RNA purification methods

Abstract

Methods for purifying RNA from a sample, comprising one or more steps of tangential flow filtration, hydroxyapatite chromatography, core bead flow-through chromatography, or any combinations thereof. These techniques are useful individually, but show very high efficiency when used in combination, or when performed in particular orders. The methods can purify RNA in a highly efficient manner without unduly compromising potency or stability, to provide compositions in which RNA is substantially cleared of contaminants. Moreover, they can be performed without the need for organic solvents.

Claims

1. A method for removing protein contaminants from a desired RNA species in an RNA-containing sample, said method comprising the steps of: removing the protein contaminants from said desired RNA species in said RNA containing sample by applying said RNA-containing sample to a core bead flow-through chromatography medium comprising porous beads having a molecular weight cut-off (MWCO)/pore size of at least 250 kDa, wherein said RNA-containing sample comprises an in vitro transcription reaction sample; and obtaining an RNA-containing sample without the protein contaminants from a flow-through of said core bead flow-through chromatography medium, wherein said protein contaminants are in a size that can enter pores in said porous beads and said desired RNA-containing species has a molecular weight cut-off/pore size that is larger than the molecular weight cut-off/pore size of the porous beads.

2. The method of claim 1, wherein the molecular weight cut-off (MWCO)/pore size of at least 250 kDa is a molecular weight cut-off/pore size of at least 300 kDa.

3. The method according to claim 1, wherein the desired RNA species is a single-stranded RNA.

4. The method according to claim 3, wherein the single-stranded RNA is an mRNA.

5. The method according to claim 3, wherein the single-stranded RNA comprises one or more nucleotides having a modified nucleobase.

6. The method according to claim 1, wherein the desired RNA species comprises a linear sequence of at least 2,000 nucleotides.

7. The method according to claim 1, wherein a salt is added to the RNA-containing sample to a final concentration between 0 mM and 500 mM.

8. The method according to claim 1, wherein the method further comprises preparing said RNA-containing sample by pre-purifying an in vitro transcription reaction sample.

9. The method according to claim 1, wherein the method is conducted using a buffer containing a potassium salt.

10. The method according to claim 1, wherein the desired RNA species obtained from the method is purified to a preparative scale.

11. The method according to claim 1, wherein the method further includes discarding materials which do not contain the desired RNA species from the RNA-containing sample.

12. The method according to claim 1, wherein the RNA-containing sample contains one or more of: plasmid DNA, deoxy-oligonucleotides, deoxynucleoside monophosphates, ribonucleoside triphosphates and proteins; and wherein the RNA-containing sample does not contain genomic DNA and/or a cell membrane or fragments thereof.

13. The method according to claim 1, wherein the RNA-containing sample contains the RNA species and plasmid DNA, deoxy-oligonucleotides, deoxynucleoside monophosphates, ribonucleoside triphosphates and proteins.

14. The method according to claim 1, wherein the desired RNA species comprises a linear sequence of at least 1,000 nucleotides.

15. The method according to claim 1, further comprises purification of said desired RNA species.

16. The method according to claim 15, wherein the purification step is conducted using tangential flow filtration.

17. The method according to claim 16, wherein the tangential flow filtration is a tangential flow filtration system having a hydrophilic membrane.

18. The method according to claim 1, wherein the in vitro transcription sample is made by an in vitro transcription of an RNA.

19. The method according to claim 1, wherein the desired RNA species obtained from the method is at least 99% pure.

20. The method according to claim 1, wherein the desired RNA species obtained from the method is free from DNA, pyrophosphates, and nucleotides.

21. The method according to claim 1, further comprises purifying the RNA-containing sample without the protein contaminants using a tangential flow filtration system.

22. The method according to claim 21, wherein said purifying the RNA-containing sample without the protein contaminants using a tangential flow filtration system is performed immediately after the obtaining step.

23. The method according to claim 1, wherein the cores of said porous beads are bound with a ligand with multiple functionalities.

24. The method according to claim 23, wherein the ligand is both hydrophobic and positively charged.

25. The method according to claim 1, wherein the core bead flow-through chromatography medium is a core bead flow-through chromatography column.

26. The method of claim 1, wherein the molecular weight cut-off (MWCO)/pore size of at least 250 kDa is a molecular weight cut-off/pore size of-at least about 700 kDa.

27. A method for preparing a pharmaceutical composition comprising a purified desired RNA species, comprising the steps of: (a) removing protein contaminants from a desired RNA species in an RNA-containing sample by applying said RNA-containing sample to a core bead flow-through chromatography medium comprising porous beads having a molecular weight cut-off (MWCO)/pore size of at least 250 kDa, wherein said RNA-containing sample comprises an in vitro transcription reaction sample, wherein said protein contaminants are in a size that can enter pores in said porous beads and said desired RNA-containing species has a molecular weight cut-off/pore size that is larger than the molecular weight cut-off/pore size of the porous beads; (b) obtaining a purified RNA-containing sample comprising said purified desired RNA species from a flow-through of said core bead flow-through chromatography medium; and (c) preparing said pharmaceutical composition by formulating said purified desired RNA species from the purified RNA-containing sample to form a pharmaceutical composition.

28. The method of claim 27, wherein said method removes undesired components comprising nucleotides from said RNA-containing sample.

29. The method according to claim 27, wherein said method further comprises purification of said purified RNA-containing sample using a hydroxyapatite chromatography.

30. The method according to claim 27, wherein said method further comprises purification of said purified RNA-containing sample using a tangential flow filtration system.

31. The method of claim 27, wherein the molecular weight cut-off (MWCO)/pore size of at least 250 kDa is a molecular weight cut-off/pore size of at least about 700 kDa.

32. The method according to claim 27, wherein said method for further comprises purification of the RNA-containing sample without the protein contaminants using a tangential flow filtration system.

33. A method for removing protein contaminants from a desired RNA species in an RNA-containing sample, said method comprising the steps of: removing the protein contaminants from said desired RNA species in said RNA containing sample by applying said RNA-containing sample to a core bead flow-through chromatography column having a stationary phase comprising porous beads having a molecular weight cut-off (MWCO)/pore size of at least 250 kDa, wherein said RNA-containing sample comprises an in vitro transcription reaction sample; and obtaining an RNA-containing sample without the protein contaminants from a flow-through of said core bead flow-through chromatography column, wherein said protein contaminants are in a size that can enter pores in said porous beads and said desired RNA-containing species has a molecular weight cut-off/pore size that is larger than the molecular weight cut-off/pore size of the porous beads.

34. The method of claim 33, wherein the molecular weight cut-off (MWCO)/pore size of at least 250 kDa is a molecular weight cut-off/pore size of at least 600 kDa.

35. The method of claim 33, wherein the molecular weight cut-off (MWCO)/pore size of at least 250 kDa is a molecular weight cut-off/pore size of at least about 700 kDa.

36. The method according to claim 35, wherein the cores of said porous beads are bound with a ligand with multiple functionalities.

37. The method according to claim 36, wherein the ligand is both hydrophobic and positively charged.

38. The method according to claim 35, wherein said method further comprises purification of the RNA-containing sample without the protein contaminants using a hydroxyapatite chromatography.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the result of RNA purification using tangential flow filtration and hydroxyapatite chromatography: in vitro transcription reaction sampleprotein removalin vitro transcription reaction sample before purification (lane 1), after tangential flow filtration (lane 2), after hydroxyapatite chromatography (lane 3), after tangential flow filtration (lane 4).

(2) FIGS. 2A-2B show the result of RNA purification using hydroxyapatite chromatography. FIG. 2A illustrates Hofmeister series of ions in order of their ability to salt out proteins. FIG. 2B shows the dynamic light scattering analysis of RNA aggregate particle size in various elution buffersx-axis: salt concentration in mM; y-axis: particle radius in nm.

(3) FIG. 3A shows the result of RNA purification using tangential flow filtration and hydroxyapatite chromatography: in vitro transcription reaction sampleprotein removalin vitro transcription reaction sample before purification (lane 1), after tangential flow purification (lane 2), after hydroxyapatite chromatography (lane 3). FIG. 3B shows the result of RNA purification using tangential flow filtration and hydroxyapatite chromatography: in vitro transcription reaction sampleDNA removalDNA per mg of RNA present in an in vitro transcription reaction sample before purification (first data point from left), after tangential flow filtration (second data point), after hydroxyapatite chromatography (third data point).

(4) FIG. 4A-4B show the result of RNA purification using core bead flow-through chromatography: in vitro transcription reaction sampleprotein removalin vitro transcription sample before purification (lane 1), flow-through after core bead flow-through chromatography (lane 2), eluate after column cleaning-in-place (lane 3).

(5) FIG. 5 shows the result of RNA purification using core bead flow-through chromatography: in vitro transcription reaction sampleeffect of salt in sample and chase buffer on protein removal.

(6) FIG. 6A shows the result of quantification of RNA or RNA plus nucleotides. FIG. 6B and FIG. 6D show the result of tangential flow filtration vs. core bead flow-through chromatographyin vitro transcription reaction samplenucleotide removal. FIG. 6C shows the result of tangential flow filtration vs. core bead flow-through chromatography vs. core bead flow-through chromatography and hydroxyapatite chromatography vs. tangential flow filtration and hydroxyapatite chromatographyin vitro transcription reaction sampleprotein removal.

(7) FIGS. 7A-7G show the result of RNA purification using combination of methods described herein: in vitro transcription reaction sampleeffect on RNA recovery, protein removal, nucleotide removal and DNA removal. FIG. 7A shows recovery of RNA measured by direct quantification by RiboGreen? assay (sample dilution 10000 fold). FIG. 7B shows the purity of RNA sample by quantitative ELISA (T7 polymerase, samples in italics are below LOQ). The graph shows detectable T7, below LOQ for most samples by ELISA. FIG. 7C shows the purity of RNA sample by quantitative ELISA (capping enzyme, samples in italics are below LOQ, 0 equals below LOD). The graph shows capping enzyme below LOD in sample P3, post CC250 and P2 and P4, post HTP. FIG. 7D shows the purity of RNA sample by SDS pagesilver staining (4 ?g RNA loaded per lane). FIG. 7E shows nucleotide removal, expressed as ratio of quantification by OD/quantification by RiboGreen? (Y-axis refers to OD/RiboGreen?). FIG. 7F shows plasmid DNA carryover, using qPCR assay on plasmid. DNA before purification: 1.0 ng/dose. Following purification: 0.6/0.7 ng/dose. Lowest concentration was found after HTP chromatography. Same TFF cartridge was used in all 4 processes/steps: possible carryover. No background (buffer) control was used in this experiment. FIG. 7G shows the level of E. coli protein contamination, using host (E. coli) protein oat polyclonal antibodies (HRP labelled) to E. coli. In this assay L.O.D. (limit of detection) is 1 ng/band, 4 ug RNA was loaded in each lane. The figure shows that host contaminant below 1 ng/protein.

(8) FIG. 8 shows the design of an experimental statistically significant approach for core bead flow-through chromatographyin vitro transcription reaction sampleoptimisation of parameterssalt concentration: 0 mM (?1) to 500 mM (1); sample dilution: no dilution (1) to 4-fold dilution (?1); flow rate (linear velocity): 50 cm/h (?1) to 500 cm/h (1).

(9) TABLE-US-00001 Factors chosen for the study and range: Range Outputs: Recovery ?1 +1 T7 removal Capping enz Removal Flow (cm/h) 50 500 Nucleotides removal Salt 0 500 Precolumn pressure Conc 0.25 1 Time

MODES FOR CARRYING OUT THE INVENTION

Example 1: Method for Quantifying RNA Yield and Nucleotide Removal

(10) RNA was quantified in samples using an RNA-specific fluorescent dye (RiboGreen?). RNA levels before and after purification were compared to calculate % RNA yield. RiboGreen? does not detect free nucleotides.

(11) Free nucleotides are found in the unpurified in vitro transcription (NT) reaction and include un-reacted precursors for RNA (ribonucleoside triphosphate) and degradation products from DNAse digestion (deoxynucleosides monophosphate). A method was developed to measure nucleotides in the presence of RNA. Pure RNA was measured with RiboGreen? (FIG. 6A, 1.sup.st bar) and by optical density (OD) at 260 nm, using 40 as a standard approximated extinction coefficient for RNA (2.sup.nd bar). A mix of nucleotides was added to the pure RNA sample in a ten-fold excess to RNA by mass. The resulting samples were measured again with RiboGreen? (3 d bar) and by OD (4.sup.th bar).

(12) The results show that the measurement by RiboGreen? is unaffected by the presence of nucleotides in the sample, while the detected OD values reflect the total concentration of RNA and nucleotides in the sample. The presence of nucleotides, as an indicator for nucleotide removal after an RNA purification step, was calculated as the ratio of the OD measurement and the RiboGreen? assay measurement. A ratio of approximately 1 indicates pure RNA, i.e. complete nucleotide complete. Ratios above 1 indicate the presence of nucleotides in the sample.

Example 2: RNA Purification and Buffer Exchange Using Tangential Flow Filtration

(13) A 10-kb RNA replicon was produced through in vitro transcription and capping with completely chemical-defined enzymes, template, substance and buffers. A KrosFlo Research IIi Tangential Flow Filtration System was used (Spectrum Laboratories) for both RNA purification and buffer exchange in one single closed system. Various parameters were tested for optimal results as indicated below: membrane chemistry, membrane pore size, membrane area, transmembrane pressure, shear rate (retentate velocity), buffer volume, buffer capacity, buffer pH, sample salt concentration, and the presence of EDTA in the buffer.

(14) TABLE-US-00002 TFF cartridge Parameters considered for Theoretical impact Conditions Condition optimization on RNA quality screened selected Membrane Interaction of membrane mPES, PS mPES from chemistry with RNA and protein (Spectrum and Spectrum RNA recovery and Watersep) protein removal Membrane Retain large MW particle 500 kD, 750 kD 500 kD pore size and remove small MW MWCO MWCO molecules 0.05 and 0.1 ?m RNA recovery and protein removal Membrane area Buffer exchange 25, 52, 115 cm.sup.2 115 cm.sup.2 efficiency Operation time

(15) TABLE-US-00003 TFF system variables Parameters considered for Theoretical impact on Conditions Condition optimization RNA quality screened selected TMP RNA/protein separation 1-5 Psi 2 Psi (transmembrane Gel layer formation pressure) Shear rate RNA integrity 1000-5000 S.sup.?1 ~800 S.sup.?1 (retentate velocity) Gel layer formation Dialysis buffer Small molecule removal 5x-10x sample 8x sample volume Buffer exchange volume volume efficiency operation time

(16) TABLE-US-00004 Purification buffer Parameters considered for Theoretical impact on Conditions Condition optimization RNA quality screened selected Buffer capacity Buffer exchange 2, 10 mM Citrate 10 mM Tris efficiency and 10, 50 mM (buffer change from Tris RNA synthesis and to formulation) Buffer pH Interaction of RNA pH 6.5, 7.0, 7.5, pH 8.0 with protein 8.0, 8.5 and 9.0 Protein aggregation Salt Interaction of RNA 150, 250 and 500 250 mM NaCl concentration with protein mM NaCl EDTA Interaction of 0, 1, 10, and 0 mM RNA with RNA 20 mM Stability binding protein RNA stability

(17) Four consistency runs were performed using the optimised conditions and demonstrated that the tangential flow filtration method purifies RNA with high recovery (>95%), purity as measured by protein removal (>90% of T7 RNA polymerase removed, as quantified by ELISA; 5 ng T7 polymerase per 75 ug RNA post purification; >95% vaccinia capping enzyme removed, as quantified by ELISA) and as measured by nucleotide removal (>99.9% of free nucleotides removed, as quantified using the assay of Example 1), potency (no change in potency after purification) and stability (RNA is stable after purification). The operation as a single closed system prevents contamination with exogenous agents such as RNase. The method is quick (approx. 70 mins total) and easy to operate.

(18) As shown in FIGS. 6B and 6D and FIG. 7E the method is particularly useful for removing free nucleotides from the sample. As shown in FIG. 5 (lane 11), FIG. 6C and FIGS. 7B, 7C and 7D, protein impurities are also efficiently removed from the sample.

Example 3: RNA Purification Using Hydroxyapatite Chromatography

(19) To test whether hydroxyapatite chromatography could be useful for the purification of large RNA, 80 ?g of lithium chloride purified 10-kb RNA (replicon) from an in vitro transcription reaction were loaded on a hydroxyapatite column and eluted with a phosphate linear gradient composed of varying proportions of Buffer A (10 mM phosphate buffer, pH 6.8) and Buffer B (500 mM phosphate buffer, pH 6.8). It was found that mRNA can be efficiently bound and recovered from a hydroxyapatite column. RNA yield/recovery were measured by loading identical amounts of lithium chloride purified mRNA from an in vitro transcription reaction on a hydroxyapatite column or fed into the chromatography system by-passing the column. Area under the elution peaks was calculated and the ratio used as an indicator of RNA yield after column pass-through compared to without column pass-through purification (1401.25 mAu/ml vs 1934.76 mAu/ml). The RNA yield was calculated as 72%. Lithium chloride purified 10-kb RNA (replicon) from an in vitro transcription reaction was loaded on a hydroxyapatite column and eluted using phosphate buffer. Collected fractions 4, 5 and 6 were loaded on a denaturing RNA gel, confirming that the optical density read is associated with RNA.

(20) To test whether RNA can be more efficiently separated from contaminants such as protein or non-digested DNA using hydroxyapatite chromatography, the elution dynamics of purified RNA were analysed in the presence of various amounts of a salt (0-1000 mM sodium chloride) in the elution buffer. Sodium chloride was added to both elution buffers A and B so to have a constant concentration throughout the phosphate gradient. The rightward shift of the RNA elution peak shows that an increasing concentration of phosphate is required for RNA elution with increasing salt concentrations. This allows for the setup of different conditions to further separate RNA from proteins or other impurities. The addition of salt to the phosphate elution buffer can therefore be exploited to optimise fractionation of RNA from impurities. It was found that mRNA yield is inversely related to the concentration of sodium chloride in the elution buffer.

(21) To test whether RNA can be more efficiently separated from (undigested template) DNA, 100 ?g of pure DNA or pure RNA were subjected to hydroxyapatite chromatography using the same parameters. A continuous gradient of a potassium phosphate elution buffer was used. Effect of elution conditions on separating DNA from RNA was determined. It was found that DNA is eluted at higher phosphate concentrations than RNA (rightward shift of the elution peak). The inventors therefore devised a step-wise elution method whereby the phosphate concentration in the elution buffer increases step-wise, rather than continuously. RNA can therefore be selectively eluted by choosing an elution buffer phosphate concentration at which RNA but not DNA or other contaminants are eluted. A test run was then performed where equal amounts of purified RNA and DNA were mixed to a total amount of 200 ?g in solution and subjected to hydroxyapatite chromatography using a step-wise elution gradient of Buffer A and B.

(22) In a gradient elution, RNA elution occurred at a buffer conductivity of around 21.04 mS/cm. DNA elution occurred at around 30.52 mS/cm. This demonstrates that in the presence of an RNA/DNA mixture, a concentration of about 180 mM potassium phosphate (or any potassium phosphate concentration resulting in a conductivity value above 21.04 mS/cm and below 30.52 mS/cm) elutes selectively RNA and not DNA. A test run was then performed where purified DNA was analysed under the same conditions as described above. No elution was observed below about 180 mM (?18% B) potassium phosphate. The results show that RNA and DNA can efficiently be separated with a step-wise elution. DNA elution can be achieved with 38% buffer B, about 380 mM potassium phosphate (or any potassium phosphate concentration resulting in a conductivity value above 30.52 mS/cm). Using tangential flow filtration and hydroxyapatite chromatography (in vitro transcription reaction sample), elution conditions for separating DNA from RNA were optimised.

(23) In comparing various phosphate buffers useful for elution of RNA during hydroxyapatite chromatography, it was found that a potassium phosphate buffer performs better than a sodium phosphate in keeping RNA in solution and is a better candidate for hydroxyapatite column elution. Dynamic light scattering experiments (FIG. 2) showed that an increasing concentration of sodium phosphate in the elution buffer during hydroxyapatite chromatography leads to an increasingly larger apparent particle size of the eluted RNA, probably due to salt-induced RNA precipitation (salting out). This effect is reduced when using potassium phosphate instead of sodium phosphate at the same concentration, with concentrations up to 500 mM. A potassium phosphate buffer was tested for RNA elution from a hydroxyapatite column and performed comparably to a sodium phosphate buffer in terms of RNA purity and recovery for this process. Potassium phosphate is therefore identified as the salt of choice for RNA purification by hydroxyapatite chromatography.

(24) Next, a non-purified in vitro transcription reaction containing 100 ?g of a 10-kb RNA replicon was analysed using hydroxyapatite chromatography. Collected fractions 1, 2 and 3 were loaded on a denaturing RNA gel. No RNA was visible on the gel. Fractions B9 (corresponding to fraction directly preceding fraction 2) and Cl (corresponding to fraction 3) were analysed by reversed phase HPLC. The elution time was compared to nucleotide standards, confirming that the observed elution peaks at OD 260 using a non-purified in vitro transcription reaction sample were mainly composed of free nucleotides from the in vitro transcription reaction.

Example 4: RNA Purification Using Tangential Flow Filtration and Hydroxyapatite Chromatography

(25) A combination of tangential flow filtration followed by hydroxyapatite chromatography was tested for improved efficiency of RNA purification from an in vitro transcription reaction sample, and in particular for the removal of nucleotides before the sample is used in hydroxyapatite chromatography. An unpurified in vitro transcription reaction containing a 10-kb RNA replicon product was used as the starting sample. FIGS. 3A and 3B show that such a combination of method allows the efficient removal of nucleotides in the tangential flow filtration step and of DNA (reduced to 5.93 ng DNA per mg purified RNA) and protein (reduced to below detection levels) in the hydroxyapatite chromatography step, enabling the recovery of pure RNA (>80%) after the hydroxyapatite chromatography step (a step-wise elution gradient as described in Example 3 was used for elution). This is particularly useful as template DNA digestion can be omitted from the overall RNA purification procedure, leading to faster operation times.

(26) FIG. 1 further confirms the efficiency of protein removal using a hydroxyapatite chromatography step, showing that the level of protein impurities is reduced to below the level of detection using silver staining (4 ?g of purified RNA were loaded per lane). An optional further step of tangential flow filtration was used to exchange the phosphate buffer in which the purified RNA is eluted following hydroxyapatite chromatography into a citrate buffer suitable for downstream applications.

(27) FIG. 6C (lane TFF0HTP0 vs. lane Input) also confirms the usefulness of an RNA purification method combining tangential flow filtration followed by hydroxyapatite chromatography for removing protein impurities from an RNA-containing sample.

Example 5: RNA Purification Using Core Bead Flow-Through Chromatography

(28) Core bead flow-through chromatography was tested for the purification of RNA. An unpurified in vitro transcription reaction (in Tris 50 mM, pH 8.0) containing a 10-kb RNA replicon product was used as the starting sample. A HiScreen Capto? Core 700 column (product code: 17-5481-15) was initially used, on a GE ?KTAa Explorer 100 FPLC system. The sample was diluted in a buffer of Tris 50 mM, pH 8.0, to a final RNA concentration of 600 ng/?l (final volume: 8.5 ml, containing 5.1 mg RNA). The sample was injected into the column and chased with Tris buffer (50 mM) until elution of the sample was complete. The flow was set at 1 ml/min (corresponding to 125 cm/h). Column cleaning-in-place (CIP) and regeneration was as per the manufacturer's instructions. It was found that RNA can be recovered in the column flow-through (e.g., in vitro transcription reaction sample, 5.1 mg RNA, RNA was eluted in flow through). FIGS. 4A and 4B show that RNA is recovered from the column flow-through at a high level of yield (FIG. 4B, lane 2 vs. lane 1) and contains lower levels of protein impurities compared to before core bead flow-through chromatography (FIG. 4A, lane 2 vs. lane 1).

(29) To test the effect of the presence of salt on removal of protein impurities using core bead flow-through chromatography, increasing concentrations of sodium chloride or sodium phosphate added to the sample upon purification and in the chase buffer were tested. Chromatographic conditions for these purifications were equivalent to the ones specified above. Flow-through fractions containing an equal amount of purified RNA (5 ?g) were analysed by polyacrylamide gel electrophoresis and silver staining. FIG. 5 shows that an increasing salt concentration positively correlates with the level of removal of proteinaceous contaminants. Salt was added to the sample and to the chase buffer. Arrows indicate two protein contaminants (T7 polymerase and large subunit of the capping enzyme). Control sample on the far right is after purification of an in vitro transcription reaction using tangential, flow filtration only. In conclusion, increasing salt, concentration facilitates the removal of protein carryover, leading to a final protein mass that is below the level of detection using a silver-stained polyacrylamide gel and 5 ?g of RNA.

(30) Conditions for core bead flow-through chromatography were further optimised, in particular the salt concentration (0-500 mM), flow rate (50-500 cm/h), and sample dilution (4-fold dilution to undiluted; before application to the column) were varied and evaluated for their effect on the level of RNA yield (recovery), protein removal (T7 polymerase and/or capping enzyme) and nucleotide removal after core bead flow-through chromatography and the pre-column pressure and operation time of the chromatography method. FIG. 8 shows the design of an experimental statistically significant approach. The value ranges for the tested variables and the output parameters that were evaluated are indicated. Model details: Response Surface Designs, Central Composite Designs, Inscribed. The starting sample was an unpurified in vitro transcription reaction sample containing the RNA of interest. Protein carryover was evaluated by silver-stained SDS page of the flow through material, and quantified by densitometry of the protein bands. Nucleotide removal and RNA yield were quantified as described in Example 1.

(31) Table 1 shows the output values RNA yield (recovery), protein removal (T7 polymerase and capping enzyme), OD260 nm values, operation time and pre-column pressure after a core bead flow-through chromatography run under different conditions (samples A-T).

(32) TABLE-US-00005 TABLE 1 Inscribed CCI Flow Inj RNA ul on Samples Flow Salt Conc cm/h Salt mM Conc inj Vol ug GEL A ?0.594 ?0.594 ?0.594 141.3 101.5 0.402 500 106.0 19.9 B 0.594 ?0.594 ?0.594 408.7 101.5 0.402 500 306.5 19.9 C ?0.594 0.594 ?0.594 141.3 398.5 0.402 500 106.0 19.9 D 0.594 0.594 ?0.594 408.7 101.5 0.402 500 306.5 19.9 E ?0.594 ?0.594 0.594 141.3 101.5 0.848 500 106.0 9.4 F 0.594 ?0.594 0.594 408.7 101.5 0.848 500 306.5 9.4 G ?0.594 0.594 0.594 141.3 398.5 0.848 500 106.0 9.4 H 0.594 0.594 0.594 408.7 398.5 0.848 500 306.5 9.4 I 0.000 0.000 0.000 275.0 250.0 0.625 500 206.3 12.8 L 0.000 0.000 0.000 275.0 250.0 0.625 600 206.3 12.8 M 0.000 0.000 0.000 275.0 250.0 0.625 500 206.3 12.8 N 0.000 0.000 0.000 275.0 250.0 0.625 500 206.3 12.8 O 1.000 0.000 0.000 50.0 250.0 0.625 500 37.5 12.8 P 1.000 0.000 0.000 500.0 250.0 0.625 500 375.0 12.8 Q 0.000 ?1.000 0.000 275.0 0.0 0.625 500 206.3 12.8 R 0.000 1.000 0.000 275.0 500.0 0.625 500 206.3 12.8 S 0.000 0.000 ?1.000 275.0 250.0 0.250 500 206.3 32.0 T 0.000 0.000 1.000 275.0 250.0 1.000 500 206.3 8.0 avg OD Ribogreen AU Flow through Pre column Samples ng/ul total recovery % ng/ul total recovery % Protein Time Pressure A 91.9 229.6 95.2 112.8 281.9 116.8 0.162 0.0176 0.140 B 103.9 259.6 107.6 119.3 298.2 123.6 0.297 0.0061 0.260 C 131.0 327.5 135.7 105.1 262.8 108.9 0.083 0.0176 0.140 D 142.9 357.1 148.0 102.3 255.7 106.0 0.100 0.0061 0.260 E 227.7 569.3 111.9 214.8 537.1 105.6 0.180 0.0083 0.160 F 254.8 636.9 125.2 237.4 593.5 116.7 0.221 0.0029 0.260 G 259.6 648.9 127.6 183.2 458.0 90.0 0.080 0.0083 0.160 H 291.1 727.8 143.1 202.4 506.1 99.5 0.122 0.0029 0.280 I 204.0 509.9 136.0 198.5 496.2 132.3 0.160 0.0058 0.200 L 206.1 515.1 137.4 191.6 479.0 127.7 0.169 0.0058 0.200 M 206.4 516.0 137.6 160.7 401.6 107.1 0.144 0.0058 0.200 N 211.8 529.5 141.2 174.3 435.7 116.2 0.104 0.0058 0.200 O 180.8 452.0 120.5 157.7 394.1 105.1 0.062 0.0320 0.070 P 210.7 526.8 140.5 197.7 494.3 131.8 0.112 0.0032 0.320 Q 140.3 350.6 93.5 178.1 445.1 118.7 0.156 0.0058 0.210 R 208.2 520.4 138.8 142.1 355.2 94.7 0.064 0.0058 0.200 S 72.0 180.0 120.0 76.4 190.9 127.3 0.018 0.0145 0.200 T 315.9 789.8 131.6 269.6 674.1 112.3 0.096 0.0036 0.200

(33) The output parameters T7 polymerase removal and capping enzyme removal in samples A-T were quantified by resolution of the core bead chromatography flow through fraction using polyacrylamide gel electrophoresis and silver-stained followed by quantification using densitometry of the protein bands. An unpurified in vitro transcription sample was used as control. Results were further analysed according to chromatography conditions: effect of salt concentration and sample dilution on RNA recovery, T7 polymerase removal (quantification in relative units) and capping enzyme removal (quantification in relative units); effect of flow rate and sample dilution on RNA recovery, T7 polymerase removal and capping enzyme removal; effect of flow rate and salt concentration on RNA recovery, T7 polymerase removal and capping enzyme removal.

(34) Using an unpurified in vitro transcription reaction as the starting sample, the maximum sample volume per column volume (CV) was determined at which protein is sufficiently removed using core bead flow-through chromatography. Effect of sample-to-column volume ratio on protein removal was determined. Samples were diluted up to a maximum sample/CV ratio of 10:1 (CV: 1 ml; ID: 0.7 cm; height: 2.5 cm, L. vel: 250 cm/h; flow: 1.6 ml/min; contact time: 36 seconds) or 64:1 (CV: 0.137 ml; ID: 0.5 cm; height: 0.7 cm, L. vel: 250 cm/h; flow: 0.82 ml/min; contact time: 10 seconds) and potassium chloride was added to a final concentration of 250 mM. The flow-through from each run was analysed by polyacrylamide gel electrophoresis and silver staining. It was found that protein break-through occurred when the sample-CV ratio exceeded about 10:1, under the conditions used. In conclusion, a sample/CV ratio of up to 10 efficiently purified RNA from protein impurities in the experimental condition used (e.g. 10 ml IVT reaction can be diluted to 40 ml and efficiently purified with a 1 ml column).

(35) Further, various sample and/or chase buffers compositions for use in core bead flow-through chromatography were compared with regards to the degree of observed RNA precipitation in these buffers, measured using dynamic light scattering and an increasing apparent particle size as an indicator of RNA precipitation. Table 2 summarizes the results of core bead flow-through chromatography: dynamic light scattering analysis of RNA aggregate particle size in the presence of various salts. The second column refers to salt concentration in mM. Numbers in columns 3-7 are particle radius in nm. The Table shows that potassium phosphate buffer (pH 6.5) and potassium chloride buffer (pH 8.0) are good candidates for an optimised flow through purification.

(36) TABLE-US-00006 TABLE 2 Tris 10 mM pH Tris 10 mM pH 8.0 + NaCl 8.0 + KCl KPO4 pH 6.5 KPO4 pH 8.0 NaPO4 pH 6.5 NaCl 0 20.3 (mM) 83 23.2 22.3 21.1 20.2 21.9 167 21.8 20.4 20.1 19.8 22.2 250 22 19.7 20 20.7 23.8 333 23.9 19.4 20.7 23.1 26.8 417 27.6 20.2 21.3 26.6 31.8 500 32.6 21.3 22.5 31.1 39.8

Example 6: RNA Purification Using Core Bead Flow-Through Chromatography and Tangential Flow Filtration

(37) Using an unpurified in vitro transcription reaction as the starting sample containing a 10-kb RNA replicon product, nucleotide and protein removal were compared using either tangential flow filtration or core bead flow-through chromatography (using potassium chloride concentrations of 0, 250 or 500 mM in the sample). FIGS. 6B and 6D show that tangential flow filtration efficiently removes nucleotide impurities. FIG. 6C shows that core bead flow-through chromatography efficiently removes protein impurities in the presence of potassium chloride. Therefore, where it is desired to remove nucleotide and protein impurities, it is desired that core bead flow-through chromatography is followed by tangential flow filtration.

Example 7: RNA Purification Using Core Bead Flow-Through Chromatography and Hydroxyapatite Chromatography

(38) The presence of additional salts such as potassium chloride in the sample and/or chase buffer may sometimes be undesired. Using an unpurified in vitro transcription reaction as the starting sample containing a 10-kb RNA replicon, protein removal was compared using core bead flow-through chromatography (without additional salt, i.e. 0 mM potassium chloride) alone or followed by hydroxyapatite chromatography (also without additional salt, i.e. 0 mM sodium chloride). FIG. 6C (lane CC0HTP0 vs. lane CC0) shows that efficient protein removal can be achieved even in the absence of additional salt, when core bead flow-through chromatography is followed by hydroxyapatite chromatography.

Example 8: Combinations of Methods for RNA Purification and Buffer Exchange

(39) Four different process streams (P1-P4) were devised for RNA purification (Table 3) and compared with regards to RNA recovery/yield and purity (FIGS. 6A-6G).

(40) TABLE-US-00007 TABLE 3 Process stream Options: Purification .fwdarw. Buffer Exchange 1 TFF (puri b.) .fwdarw. TFF (formulation b.) 2 TFF .fwdarw. LC (hydroxyhapatite) .fwdarw. TFF (formulation b.) (no salts) 3 GE Core beads (250 KCl) .fwdarw. TFF (formulation b.)/SEC 4 GE Core .fwdarw. LC (hydroxyhapatite) .fwdarw. TFF (formulation b.) beads (no salts)

(41) An in vitro reaction containing a 10-kb RNA replicon of interest was used as the starting sample.

(42) RNA purity was related to the level of protein (T7 polymerase, capping enzyme, RNase inhibitor, pyrophosphatase, E. coli proteins carried over from DNA template amplification), plasmid DNA and nucleotide after each step. RNA recovery and nucleotide levels were measured using the methods of Example 1. Protein levels were measured using ELISA or polyacryl amid gel electrophoresis followed by silver staining or antibody-based detection (western blot). DNA levels were measured by quantitative PCR.

(43) A step of tangential flow filtration can be used to exchange buffer but where this results in increased purity it is also a purification step.

(44) For purposes of comparison, a step of DNA digestion using DNase was performed for all processes after IVT and before applying the sample to the chromatography/filtration system. However, it should be noted that this step is not mandatory for example where hydroxyapatite chromatography is used.

(45) FIGS. 7A-7G show that protein carryover (T7 and capping enzyme) is observed only with process 1. Hydroxyapatite chromatography and core bead chromatography can remove protein carry over efficiently. Traces are detected after purification, below the level of detection of the ELISA assay. Core bead flow-through purification followed by tangential flow filtration is easier to operate that hydroxyapatite chromatography. RNA yield was: P1: 74.8%, P2: 37.3%, P3: 76.2%, P4: 60.7% (RNA recovery is summarized in Table 4). Nucleotide removal was complete in all processes in the final product. Process time ranges from 45 mins to 84 mins for all processes. DNA concentration in the final product was 0.6 ng DNA per 75 ?g RNA. The level of E. coli protein contamination was below the detection level of the western blot method used. The apparent RNA particle size as measured by dynamic light scattering in the final product was 40-45 nm radius for all processes.

(46) TABLE-US-00008 TABLE 4 Step Overall Step: recovery recovery P1 1 - TFF250 13.8 2 - TFFfb 543.4 74.8 P2 1 - TFF0 81.9 2 - HTP0 77.0 63.0 3 - TFFfb 59.2 37.3 P3 1 - CC250 88.7 2 - TFFfb 85.9 76.2 P4 1 - CC0 90.1 2 - HTP0 88.8 79.9 3 - TFFfb 75.9 60.7

Example 9: Large-Scale Purification of RNA

(47) A combination of tangential flow filtration followed by hydroxyapatite chromatography was used for preparative RNA purification from an in vitro transcription reaction sample. An unpurified in vitro transcription reaction containing 6 mg of a 10-kb RNA capped replicon product was used as the starting sample. Tangential flow filtration was performed using 10 mM Tris pH 8.0. The RNA-containing fraction was retained. Potassium chloride was added to the sample at a final concentration of 500 mM, and the sample was applied to the hydroxyapatite column (CHT? Ceramic Hydroxyapatite Type II, 40 ?m particle size, Biorad, in a GE Hi Scale 26 column, 20 cm height, 100 ml; run on a GE ?KTA explorer 100; flow 10 ml/min; linear velocity 300 cm/h). Elution buffers were buffer A (10 mM potassium phosphate, pH 6.5) and buffer B (1M potassium phosphate, pH 6.5). RNA was selectively eluted with 18% buffer B (180 mM potassium phosphate). The results demonstrate that this method achieves large-scale, preparative RNA purification with high yield and purity.

(48) A combination of core bead flow-through chromatography followed by TFF was used for preparative RNA purification from an in vitro transcription reaction sample. An unpurified in vitro transcription reaction containing 120 mg of a 10-kb capped RNA replicon product was used as the starting sample. The sample was, diluted 4-fold, then potassium chloride to 250 or 500 mM was optionally added, and the sample was applied to a core bead flow-through column using Capto? Core 700 beads. Chromatography was performed at a linear flow rate of 275 cm/h (volumetric 25 ml/min) with a contact time of 2.21. The RNA-containing flow-through was then further purified, concentrated 2-fold, and buffer-exchanged into final formulation buffer (all in one procedure) using TFF (hollow-fibre module, 500 kDa cut-off, mPES).

(49) The process was tested with 100 ml capped IVT RNA (about 120 mg), using a 50 ml Captocore column (Captocore 700, 2.6 cm internal diameter, 10 cm height run at the conditions described above, flow 25 ml/min) and a 790 cm 2 TFF cartridge (same conditions, flow 200 ml/min). The final material had comparable characteristics to the smaller scale process in terms of activity, purity and yield. Even in preliminary experiments the process had a yield of about 80% per step, giving a recovery of 65% overall, and was completed in 70 (12 minutes for the Captocore step, 58 minutes for TFF).

(50) The following table shows suitable process parameters for four available columns which can cope with sample volumes of from 10 to 1000 ml:

(51) TABLE-US-00009 sample Linear Contact Internal Column Dilution Process Volume velocity time diameter Area Height volume Sample/ final Flow time (ml) (cm/h) (min) (cm) (cm2) (cm) (ml) CV volume (ml) (ml/min) (min) GE HiScreen 10 275 2.21 0.77 0.47 10.13 4.71 2.32 40 2.1 19 GE HiScale 26/20 100 275 2.21 2.60 5.31 10.13 53.75 1.86 400 24.3 16 GE HiScale 26/20 200 275 2.21 2.60 5.31 10.13 53.75 3.72 800 24.3 33 Spectra/Chrom 50/100 1000 275 2.21 5.00 19.63 10.13 198.78 5.03 4000 89.9 44
The table shows flow rate as a linear velocity, which means that the columns' internal diameters are irrelevant in defining the in method. Linear velocity can be maintained constant in the scaled-up processes. The different column diameter is used to calculate the flow rate in so as to keep the linear velocity constant and thus to maintain the same contact time (i.e. the time that the sample stays in the column).

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