Lyophilization works by freezing the material and then reducing the surrounding pressure to allow the frozen water in the material to sublimate directly from the solid phase to the gas phase. It usually comprises three stages: freezing, primary drying, and secondary drying.
  • the material is cooled to below its triple point, the lowest temperature at which the solid and liquid phases of the material can coexist. This ensures that sublimation rather than melting will occur in the following steps. Larger crystals are easier to freeze-dry. To produce larger crystals, the product should be frozen slowly or can be cycled up and down in temperature. This cycling process is called annealing. Usually, the freezing temperatures are between -50 °C and -80 °C (-58 °F and -112 °F). The freezing phase is the most critical in the whole freeze-drying process. During the primary drying phase, the pressure is lowered to the range of a few millibars, and enough heat is supplied to the material for the ice to sublime.
  • the amount of heat necessary can be calculated using the sublimating molecules' latent heat of sublimation.
  • this phase may be slow (in some instances even up to several days), because, if too much heat is added, the material's structure could be altered.
  • pressure is controlled through the application of partial vacuum. The vacuum speeds up the sublimation, making it useful as a deliberate drying process.
  • the secondary drying phase aims to remove unfrozen water molecules, since the ice was removed in the primary drying phase. This part of the freeze-drying process is governed by the material's adsorption isotherms. In this phase, the temperature is raised higher than in the primary drying phase, and can even be above 0 °C, to break any physico-chemical interactions that have formed between the water molecules and the frozen material. Usually the pressure is also lowered in this stage to encourage desorption.
  • the final residual water content in the product is extremely low, around 1% to 4%, which leads to prolonged shelf life at room temperature, so long as the product is protected from reabsorption of moisture.
  • Resistivity The term grasping during lyophilization, specifically during primary drying, to avoid melting of samples. If a sensor drops under a designated value, heating is switched off and the freezing of samples starts again.
  • Co-solvent A solvent is a substance that dissolves a solute (a chemically distinct liquid, solid or gas, e.g. R A) resulting in a solution.
  • a solvent is usually a liquid but can also be a solid or a gas.
  • the quantity of solute that can dissolve in a specific volume of solvent varies with temperature.
  • the term corpco-solvent describes a second solvent that is added (usually concomitantly) with a first solvent, for example pre-mixed. For the present invention, the mixture of the first solvent ddH 2 0 and the second solvent (i.e. the "co-solvent”) t-Butanol is particularly preferred.
  • Freeze point The "freeze point” or “freezing point” or “crystallization point” is the temperature at which a liquid changes state from liquid to solid.
  • Multi well plate describes a plate with 6, 12, 24, 48, 96, 192, 384, or 1536 wells for sample containment. Some multi well plates have 3456 or 9600 wells. Multiwell plates may be deep well plates, which typically can hold larger volumes than standard well plates, e.g.1 to 2 ml volumes in a 96 well plate format. Multi well plates most commonly are made from plastic materials, e.g. polystyrene, polypropylene, polycarbonate, cyclo-olefms, but can also be made from glass or quartz, or from plastic coated with a thin layer of glass or quartz. Multi well plates allow for processing of several samples in parallel, keeping conditions and handling times consistent for comparison between samples. They make possible high throughput settings, e.g. high throughput screening, sequencing, purification, formulation, etc.
  • High throughput setting robot-assisted setting: The term "high throughput setting" describes any setting in which several, often up to thousands, of samples are quickly processed in parallel using multi well plates, with consistent experimental or production conditions. This leads to the advantages of consistency of results or products across large numbers of samples and shortening of experimental and production processes.
  • Dilution/ diluting The terms “dilution” and “diluting” describe the reduction of the concentration of a compound, e.g. RNA, in a liquid sample, typically by increasing the volume of the sample by addition of a diluent, e.g. water.
  • a diluent e.g. water.
  • Readily soluble The solubility of lyophilized RNA samples can be measured by determining the RNA content of samples that had been re-dissolved in water under identical conditions, and the coefficient of variation is determined.
  • the ratio of samples that show a higher coefficient of variation than 5 % is used as a measure for the "insolubility" of the RNA samples ("CV"), i.e. a sample with a CV of ⁇ 5% is considered “readily soluble”.
  • the term “therapy” is used to designate the administration of nucleic acids, e.g. RNA, to a subject in need thereof, for example for the purpose of immunization or prophylaxis or treatment of a medical condition.
  • the term “therapeutic” is used to describe nucleic acids, e.g. RNA, that are suitable for such a therapy.
  • Such therapeutic nucleic acids comprise e.g. mRNA molecules encoding antigens for use as vaccines (Fotin-Mleczek et al. 2012. J. Gene Med. 14(6):428-439), RNA molecules for replacement therapies (Thess, et al.
  • RNA molecules for the provision of RNA-coded antibodies for passive immunization or cancer immunotherapies e.g., WO2008083949
  • noncoding immunostimulatory RNA molecules e.g., WO2009095226
  • other noncoding RNAs such as microRNAs or RNAs suitable for genome editing (e.g., CRISPR/Cas9 guide RNAs).
  • oligonucleotides with defined chemical structure provides a rapid and inexpensive access to custom-made oligonucleotides of any desired sequence.
  • enzymes synthesize DNA and RNA only in the 5' to 3' direction
  • chemical oligonucleotide synthesis does not have this limitation, although it is most often carried out in the opposite, i.e. the 3' to 5' direction.
  • the process is implemented as solid-phase synthesis using the phosphoramidite method and phosphoramidite building blocks derived from protected nucleosides (A, C, G, and U), or chemically modified nucleosides.
  • the building blocks are sequentially coupled to the growing oligonucleotide chain on a solid phase in the order required by the sequence of the product in a fully automated process.
  • the product is released from the solid phase to the solution, deprotected, and collected.
  • the occurrence of side reactions sets practical limits for the length of synthetic oligonucleotides (up to about 200 nucleotide residues), because the number of errors increases with the length of the
  • oligonucleotide being synthesized. Products are often isolated by HPLC to obtain the desired oligonucleotides in high purity.
  • oligonucleotides find a variety of applications in molecular biology and medicine. They are most commonly used as antisense oligonucleotides, small interfering RNA, primers for DNA sequencing and amplification, probes for detecting complementary DNA or RNA via molecular hybridization, tools for the targeted introduction of mutations and restriction sites, and for the synthesis of artificial genes.
  • DNA is the usual abbreviation for deoxyribonucleic acid. It is a nucleic acid molecule, i.e. a polymer consisting of nucleotide monomers. These nucleotides are usually deoxy- adenosine-monophosphate, deoxy-thymidine-monophosphate, deoxy-guanosine- monophosphate and deoxy-cytidine-monophosphate monomers or analogs thereof which are - by themselves - composed of a sugar moiety (deoxyribose), a base moiety and a phosphate moiety, and polymerize by a characteristic backbone structure.
  • the backbone structure is, typically, formed by phosphodiester bonds between the sugar moiety of the nucleotide, i.e. deoxyribose, of a first and a phosphate moiety of a second, adjacent monomer.
  • the specific order of the monomers i.e. the order of the bases linked to the sugar/phosphate-backbone, is called the DNA-sequence.
  • DNA may be single-stranded or double-stranded.
  • the nucleotides of the first strand typically hybridize with the nucleotides of the second strand, e.g. by A/T -base-pairing and G/C-base-pairing.
  • in vitro transcription relates to a process wherein RNA is synthesized in a cell-free system (in vitro). DNA is used as template for the generation of RNA transcripts.
  • the promoter also referred to herein as "promoter sequence” for controlling in vitro transcription can be any promoter for any DNA dependent RNA polymerase (referred to herein also as "RNA polymerase").
  • RNA polymerase DNA dependent RNA polymerase
  • Particular examples of DNA dependent RNA polymerases are the T7, T3, and SP6 RNA polymerases.
  • a DNA template for in vitro RNA transcription may be obtained by cloning of a nucleic acid, in particular cDNA corresponding to the target RNA to be in vitro transcribed, and introducing it into an appropriate DNA for in vitro transcription, for example into plasmid DNA.
  • the cDNA may be obtained by reverse transcription of mRNA, chemical synthesis, or oligonucleotide cloning.
  • the DNA template for in vitro RNA synthesis may also be obtained by gene synthesis.
  • RNA polymerase such as bacteriophage-encoded RNA polymerases
  • NTPs ribonucleoside triphosphates
  • a cap analog as defined below e.g. m7G(5')ppp(5')G (m7G)
  • RNA-dependent RNA polymerase capable of binding to the promoter sequence within the DNA template (e.g. T7, T3 or SP6 RNA polymerase);
  • RNase ribonuclease
  • pyrophosphatase to degrade pyrophosphate, which may inhibit transcription
  • MgCl 2 which supplies Mg 2+ ions as a co-factor for the polymerase
  • the (transcription) buffer may be selected from the group consisting of 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES) and tris(hydroxymethyl)aminomethane (Tris).
  • HEPES 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid
  • Tris tris(hydroxymethyl)aminomethane
  • the buffer is used at a concentration from 10 to 100 mM, 10 to 75 mM, 10 to 50 mM, 10 to 40 mM, 10 to 30 mM or 10 to 20 mM.
  • the pH value of the buffer can be adjusted with, for example, NaOH, KOH or HC1.
  • the buffer has a pH value from 6 to 8.5, from 6.5 to 8.0, from 7.0 to 7.5, even more preferred 7.5.
  • Most preferred is a buffer selected from the group consisting of 80 mM HEPES/KOH, pH 7.5 and 40 mM Tris/HCl, pH 7.5.
  • the RNA polymerase is preferably selected from the group consisting of T3, T7 and SP6 RNA polymerase.
  • the concentration of the RNA polymerase is from about 1 to 100 nM, 1 to 90 nM, 1 to 80 nM, 1 to 70 nM, 1 to 60 nM, 1 to 50 nM, 1 to 40 nM, 1 to 30 nM, 1 to 20 nM, or about 1 to 10 nM. Even more preferred, the concentration of the RNA polymerase is from about 10 to 50 nM, 20 to 50 nM, or 30 to 50 nM. Most preferred is a RNA polymerase concentration of about 40 nM.
  • the concentration of the RNA polymerase is between 1 and 1000 U/ ⁇ g template DNA, preferably between 10 and 100 U/ ⁇ g DNA, particularly if plasmid DNA is used as template DNA.
  • the in vitro transcription is preferably performed in the presence of pyrophosphatase.
  • the concentration of the pyrophosphatase is from about 1 to 20 units/ml, 1 to 15 units/ml, 1 to 10 units/ml, 1 to 5 units/ml, or 1 to 2.5 units/ml. Even more preferred the concentration of the pyrophosphatase is about 5 units/ml.
  • the in vitro transcription reaction mixture preferably comprises Mg 2+ ions.
  • the Mg 2+ ions are provided in the form of MgCl 2 or Mg(OAc) 2 .
  • the initial free Mg 2+ concentration is from about 1 to 100 mM, 1 to 75 mM, 1 to 50 mM, 1 to 25 mM, or 1 to 10 mM.
  • the initial free Mg 2+ concentration is from about 10 to 30 mM or about 15 to 25 mM. Most preferred is an initial free Mg 2+ concentration of about 24 mM.
  • the person skilled in the art will understand that the choice of the Mg 2+ concentration is influenced by the initial total NTP concentration.
  • the in vitro transcription reaction mixture preferably comprises a reducing agent
  • the reducing agent is selected from the group consisting of dithiothreitol (DTT), dithioerythritol (DTE), Tris(2- carboxyethyl)phosphine (TCEP) and ⁇ -mercaptoethanol.
  • DTT dithiothreitol
  • DTE dithioerythritol
  • TCEP Tris(2- carboxyethyl)phosphine
  • concentration of the reducing reagent is from about 1 to 50 mM, 1 to 40 mM, 1 to 30 mM, or 1 to 20 mM, or 1 to 10 mM. Even more preferred the concentration of the reducing reagent is from 10 to 50 mM or 20 to 40 mM. Most preferred is a concentration of 40 mM of DTT.
  • the in vitro transcription reaction mixture preferably comprises a polyamine.
  • the polyamine is selected from the group consisting of spermine and spermidine.
  • concentration of the polyamine is from about 1 to 25 mM, 1 to 20 mM, 1 to 15 mM, 1 to 10 mM, 1 to 5 mM, or about 1 to 2.5 mM. Even more preferred the concentration of the polyamine is about 2 mM. Most preferred is a concentration of 2 mM of spermidine.
  • the in vitro transcription reaction mixture preferably comprises a ribonuclease inhibitor.
  • concentration of the ribonuclease inhibitor is from about 1 to 500 units/ml, 1 to 400 units/ml, 1 to 300 units/ml, 1 to 200 units/ml, or 1 to 100 units/ml. Even more preferred the concentration of the ribonuclease inhibitor is about 200 units/ml.
  • the total NTP concentration in the in vitro transcription reaction mixture may be between 1 and 100 mM, preferably between 10 and 50 mM, and most preferably between 10 and 20 mM.
  • the term total nucleotide concentration means the total concentration of NTPs, e.g. the sum of the concentrations of ATP, GTP, CTP, UTP, and/or cap analog present initially in the in vitro transcription when the various components of the reaction have been assembled in the final volume for carrying out the in vitro transcription reaction.
  • the nucleotides will be incorporated into the RNA molecule and consequently the total nucleotide concentration will be progressively reduced from its initial value.
  • the single nucleotides are provided in a concentration between 0.1 and 10 mM, preferably between 1 and 5 mM and most preferably in a
  • the in vitro transcription reaction mixture preferably further comprises a cap analog.
  • concentration of GTP is preferably reduced compared to the other nucleotides (ATP, CTP and UTP).
  • the cap analog is added with an initial concentration in the range of about 1 to 20 mM, 1 to 17.5 mM, 1 to 15 mM, 1 to 12.5 mM, 1 to 10 mM, 1 to 7.5 mM.
  • the cap analog is added in a concentration of 5.8 mM and the GTP concentration is reduced to a concentration of 1.45 mM whereas ATP, CTP and UTP are comprised in the reaction in a concentration of 4 mM each.
  • the ribonucleoside triphosphates (NTPs) GTP, ATP, CTP and UTP or analogs thereof may be provided with a monovalent or divalent cation as counterion.
  • the monovalent cation is selected from the group consisting of Li + , Na + , K + , NH4 + or tris(hydroxymethyl)- amino methane (Tris).
  • the divalent cation is selected from the group consisting of Mg 2+ , Ba 2+ and Mn 2+ .
  • a part or all of at least one ribonucleoside triphosphate in the in vitro transcription reaction mixture may be replaced with a modified nucleoside triphosphate (as defined herein).
  • said modified nucleoside triphosphate is selected from the group consisting of pseudouridine-5 '-triphosphate, l-methylpseudouridine-5 '-triphosphate, 2- thiouridine-5 '-triphosphate, 4-thiouridine-5 '-triphosphate and 5-methylcytidine-5 '- triphosphate.
  • modified nucleotides which can be used in this context are listed below.
  • the DNA template can optionally be removed using methods known in the art comprising DNase I digestion.
  • DNase I digestion it is particularly preferred to add 6 ⁇ DNAse I (1 mg/ml) and 0.2 ⁇ CaCb solution (0.1 M) / ⁇ g DNA template to the transcription reaction, and to incubate it for at least 3 h at 37°C.
  • Nucleoside/Nucleotide are glycosylamines that correspond to nucleotides without a phosphate group.
  • a nucleoside consists simply of a nucleobase (also termed a nitrogenous base) and a 5-carbon sugar (either ribose or deoxyribose), whereas a nucleotide is composed of a nucleobase, a five-carbon sugar, and one or more phosphate groups.
  • the base is bound to either ribose or deoxyribose via a beta-glycosidic linkage.
  • Examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine and inosine.
  • Modified nucleoside triphosphate refers to chemical modifications comprising backbone modifications as well as sugar modifications or base modifications. These modified nucleoside triphosphates are also termed herein as (nucleotide) analogs, modified nucleosides/nucleotides or nucleotide/nucleoside modifications.
  • modified nucleoside triphosphates as defined herein are nucleotide analogs/modifications, e.g. backbone modifications, sugar modifications or base
  • a backbone modification in connection with the present invention is a modification, in which phosphates of the backbone of the nucleotides are chemically modified.
  • a sugar modification in connection with the present invention is a chemical modification of the sugar of the nucleotides.
  • a base modification in connection with the present invention is a chemical modification of the base moiety of the nucleotides.
  • nucleotide analogs or modifications are preferably selected from nucleotide analogs which are applicable for transcription and/or translation.
  • modified nucleosides and nucleotides which may be used in the context of the present invention, can be modified in the sugar moiety.
  • the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy” substituents.
  • R H, alkyl, cycloalkyl, aryl
  • “Deoxy” modifications include hydrogen, amino (e.g. NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); or the amino group can be attached to the sugar through a linker, wherein the linker comprises one or more of the atoms C, N, and O.
  • the sugar group can also contain one or more carbons that possess the opposite
  • a modified nucleotide can include nucleotides containing, for instance, arabinose as the sugar.
  • the phosphate backbone may further be modified in the modified nucleosides and nucleotides.
  • the phosphate groups of the backbone can be modified by replacing one or more of the oxygen atoms with a different substituent.
  • the modified nucleosides and nucleotides can include the full replacement of an unmodified phosphate moiety with a modified phosphate as described herein.
  • modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.
  • Phosphorodithioates have both non-linking oxygens replaced by sulfur.
  • the phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylene-phosphonates).
  • the modified nucleosides and nucleotides can further be modified in the nucleobase moiety.
  • nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine and uracil.
  • the nucleosides and nucleotides described herein can be chemically modified on the major groove face.
  • the major groove chemical modifications can include an amino group, a thiol group, an alkyl group, or a halo group.
  • the nucleotide analogs/modifications may be selected from base modifications, which are preferably selected from 2-amino-6-chloropurineriboside-5'-triphosphate, 2-Aminopurine- riboside-5 '-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-Amino-2'-deoxycytidine- triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'- Fluorothymidine-5 '-triphosphate, 2'-0-Methyl inosine-5 '-triphosphate 4-thiouridine-5'- triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5- bromocytidine-5 '-triphosphate, 5-bromouridine-5'-triphosphate, 5-Bromo-2'-deoxycytidine-5'- triphosphate,
  • nucleotides for base modifications selected from the group of base-modified nucleotides consisting of 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate.
  • Modified nucleosides include pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza- uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3- methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl- uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl- pseudouridine, 4-thio- 1-methyl-pseudouridine, 2-thio-l-methyl-pseudouridine, 1 -methyl- 1- deaza-pseudouridine, 2-thio- 1
  • dihydropseudouridine 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2- methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio- pseudouridine.
  • Modified nucleosides further include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5- methyl- cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-l- methyl-l-deaza-pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocytidine, zebularine, 5-aza- zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-
  • Modified nucleosides also include 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7- deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6- diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6- dimethyladeno
  • modified nucleosides include inosine, 1 -methyl- ino sine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza- guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2- dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.
  • the nucleotide can be modified on the major groove face and can include replacing hydrogen on C-5 of uracil with a methyl group or a halo group.
  • a modified nucleoside is 5 '-O-(l-Thiophosphate)- Adenosine, 5'-0-(l-Thiophosphate)-Cytidine, 5'-0-(l- Thiophosphate)-Guanosine, 5'-0-(l-Thiophosphate)-Uridine or 5'-0-(l-Thiophosphate)- Pseudouridine.
  • the modified nucleotides may include nucleoside modifications selected from 6-aza-cytidine, 2-thio-cytidine, a-thio-cytidine, Pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, Nl-methyl-pseudouridine, 5,6-dihydrouridine, a-thio -uridine, 4-thio-uridine, 6-aza-uridine, 5- hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine, a-thio- guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine, 7-deaza-guanosine, Nl- methyl-adenosine, 2-amino-6-Chloro-purine, N6-methyl-2-amino-purine, Pseudo-
  • RNA integrity The term "integrity" describes whether the complete RNA molecule is obtained and maintained. Low integrity could be due to, amongst others, degradation, cleavage, incorrect basepairing, incorporation of modified nucleotides or the modification of already incorporated nucleotides, lack of or incomplete capping, lack of or incomplete polyadenylation, or incomplete transcription. It can be measured, e.g., by capillary
  • Lyoprotectant typically refers to an excipient, which partially or totally replaces the hydration sphere around the RNA molecule and thus prevents catalytic and/or hydrolytic processes leading to damage of the RNA or a less stable RNA lyophilisate.
  • Suitable lyoprotectants in the context of the invention comprise monosaccharides, such as e.g. glucose, fructose, galactose, sorbose, mannose, etc., and mixtures thereof; disaccharides, such as e.g. lactose, maltose, sucrose, trehalose, cellobiose, etc., and mixtures thereof;
  • polysaccharides such as raffmose, melezitose, maltodextrins, dextrans, dextrins, cellulose, starches, etc., and mixtures thereof; and alditols, such as glycerol, mannitol, xylitol, maltitol, lactitol, xylitol sorbitol, pyranosyl sorbitol, myoinositol, etc., and mixtures thereof.
  • a sugar that is preferred in this context has a high water displacement activity and a high glass transition temperature.
  • a suitable sugar is preferably hydrophilic but not hygroscopic.
  • the sugar preferably has a low tendency to crystallize, such as trehalose.
  • a lyoprotectant of the inventive method is preferably selected from the group consisting of mannitol, sucrose, glucose, mannose and trehalose. Trehalose is particularly preferred as a lyoprotectant.
  • any of the below defined further components may be used as lyoprotectant.
  • Particularly alcohols such as PEG, mannitol, sorbitol, cyclodextran, DMSO, amino acids and proteins such as prolin, glycine, phenylanaline, arginine, serine, albumin and gelatine may be used as lyoprotectant.
  • metal ions, surfactans and salts as defined below may be used as lyoprotectant.
  • polymers may be used as lyoprotectant, particularly po lyviny lpyrro lidone .
  • the weight ratio of R A to the lyoprotectant is preferably in a range from about 1 :2000 to about 1 : 10, more preferably from about 1 : 1000 to about 1 : 100, more preferably a in a range from about 1 :250 to about 1 : 10 and more preferably in a range from about 1 : 100 to about 1 : 10 and most preferably in a range from about 1 : 100 to about 1 :50.
  • a preferred complexing agent in the present invention is a "cationic or polycationic compound".
  • This compound in the context of the present invention comprises cationic or polycationic polymers, cationic or polycationic peptides or proteins, e.g.
  • protamine cationic or polycationic polysaccharides and/or cationic or polycationic lipids.
  • Cationic or polycationic compounds being particularly preferred agents in this context include protamine, nucleoline, spermine or spermidine, or other cationic peptides or proteins, such as poly-L-lysine (PLL), poly-arginine, basic polypeptides, cell penetrating peptides (CPPs), including HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22 derived or analog peptides, HSV VP22 (Herpes simplex), MAP, KALA or protein
  • transduction domains PTDs
  • PpT620 prolin-rich peptides
  • arginine-rich peptides lysine- rich peptides
  • MPG-peptide(s) Pep-1, L-oligomers
  • Calcitonin peptide(s) Antennapedia- derived peptides (particularly from Drosophila antennapedia), pAntp, plsl, FGF, Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(l), pVEC, hCT-derived peptides, SAP, or histones.
  • the mRNA/RNA according to the invention is complexed with one or more polycations, preferably with protamine or oligofectamine, most preferably with protamine. In this context, protamine is particularly preferred.
  • cationic or polycationic compounds may include cationic polysaccharides, for example chitosan, polybrene, cationic polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g.
  • cationic polysaccharides for example chitosan, polybrene, cationic polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g.
  • PEI polyethyleneimine
  • DOTMA [l-(2,3- sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Choi, BGTC, CTAP, DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol- amine, DOSPA, DODAB, DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP: dioleoyloxy-3- (trimethylammonio)propane, DC-6-14: 0,0-ditetradecanoyl-N-(- trimethylammonioacetyl)diethanolamine chloride, CLIPl : rac-[(2,3-dioctadecyloxypropyl)(2- hy droxy ethyl)]
  • modified polyaminoacids such as alpha-aminoacid-polymers or reversed polyamides, etc.
  • modified poly ethylenes such as PVP (poly(N-ethyl-4-vinylpyridinium bromide)), etc.
  • modified acrylates such as pDMAEMA (poly(dimethylamino ethyl methylacrylate)), etc.
  • modified amidoamines such as pAMAM (poly(amidoamine)), etc.
  • modified polybetaaminoester (PBAE) such as diamine end modified 1,4 butanediol diacrylate-co-5-amino-l-pentanol polymers, etc.
  • dendrimers such as polypropylamine dendrimers or pAMAM based dendrimers, etc.
  • polyimine(s) such as PEI:
  • poly(ethyleneimine), poly(propyleneimine), etc. polyallylamine, sugar backbone based polymers, such as cyclodextrin based polymers, dextran based polymers, chitosan, etc., silan backbone based polymers, such as PMOXA-PDMS copolymers, etc., blockpolymers consisting of a combination of one or more cationic blocks (e.g. selected from a cationic polymer as mentioned above) and of one or more hydrophilic or hydrophobic blocks (e.g. polyethyleneglycole); etc.
  • cationic blocks e.g. selected from a cationic polymer as mentioned above
  • hydrophilic or hydrophobic blocks e.g. polyethyleneglycole
  • the ratio of the RNA molecules as defined herein to the cationic or polycationic compound and/or the polymeric carrier, preferably as defined herein, is selected from a range of about 6: 1 (w/w) to about 0,25: 1 (w/w), more preferably from about 5: 1 (w/w) to about 0,5 : 1 (w/w), even more preferably of about 4: 1 (w/w) to about 1 : 1 (w/w) or of about 3 : 1 (w/w) to about 1 : 1 (w/w), and most preferably a ratio of about 3 : 1 (w/w) to about 2: 1 (w/w).
  • the ratio of the RNA molecules as defined herein to the cationic or polycationic compound and/or the polymeric carrier, preferably as defined herein, in the component of the complexed RNA molecules may also be calculated on the basis of the nitrogen/phosphate ratio (N/P-ratio) of the entire complex.
  • an N/P -ratio is preferably in the range of about 0.1-10, preferably in a range of about 0.3-4 and most preferably in a range of about 0.5-2 or 0.7-2 regarding the ratio of RNA molecules : cationic or polycationic compound and/or polymeric carrier, preferably as defined herein, in the complex, and most preferably in a range of about 0.7-1,5, 0.5-1 or 0.7-1, and even most preferably in a range of about 0.3-0.9 or 0.5-0.9, preferably provided that the cationic or polycationic compound in the complex is a cationic or polycationic cationic or polycationic protein or peptide and/or the polymeric carrier as defined above.
  • RNA is to be used in sensitive applications, such as e.g. in therapy.
  • the inventors replaced the state-of-the art precipitation step, which is commonly performed after HPLC purification of RNA, by a lyophilization step to remove the TEAA contaminant from the RNA.
  • the lyophilization step was further optimized by adding a co-solvent, namely tert-Butanol (also referred to as "t-BuOH” herein), which inter alia dramatically decreases the duration of the lyophilization process and increases the recovery of the lyophilized RNA.
  • An organic solvent which may be present in addition to the TEAA (acetonitrile is a prominent organic solvent often used together with TEAA) may be removed using rotary vacuum concentration (RVC) and/or evaporation.
  • RVC rotary vacuum concentration
  • RNA lyophilized according to the present invention may also be referred to as "formulated RNA" (since it is ready to be formulated into a therapeutic dosage form, see above) and, accordingly, the present method may either be referred to as method of purifying (since TEAA is removed) or as method of formulating RNA (since lyophilized RNA "ready to be used” is provided) or as a combination of both.
  • formulated RNA since it is ready to be formulated into a therapeutic dosage form, see above
  • the present method may either be referred to as method of purifying (since TEAA is removed) or as method of formulating RNA (since lyophilized RNA "ready to be used” is provided) or as a combination of both.
  • Example 1 RNA production, RNA purification by HPLC, and removal of acetonitrile from HPLC-purified RNA by rotary vacuum concentration in multi well plates: 1. Transcription and HPLC purification of RNA:
  • RNA templates were linearized and transcribed in vitro using T7 RNA polymerase (Thermo Fisher Scientific Inc.) according to standard procedures in the presence of a suitable buffer, a nucleotide mixture and a cap analog (m7GpppG). Subsequently, the obtained RNA products (ranging from about 1700 to 2000 nucleotides in size) were purified using HPLC following the manufacturer's instructions (PureMessenger®; CureVac AG, Tubingen, Germany; see WO2008077592). Due to the elution with a buffer comprising triethylammonium acetate (TEAA) and acetonitril, the samples comprising the RNA comprise also TEAA and acetonitril. 2. Removal of acetonitrile by rotary vacuum concentration
  • RNA concentrations ranged from 0.2 to 2 g/1.
  • NaCl (6 mmol/g RNA) was added to provide a counter-ion, which facilitates the removal of (potentially) bound TEAA from the RNA.
  • Acetonitrile was then removed by subjecting the HPLC samples to rotary vacuum concentration (RVC) using a RVC-2-33IR (Christ, Germany) and the program provided in Table 1.
  • RNA solutions were analysed for residual acetonitrile via headspace gas chromatography-flame ionization detection (GC FID), performed by an analytical laboratory according to standard methods (SAS Hagmann GmbH, Horb, Germany). All rotary vacuum concentrated HPLC samples were substantially free from acetonitrile ( ⁇ 40 ppm) and subject to a further purification step as outlined in Example 2.
  • Table 1 Program for rotary vacuum concentration of RNA to remove acetonitrile
  • Example 1 shows that the RVC step resulted in acetonitrile-free RNA.
  • Example 2 Lyophilization of naked RNA in multi well plates to remove TEAA:
  • the samples were diluted with double distilled H 2 0 (ddH 2 0) to obtain a TEAA concentration of 0.1 M and an RNA concentration of 0.2 - 2 g/1 in the resulting diluted samples.
  • All lyophilization cycles were performed in an Epsilon 2-6D LSCplus (Christ, Germany) using again the deep well plates (multi well, polypropylene).
  • An aluminium thermo block was used to improve heat transfer between shelf and plate.
  • 1 ml of a sample was subjected to lyophilization (freezing, primary drying, secondary drying) using the program provided in Table 2.
  • the LyoControl RX sensor was set to 80% resistivity to avoid melting of samples.
  • Example 6 After one lyophilization cycle, the samples were dissolved in 600 ⁇ water for injection and incubated in the rotatory vacuum concentrator (RVC-2-33IR, Christ) at 80°C and 100 rpm for 20 min to dissolve the RNA for the next lyophilization cycle. The procedure was repeated twice to arrive at three cycles in total (final protocol: LYO; RCV (80°C); LYO; RCV (80°C) LYO; RCV (80°C)). For analysis purposes, the final lyophilized samples were dissolved in ddH 2 0 (in the last RCV (80°C) step) and tested for residual TEAA (see Figure 1), RNA integrity (see Figure 2) and the total RNA recovery rate (see Figure 6, left column). A detailed description of the analytic methods is provided in Example 6.
  • Examples 1 and 2 show that the procedure yielded TEAA- and acetonitrile-free RNA with recovery rates of about 80% and RNA integrity of about 80%. Furthermore, using the present method, it was possible to achieve a homogeneous lyophilization of all samples in the multi well plate.
  • Example 3 Lyophilization of naked RNA in single vials to remove TEAA: Individual samples in six separate glass vials, rather than multi well plates as in Example 2, were processed as described above in Example 2, and the RNA yield before and after each of the three LYO; RCV (80 °C) cycles was determined (see Figure 3). No loss of RNA was detected over all three cycles, which is difficult to achieve by the alternative method of precipitation.
  • Example 3 shows that the lyophilization method is generally preferred over a precipitation method since no loss of RNA was detected.
  • Example 4 Lyophilization of naked RNA in glass or glass-coated plates to remove TEAA:
  • TEAA removal by lyophilization resulted in an RNA recovery of about 80% in polystyrene multi well plates (see Example 2).
  • Multi well plates made from plastic materials like polypropylene show low heat conductivity compared to glass multi well plates.
  • Glass or glass-coated plates show better heat transfer rates, which are likely to reduce the duration of the lyophilization cycle and are likely to lead to higher RNA recovery rates.
  • the method described in Example 2 can therefore be performed with glass or glass-coated plates using a shortened lyophilization program (i.e. less incubation times and/or less lyophilization cycles).
  • Example 4 shows that the lyophilization program might even be further shortened, e.g. if different materials and in particular glass or glass-coated plates are used.
  • Example 5 Co-solvent lyophilization of naked RNA in multi well plates to remove TEAA:
  • t-Butanol (t-BuOH) was used as a co-solvent.
  • t-BuOH has a high vapor pressure, freezes completely and quickly, and sublimes during primary drying of the lyophilization cycle.
  • aqueous solutions of TEAA and t-BuOH was determined using a LyoRX resistivity sensor (Epsilon 2-6D LSCplus, Christ) according to the manual of the LyoRX resistivity sensor. These experiments were performed in the absence of RNA. The results were used to determine the freeze-point of aqueous solutions of TEAA and t-BuOH (see Table 3). Table 3: Sample composition for freeze-point determination of aqueous solutions of TEAA and t-BuOH.
  • the freeze point should not be exceeded during the freezing step of the lyophilization cycle. Based on these results, the t-BuOH concentration for the following experiments was set to 20% since the freeze points at this concentration were the lowest (i.e. about -30°C) under almost all conditions tested.
  • RNA samples from RVC were diluted with water for injection (WFI) and t-BuOH to obtain samples containing RNA amounts from 0.2-1.5 g/1 (f.c), liquid volumes from 500- 1000 ⁇ and 20 % (m/V, f.c.) t-BuOH prepared according to Table 3. All samples were transferred into a 96 deep well plate (see Table 4).
  • Table 4 Sample composition for RNA co-solvent lyophilization to remove TEAA.
  • the vacuum for the primary drying phase was adjusted according to the vapour pressure over ice, to ensure that samples would not thaw in the primary drying phase (the sample temperature should be kept below the freeze point).
  • the optimal vacuum was calculated according to the vacuum pressure over ice after first determining the freeze point (see Table 3 and Figure 4). Additionally, the LyoControl RX sensor was set to 80% resistivity to avoid melting of samples, i.e. in case of the sensor dropping under 80%>, the shelf heating is switched off and the freezing of samples starts again. The shelf heating continued when all samples were frozen. To determine the end of the primary drying phase, a periodic pressure increase test with a progression condition of 20% was set. After the secondary drying phase, the samples were re-suspended in 500 ⁇ WFI.
  • the samples were shaken at 750 rpm under heating at 45°C for 30 minutes (compared to 80°C without co- solvent; see Example 2).
  • the lyophilization cycle was repeated with the programs used for the first cycle (LYO - re-suspension step - LYO).
  • the lyophilized samples were dissolved in ddH20 and analyzed for residual TEAA, RNA integrity, total RNA recovery rate and residual t-BuOH (a detailed description of analytic methods is provided in Example 6).
  • the co-solvent lyophilization method shortened the TEAA removal from about 8 workdays (using a single-solvent lyophilization method) to only three workdays (see Figure 5) with RNA recovery rates of nearly 100 % (see Figure 6). Furthermore, various other product parameters such as RNA integrity, dissolvability and RNA recovery rates could be improved using co-solvent lyophilization (as outlined in Example 6). Program 4 (of Table 5 shown below) yielded the most preferable results, and represents a particularly preferred embodiment of the present invention.
  • Example 5 shows that the lyophilization step can be shortened and improved if a co-solvent, preferably t-BuOH, is added.
  • a co-solvent preferably t-BuOH
  • RNA integrity after normalization was analyzed by capillary electrophoresis (Fragment Analyzer Advanced Analytical) with a DNF-471 Standard Sensitivity RNA Kit according to the manufacturer's instructions.
  • the "% (Cone.)” given by the software PROsizeTM with the minimal peak height set to 15 RFU and peak width set to 3 sec was used as a value to measure the RNA integrity.
  • the integrity values of samples after single solvent lyophilization and heat treatment were stable (about 80% RNA integrity), showing that the herein established protocol is a suitable method for purification of HPLC samples.
  • the residual TEAA content after lyophilization was detected via headspace GC FID by a commercial analytic supplier (SAS Hagmann GmbH according to Eu.Pharm. 7.0) with the RNA concentration of the samples after lyophilization adjusted to 1 g/1.
  • the TEAA content decreased to about 0.5 g TEAA/ g RNA (see Figure 1) after about 50 h of lyophilization.
  • three cycles were needed to yield levels of about 0.5 g TEAA per g RNA (see Figure 8, left column).
  • the TEAA content after the much faster co-solvent lyophilization process was comparable to or less than the TEAA content after single solvent lyophilization ( Figure 8). Again, co-solvent lyophilization is preferred.
  • RNA content after solvation of the lyophilization cake in typically 600 ⁇ WFI was measured via Nanodrop (Thermo scientific).
  • the RNA recovery expressed as "YRNA" of every run was calculated by comparison of the total RNA yield before and after the lyophilization cycle according to the following formula, where "CRNAI/2" is taken as RNA concentration before/after lyophilization in mg/ml and V 1/2 as sample volume before after lyophilization in ml:
  • RNA recovery rate was about 76% (see Figure 6, left column). RNA recovery rates of up to 100 % were achieved with co-solvent lyophilization in plastic-based multi well plates, with programs 2-4 (see Figure 6, right columns as indicated).
  • the residual t-BuOH content was determined by Headspace GC FID by SAS Hagmann GmbH with a RNA concentration of lg/1. Residual t-BuOH was reduced to levels below 0.05 g t-BuOH per g RNA for all programs tested (see Figure 9).
  • the lyophilized RNA needs to be dissolved in ddH20 for further processing (e.g., LNP encapsulation, protamine complexation etc.).
  • the solubility was measured by determining the RNA content of at least 16 samples on at least two different timepoints. For each sample, the coefficient of variation was determined. The ratio of samples that showed a higher coefficient of variation than 5 % was used as a measure for the
  • RNA samples were lyophilized either by single solvent lyophilization or by co-solvent lyophilization and incubated at 50 or 45°C, respectively, for 30 min. Thereafter, the "insolubility" was measured (see Figure 10).

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Abstract

La présente invention concerne un procédé de purification et/ou de formulation d'ARN à partir d'échantillons comprenant l'ARN et TEAA, le procédé pouvant être mis en oeuvre dans des plaques multipuits. La présente invention concerne en outre un ARN purifié et/ou formulé avec un tel procédé.
PCT/EP2017/052074 2017-01-31 2017-01-31 Purification et/ou formulation d'arn WO2018141371A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008077592A1 (fr) 2006-12-22 2008-07-03 Curevac Gmbh Procédé de purification d'arn à l'échelle préparative par hplc
WO2008083949A2 (fr) 2007-01-09 2008-07-17 Curevac Gmbh Anticorps codé par un arn
WO2009095226A2 (fr) 2008-01-31 2009-08-06 Curevac Gmbh Acides nucléiques de formule (i) (nuglxmgnnv)a et leurs dérivés sous forme d'immunostimulant/adjuvant
WO2013052523A1 (fr) 2011-10-03 2013-04-11 modeRNA Therapeutics Nucléosides, nucléotides et acides nucléiques modifiés, et leurs utilisations
WO2016165831A1 (fr) 2015-04-17 2016-10-20 Curevac Ag Lyophilisation de l'arn
WO2016180430A1 (fr) 2015-05-08 2016-11-17 Curevac Ag Procédé de production d'arn

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008077592A1 (fr) 2006-12-22 2008-07-03 Curevac Gmbh Procédé de purification d'arn à l'échelle préparative par hplc
WO2008083949A2 (fr) 2007-01-09 2008-07-17 Curevac Gmbh Anticorps codé par un arn
WO2009095226A2 (fr) 2008-01-31 2009-08-06 Curevac Gmbh Acides nucléiques de formule (i) (nuglxmgnnv)a et leurs dérivés sous forme d'immunostimulant/adjuvant
WO2013052523A1 (fr) 2011-10-03 2013-04-11 modeRNA Therapeutics Nucléosides, nucléotides et acides nucléiques modifiés, et leurs utilisations
WO2016165831A1 (fr) 2015-04-17 2016-10-20 Curevac Ag Lyophilisation de l'arn
WO2016180430A1 (fr) 2015-05-08 2016-11-17 Curevac Ag Procédé de production d'arn

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BRUNELLE ET AL., METHODS ENZYMOL., vol. 530, 2013, pages 101 - 114
FOTIN-MLECZEK ET AL., J. GENE MED., vol. 14, no. 6, 2012, pages 428 - 439
GEALL ET AL., SEMIN. IMMUNOL., vol. 25, no. 2, 2013, pages 152 - 159
THESS ET AL., MOLECULAR THERAPY, 2015

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