Method for producing and purifying RNA, comprising at least one step of tangential flow filtration
11667910 · 2023-06-06
Assignee
Inventors
- Andreas Funkner (Tübingen, DE)
- Stefanie Dorner (Tübingen, DE)
- Stefanie Sewing (Tübingen, DE)
- Johannes Kamm (Tübingen, DE)
- Norbert Broghammer (Tübingen, DE)
- Thomas KETTERER (Gomaringen, DE)
- Thorsten MUTZKE (Reutlingen, DE)
Cpc classification
C12N15/1017
CHEMISTRY; METALLURGY
International classification
C12N15/10
CHEMISTRY; METALLURGY
B01D61/14
PERFORMING OPERATIONS; TRANSPORTING
C12P19/34
CHEMISTRY; METALLURGY
Abstract
The present invention relates to method for producing and purifying RNA comprising the steps of providing DNA encoding the RNA; transcription of the DNA into RNA; and conditioning and/or purifying of the solution comprising transcribed RNA by one or more steps of tangential flow filtration (TFF).
Claims
1. A method for producing purified RNA, comprising the steps of: A1) providing plasmid DNA encoding a RNA of 500 to 10000 nucleotides in length; A2) linearizing the DNA with a restriction endonuclease to produce linearized DNA; B) transcribing the linearized DNA to yield transcribed RNA, wherein said transcribing is in a solution comprising: nucleoside triphosphates (NTPs); T7 polymerase, spermidine, salts and a HEPES or TRIS buffer; and C) purifying the transcribed RNA by performing at least one step of tangential flow filtration (TFF) using a TFF membrane cassette, thereby producing purified RNA, wherein the method comprises at least one step of DNA or RNA purification using chromatography.
2. The method of claim 1, wherein the at least one step of DNA or RNA purification using chromatography comprises using anion exchange chromatography.
3. The method of claim 1, wherein at least one step of TFF is performed after the chromatography.
4. The method of claim 1, wherein C) purifying the transcribed RNA comprises at least two steps of TFF.
5. The method of claim 4, wherein the at least two steps of TFF are both using a TFF membrane cassette.
6. The method of claim 1, wherein the TFF membrane cassette comprises a cellulose-based TFF membrane.
7. The method of claim 6, wherein C) purifying the transcribed RNA comprises performing at least one step of TFF with an aqueous salt solution.
8. The method of claim 7, wherein the aqueous salt solution is a NaCl solution or an organic salt solution.
9. The method of claim 7, wherein performing at least one step of TFF comprises using a TFF membrane with a molecular weight cutoff of ≤500 kDa.
10. The method of claim 1, further comprising treating the solution with DNAse after said transcribing.
11. The method of claim 1, wherein the RNA is a mRNA.
12. The method of claim 9, wherein the transcribing is in a buffer comprising 0.1 mM to 10 mM spermidine.
13. The method of claim 12, wherein the method produces purified RNA with a reduced level of spermidine relative to the level of spermidine in step B.
14. The method of claim 11, wherein the mRNA comprises a modified nucleotide.
15. The method of claim 14, wherein the modified nucleotide is 1-methyl-pseudouridine.
16. The method of claim 13, wherein the transcribing is in a buffer comprising a HEPES buffer.
17. The method of claim 13, wherein performing at least one step of TFF comprises using a TFF membrane with a molecular weight cutoff of about 100 kDa to 300 kDa.
18. The method of claim 13, wherein the membrane cassette of step C2) has a MWCO of about 300 kDa.
19. The method of claim 13, wherein the RNA is 500 to 5000 nucleotides in length.
20. The method of claim 19, wherein the transcribing is in a buffer comprising a cap analog to produce a capped RNA.
21. The method of claim 20, wherein the capped RNA is a CAP1 capped RNA.
22. The method of claim 21, wherein the restriction endonuclease is a type II restriction endonuclease.
23. The method of claim 17, wherein the method further comprises formulating the purified RNA.
24. The method of claim 22, further comprising a filling step.
25. The method of claim 17, wherein the TFF membrane cassette comprises a stabilized cellulose-based TFF membrane.
26. The method of claim 23, wherein formulating the purified RNA comprises complexing the RNA with a cationic compound.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1)
(2)
(3)
(4)
(5)
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(7)
(8)
(9) The flux rates for three different TFF cassettes were in a range of 126 to 140 l/h/m.sup.2.
(10)
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(13) For concentration and diafiltration of the linearization reaction TFF membranes made from different material, with a MWCO of 100 kDa and membrane area of 200 cm.sup.2 from different suppliers (The PES-based membranes Sartocon Slice 200 from Sartorius and the NovaSet-LS ProStream (Low Binding mPES) from NovaSep and the cellulose-based membrane Sartocon Slice 200, Hydrosart from Sartorius) were tested with a high membrane load (5.6 and 6 g plasmid DNA/m.sup.2).
(14) Flux rates in the concentration step are shown in
(15) All tested membranes showed similar results. During concentration of the linearization reaction (
(16)
(17) Only a negligible amount of plasmid DNA is visible in the permeate of the concentration step and of the diafiltration step.
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EXAMPLES
Example 1—Materials
(25) The following materials of Table 1 were used in the subsequent experimental section:
(26) TABLE-US-00001 TABLE 1 Materials Equipment Manufacturer Vivaflow 50, PES, 100 kDa Sartorius Sartocon Slice 200 100 kDa, PES Sartorius Sartocon Slice 200 100 kDa, Hydrosart Sartorius (cellulose-based membrane) Sartcocon Slice 200 300 kDa, PES Sartorius Omega Centramate T OS100T02, PES PALL GmbH 100 kDa NovaSet-LS ProStream (Low Binding mPES), NovaSep 100 kDa Hollow fibre module, 100 kDa, PES GE-Healthcare Spin-Filter Nanosep ® (100 kDa), PES Pall GmbH Spin-Filter Nanosep ® (300 kDa), PES Pall GmbH Spin-Filter Nanosep ® (1000 kDa), PES Pall GmbH Spin-Filter Vivaspin ® 500 (100 kDa), PES Sartorius Spin-Filter Vivaspin ® 500 (300 kDa), PES Sartorius Spin-Filter Vivaspin ® 500 (1000 kDa), PES Sartorius Vivaflow 50 Modul, PES, 100 kDa Sartorius Sartoflow Slice 200, PES, 100 kDa Sartorius
(27) General Methods:
Example 2—Linearization of Plasmid DNA
(28) The following conditions were used for linearization of the plasmid DNA:
(29) 1 μg plasmid DNA
(30) 0.5 μl reaction buffer
(31) 3 Units restriction enzyme EcoRI
(32) Add. 5 μl with WFI (water for injection)
(33) The reaction was incubated for 3 hours at 37° C. and stopped by heat-inactivation of the restriction enzyme (65° C., 30 minutes).
Example 3—General Description of the TFF Process
(34) All tubes and the retentate vessel were cleaned with 75% EtOH and water and were assembled.
(35) The membrane cassette was fixed into the corresponding holder, according to the manufacturer's instruction, and respectively the hollow fibre membrane was connected to the system.
(36) Afterwards, the system and membrane was flushed with at least 1 L water, 1 L 1 M or 0.5 M NaOH for 1 hour (for removal of potential contaminants, like RNases) and was washed again with water, until pH value in the permeate was neutral. Subsequently, the whole system was flushed with water for injection (WFI) or diafiltration solution or buffer.
(37) 3.1—Concentration Step
(38) DNA/RNA-solution was filled into the retentate vessel, and was optionally concentrated to the required concentration, by setting the indicated pressures.
(39) 3.2—Diafiltration Step
(40) After the optional concentration step, diafiltration was started. Therefore the diafiltration tube was placed into the diafiltration solution or buffer. During diafiltration, the amount of permeate which left the system was automatically replaced by diafiltration solution or buffer, due to the emerging vacuum. When the required diafiltration volume (dv) was reached, a different diafiltration solution or buffer was optionally added to the system, and a second diafiltration step was carried out. Before ending of the TFF step, the retentate was optionally again concentrated to the required volume, before withdrawal of the retentate from the system. Subsequently the system was flushed with 25 mL WFI (water for injection) or buffer (permeate valve closed). Flushing liquid was optionally pooled with the TFF retentate. Optionally, RNA/DNA concentration was measured and recovery of nucleic acid calculated.
(41) 3.3—System/Membrane Maintenance
(42) After usage of the membranes, the cassette was flushed with 0.5 L water, subsequently with 0.5 M or 1 M NaOH for 1 hour, and again with water, until pH in the permeate was neutral. Afterwards, the water permeate flux value was determined to verify the cleanness of the membrane. At the end, the membrane was removed from the TFF system and stored in either 0.1 M NaOH or in 20% EtOH. The TFF system was afterwards cleaned with 75% EtOH and water and stored dryly.
Example 4—In Vitro Transcription
(43) 4.1—In vitro transcription
(44) The linearized DNA plasmids were transcribed in vitro using T7 polymerase. The in vitro transcription was performed in the presence of a CAP analog (m7GpppG). The in vitro transcription was carried out in 5.8 mM m7G(5′)ppp(5′)G Cap analog, 4 mM ATP, 4 mM CTP, 4 mM UTP, and 1.45 mM GTP, 50 μg/ml DNA plasmid, 80 mM HEPES, 24 mM MgCl.sub.2, 2 mM Spermidine, 40 mM DTT, 100 U/μg DNA T7 RNA polymerase, 5 U/μg DNA pyrophosphatase, and 0.2 U/μl RNAse inhibitor.
(45) The in vitro transcription reaction was incubated for 4 hours at 37° C.
(46) After transcription the reaction was stopped by adding ETDA to a final concentration of 25 mM.
(47) 4.2—DNA Template Removal: DNase I Treatment
(48) To digest DNA template 6 μl DNAse I (1 mg/ml) and 0.2 μl CaCl.sub.2) solution (0.1 M)/μg plasmid DNA were added to the transcription reaction, and incubated for 3 h at 37° C.
Example 5—HPLC Purification of the RNA
(49) The RNA was purified using PureMessenger® (CureVac, Tubingen, Germany; WO2008/077592A1).
(50) Briefly, the TFF conditioned transcription reaction was purified using Reversed-Phase High pressure liquid chromatography (RP-HPLC). The RP-HPLC was performed with a macroporous styrene/divinylbenzene column (particle size 30 μm, pore size 4000 Å) and column dimensions of 21.2 mm×250 mm (volume 88.25 ml).
(51) 1 g/l RNA in 100 mM triethylammonium acetate (TEAA) was prepared, filtered with a 5 μm PVDF filter and used for preparative RP-HPLC. After mounting the column (stored in 88% acetonitrile), the storage solution was washed out with ultra-pure water. Next, the RNA sample was loaded onto the column and eluted with an eluent B/eluent A—gradient (eluent A: 100 mM triethylammonium acetate (TEAA) in (water for injection (WFI), pH 7.0; eluent B: 100 mM TEAA in 25% acetonitrile) starting with 100% eluent A, and ending with 100% eluent B.
(52) During the elution procedure, fractions were automatically collected. Subsequently fractions were analyzed for RNA content by photometrical determination (A260) and for RNA integrity by agarose gel electrophoresis or analytical HPLC.
Example 6—Analytical Methods
(53) 6.1—RNA Gel Electrophoresis
(54) RNA was separated in formaldehyde-containing agarose gels (0.7% w/w formaldehyde, 1.2% w/v agarose) in 3-Morpholinopropane sulfonic acid buffer (for details on method see Sambrook, Russel: Molecular Cloning: A Laboratory Manual, vol. 3, Cold Spring Harbor Laboratory, 2000.). RNA samples were denatured in RNA sample buffer (Thermo scientific) at 80° C. for 5 min before loading on the gel. 1 μg of RNA was loaded per lane.
(55) 6.2—Protein Gel Electrophoresis (SDS-PAGE)
(56) SDS-PAGE was performed with ready-to-use 12% Mini-PROTEAN TGX gels (Bio-Rad). 4× Laemmli sample loading buffer and 10×SDS-PAGE running buffer were purchased from Bio-Rad. Samples were mixed with 4× loading buffer and incubated at 95° C. for 5 min. Sample load was normalized to 10 μg RNA per lane. A voltage of 150 mV (corresponding to approx. 35 mA per gel) was applied until the smallest marker band reached the lower end of the gel. Visualization of protein bands was performed with ready-to-use Simply Blue Safe Stain (Invitrogen) according to the protocol of the manufacturer. Alternatively, the Pierce Silver Stain Kit (Thermo Scientific) was used in order to increase staining sensitivity.
(57) 6.3—Quantification of RNA-Bound Spermidine
(58) Spermidine was quantified by a modified protocol as described in Flores et al. (Plant Physiol. (1982) 69, 701-706)). Briefly, spermidine is benzoylated under alkaline conditions followed by extraction with diethylether. Benzoylated spermidine is detected and quantified by HPLC or mass spectrometry. Hexamethylene diamine is used as an internal standard.
(59) 6.4—Determination of Residual Solvents
(60) The content of residual solvents in the RNA sample was determined using quantitative gas-chromatography with flame ionization detector (GC-FID).
(61) Pilot Tests:
Example 7—Pore Screening Experiments
(62) 7.1—MWCO Screening with Plasmid DNA.
(63) For determination of suitable MWCO (molecular weight cut-off) of membranes used in TFF, a MWCO-screening was conducted. Therefore spin filters were used, as they require small volumes. Spin filters from two different manufacturers (Spin-Filter Nanosep® (100 kDa, 300 kDa and 1000 kDa), PES from PALL GmbH and Spin-Filter Vivaspin® 500 (100 kDa, 300 kDa and 1000 kDa), PES from Sartorius) were tested with a MWCO of 100, 300 and 1000 kDa. Prior to use, the spin filters were flushed with 500 μl WFI (water for injection), and WFI was removed from the permeate compartment completely. Subsequently, 300 μl of three different linearization reactions (see Example 2) were added and centrifuged at room temperature, according to the manufacturer's instructions. After approximately half the volume had passed the membrane, centrifugation was stopped and the exact volume in the permeate and retentate chamber was determined. Retentate chamber was then flushed with 100 μl WFI and combined with the retentate. Beside volume measurement, also the concentration of DNA was determined in the starting solution and retentate solution. DNA concentration was determined photometrically by measuring the absorption at 260 nm.
(64) The samples of linearization P1040 and the resulting filtration samples are shown in
(65) 7.2—MWCO Screening with RNA
(66) The experiment was conducted in the same way, as described for MWCO screening with pDNA (Example 7.1). Prior to use, the spin filters were flushed with 500 μl WFI, and WFI was removed from the permeate compartment completely. After that, 300 μl of three different transcription reactions were added and centrifuged at room temperature, according to the manufacturer's instructions. After approximately half the volume had passed the membrane, centrifugation was stopped and the exact volume in the permeate and retentate chamber was determined. Retentate chamber was then flushed with 100 μl WFI and combined with the retentate. Beside volume measurement, also the concentration of RNA was determined photometrically by measuring the absorption at 260 nm in the starting solution and retentate solution.
(67) Furthermore in
Example 8—Parameter Screening of Different Membranes
(68) The in vitro transcription reaction R2587 (according to Example 4) already diafiltrated in WFI was used for parameter screening. The different membranes (Sartocon Slice 200 100 kDa, PES from Sartorius, Sartocon Slice 200 100 kDa, Hydrosart (cellulose-based membrane) from Sartorius and NovaSet-LS ProStream (Low Binding mPES), 100 kDa from Novasep) were screened in respect to permeate flux versus TMP and dp, respectively. The sample load was 0.1 mg RNA/cm.sup.2 membrane. The Hydrosart membrane (cellulose-based membrane from Sartorius) showed the highest permeate flow rate compared to the other membranes (polyethersulfone (PES)-based membranes from Sartorius and NovaSep) tested. As can be seen from the results as shown in Table 2 a pressure difference over the membrane (dp) of at least 0.5 bar and a transmembrane pressure (TMP) of at least 0.75 bar are necessary to reach a FLUX rate of at least 100 l/h/m.sup.2.
(69) TABLE-US-00002 TABLE 2 FLUX rates resulting from different parameters selected for screening experiments Sartorius, NovaSet, PES, 100 mPES, 100 Hydrosart, kDa kDa 100 kDa p1 p2 p3 dp TMP FLUX FLUX FLUX [bar] [bar] [bar] [bar] [bar] [l/h/m.sup.2] [l/h/m.sup.2] [l/h/m.sup.2] 0.5 0 0 0.5 0.25 45 54.6 72 1 0.5 0 0.5 0.75 133.8 141.6 171 1.5 1 0 0.5 1.25 164.4 172.8 235.8 1 0 0 1 0.5 87.6 85.2 135.9 1.5 0.5 0 1 1 180 178.8 233.1 2 1 0 1 1.5 238.8 215.4 307.8 1.5 0 0 1.5 0.75 138.6 120 198.9 2 0.5 0 1.5 1.25 228.6 247.2 298.8 2.5 1 0 1.5 1.75 280.8 308.4 381.6 2 0 0 2 1 185.4 181.2 270 2.5 0.5 0 2 1.5 270 308.1 352.8
(70) Based on these experiments the following parameters were chosen for TFF of the transcription reaction:
(71) TABLE-US-00003 TABLE 3 Selected parameters for TFF of the transcription reaction Feed Retentate Permeate pressure pressure pressure (p1) (p2) (p3) dp TMP 1.5 bar 0.5 bar 0 bar 1 bar 1 bar (= 0.15 MPa) (= 0.05 MPa) (= 0 MPa) (= 0.1 MPa) (= 0.1 MPa)
Example 9—TFF with Higher Membrane Load
(72) 9.1—TFF of Transcription Reaction Using a Hollow Fibre Module
(73) An RNA in vitro transcription reaction (R2587 with 3775 nt), diafiltrated in WFI was used to perform a flow rate screening in hollow fibre membrane (Hollow fibre module, 100 kDa, PES, 50 cm.sup.2 from GE Healthcare). The membrane load was about 2.0 mg RNA/cm.sup.2. As shown in
(74) 9.2—TFF Using TFF Cassette Modules
(75) An RNA in vitro transcription reaction (R2312 with 1885 nt), diafiltrated in WFI was used to perform a TFF using the following TFF parameters (Table 4):
(76) TABLE-US-00004 TABLE 4 TFF parameters using TFF membrane cassette modules Feed Retentate Permeate pressure pressure pressure (p1) (p2) (p3) dp TMP 0.15 MPa 0.05 MPa 0 MPa 0.1 MPa 0.1 MPa
(77) The membrane load was about 4.5 mg RNA/cm.sup.2. The Sartorius PES, 100 kDa, the NovaSet-LS ProStream (Low Binding mPES), 100 kDa from Novasep as well as the Hydrosart Sartorius 100 kDa (cellulose-based membrane) cassettes were used as TFF modules, respectively. The obtained permeate flux rates were higher than for the hollow fibre membranes with values between about 125-140 l/h/m.sup.2 as shown in
Example 10—RNA Stability During TFF
(78) An mRNA containing sample (R2564; 2083 nt) was diafiltrated with WFI for several hours at room temperature using the Sartorius PES, 100 kDa membrane cassette and the TFF parameters as described above in Example 9.2. The RNA stability after transcription reaction was compared with the stability of RNA after subsequent TFF. The stability, i.e. the RNA integrity (relative area of full-length product) was determined by analytical RP-HPLC.
(79) The results are summarized in
(80) The stability data showed that mRNA in transcription reaction mix was only stable if stored at −20° C. for up to 60 days (85-90%); at 5° C. integrity started decreasing slowly from the beginning (it sank from 85% to 61% in 61 days). A very rapid decrease in integrity was measured (decline from 81% to 51% over 14 days) if the mRNA was stored at room temperature. On the other hand, mRNA after TFF of the transcription reaction mix as described above was stable over 30 days if stored at −20° C. and at 5° C. If the mRNA was stored at room temperature it still showed high integrities for at least 7 days and then slowly decreased down to 80% integrity in 33 days. From this experiment it can be concluded, that a higher degree of RNA stability was achieved, by TFF of mRNA in WFI in comparison to the stability of RNA in the in vitro transcription reaction without purification by TFF.
Example 11-TFF of RNA Containing RP-HPLC Pool
(81) 11.1—TFF Parameters for Diafiltration of RP-HPLC Pool
(82) RP-HPLC purified RNA samples (as described in Example 5) in WFI, 0.1 M TEAA, 13% acetonitrile were used for the parameter screening.
(83) We screened the different membranes (Hydrosart (cellulose-based membrane) from Sartorius, Omega Centramate T OS100T02, PES 100 kDa from PALL, and PES-based membranes with a MWCO of 100 kDa and 300 kDa from Sartorius) in respect to TMP vs. permeate flux and at different RNA concentrations (Table 5). The different membranes behaved similar during TMP screening experiments. In general, the higher the dp and TMP the higher the measured FLUX is (Table 5). At higher TMP values the process tends to be controlled by formation of the cake layer (maximum permeate flux reached, permeate flux independent of TMP).
(84) TABLE-US-00005 TABLE 5 Results of the parameter screening Sartorius Sartorius. PALL. RNA PES 100 Hydrosart. PES 300 Centramate. conc p1 p2 p3 dp TMP kDa 100 kDa kDa 100 kDa [μg/μl] [bar] [bar] [bar] [bar] [bar] [l/h/m.sup.2] [l/h/m.sup.2] [l/h/m.sup.2] [l/h/m.sup.2]] 0.1 0.5 0 0 0.5 0.25 43.2 73.8 49.2 94.8 0.1 1 0.5 0 0.5 0.75 104.4 160.2 120 182.4 0.1 1.5 1 0 0.5 1.25 118.8 171 129.6 204 0.1 1 0 0 1 0.5 46.8 126.9 123.6 127.8 0.1 1.5 0.5 0 1 1 106.8 200.7 182.4 207.6 0.1 2 1 0 1 1.5 130.2 211.5 194.4 258 0.1 1.5 0 0 1.5 0.75 68.4 178.2 144 154.8 0.1 2 0.5 0 1.5 1.25 118.8 225.9 204 242.4 0.1 2.5 1 0 1.5 1.75 151.2 248.4 219.6 304.8 0.1 2 0 0 2 1 86.4 205.2 169.2 199.2 0.1 2.5 0.5 0 2 1.5 129.6 260.1 224.4 282 0.1 3 1 0 2 2 165.6 0 0 Sartorius Sartorius. PALL. RNA- PES 100 Hydrosart. PES 300 Centramate. conc p1 p2 p3 dp TMP kDa 100 kDa kDa 100 kDa [μg/μl] [bar] [bar] [bar] [bar] [bar] [l/h/m.sup.2] [l/h/m.sup.2] [l/h/m.sup.2] [l/h/m.sup.2]] 1 0.5 0 0 0.5 0.25 22.8 62.4 42 56.4 1 1 0.5 0 0.5 0.75 65.7 95.4 78 108 1 1.5 1 0 0.5 1.25 76.5 97.2 85.2 117.6 1 1 0 0 1 0.5 42.3 105.6 74.4 84 1 1.5 0.5 0 1 1 87.3 138 106.8 133.2 1 2 1 0 1 1.5 103.5 135.6 112.8 142.8 1 1.5 0 0 1.5 0.75 59.4 136.8 99.6 112.8 1 2 0.5 0 1.5 1.25 99.9 160.8 124.8 159.6 1 2.5 1 0 1.5 1.75 130.8 165.6 130.8 176.4 1 2 0 0 2 1 75.6 166.2 121.2 139.2 1 2.5 0.5 0 2 1.5 118.8 178.8 138 184.8 1 3 1 0 2 2 Sartorius Sartorius. PALL. RNA- PES 100 Hydrosart. PES 300 Centramate. conc p1 p2 p3 dp TMP kDa 100 kDa kDa 100 kDa [μg/μl] [bar] [bar] [bar] [bar] [bar] [l/h/m.sup.2] [l/h/m.sup.2] [l/h/m.sup.2] [l/h/m.sup.2]] 1.5 0.5 0 0 0.5 0.25 22.8 57.6 38.4 48.6 1.5 1 0.5 0 0.5 0.75 69.6 85.5 67.2 95.4 1.5 1.5 1 0 0.5 1.25 74.4 90 69.6 109.8 1.5 1 0 0 1 0.5 38.4 104.4 67.2 84.6 1.5 1.5 0.5 0 1 1 78 126 92.4 135 1.5 2 1 0 1 1.5 98.4 118.8 93.6 165 1.5 1.5 0 0 1.5 0.75 46.8 127.8 88.8 117 1.5 2 0.5 0 1.5 1.25 93.6 136.8 114 168 1.5 2.5 1 0 1.5 1.75 114 126 189 1.5 2 0 0 2 1 73.2 117 147 1.5 2.5 0.5 0 2 1.5 110.4 133.2 180 1.5 3 1 0 2 2
(85) Although higher dp and TMP values might lead to an increase in FLUX rates, the following parameters for large scale experiments were selected (Table 6):
(86) TABLE-US-00006 TABLE 6 selected parameters for TFF of RP-HPLC pool Feed Retentate Permeate pressure pressure pressure (p1) (p2) (p3) dp TMP 0.1-0.2 MPa 0.05-0.1 MPa 0 MPa 0.05-0.1 MPa 0.075-0.15 MPa
(87) The ranges for TMP and dp as shown in Table 6 were selected because under these conditions the process is not completely cake layer driven. Although higher dp values (specifically higher μl values) lead to further increase of the FLUX rate, application in large scale processes are impeded due to restrictions in pump force. Moreover, lower shear force (lower dp and TMP values) is preferred in respect to RNA stability.
(88) 11.2—Spermidine Depletion Via TFF
(89) In first experiments, the concentrated RP-HPLC pool was diafiltrated with water (WFI). In this case a relatively high residual spermidine concentration in the final RNA solution was observed. To eliminate the spermidine an additional diafiltration step before final diafiltration into water was introduced. Different diafiltration solutions (Table 7) were screened. Approximately 5-10 mL of a RNA (R2564) containing solution after RP-HPLC purification was diafiltrated with 100-200 mL diafiltration solution, followed by diafiltration with 100-200 mL water. Here, single-use Vivaflow PES-based membrane cassettes (Sartorius) with a MWCO between 10-100 kDa were applied. Samples were analyzed after 10, 20, 30 and 40 diafiltration exchange volumes, respectively. Finally, the retentate was concentrated to approx. 0.5 g/L and the amount of spermidine was determined as described in Example 6.3. As a control, the RNA was not conditioned using TFF but precipitated by lithium chloride precipitation (see e.g. Sambrook et al., Molecular Cloning, a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press 1989.) and the amount of contaminating spermidine was determined as described in Example 6.3.
(90) The results are summarized in Table 7.
(91) TABLE-US-00007 TABLE 7 Spermidine concentration after TFF using different diafiltration solutions Diafiltration buffer Spermidine concentration composition (μg spermidine/mg RNA) water 100.81 20 mM sodium phosphate pH 6.2 53.31 0.5M sodium chloride 0.01 0.2M sodium chloride 0.08 Lithium chloride precipitation 0.04
(92) Diafiltration of the RP-HPLC pool using TFF with pure water or 20 mM sodium phosphate did not efficiently remove RNA-bound spermidine. Application of high salt diafiltration solutions, e.g. NaCl based solutions, resulted in substantially complete depletion of RNA-bound spermidine. Furthermore, salts (e.g. TEAA), organic solvents (TEA, ACN) were efficiently removed.
(93) Subsequent optimization experiments have demonstrated that the NaCl concentration could be reduced to at least 0.2 M in order to increase permeate flow rates during diafiltration without effecting spermidine depletion efficiency. Direct addition of NaCl to the concentrated RP-HPLC pool (final concentration ˜0.5 M or 0.2 M, respectively) resulted in faster spermidine depletion by TFF (less diafiltration solution is needed to reach the spermidine depletion). Application of higher concentrations of NaCl in the diafiltration solution are not advisable since this might lead to RNA precipitation and, consequently, blocking of the TFF membrane. The spermidine depletion step can be performed after RP-HPLC purification or directly after in vitro transcription.
(94) 11.3.—Test of Different Membranes for Spermidine Depletion Step:
(95) Two membranes (Novasep mPES 100 kDa and the cellulose-based Sartorius Hydrosart 100 kDa) were further analyzed. We tested both membranes at higher sample load (approx. 2 mg RNA/cm.sup.2 membrane) for diafiltration. For concentration and diafiltration the following parameters were chosen: dp=1 bar and TMP=1.5 bar, membrane load: 2.0 mg mRNA/cm.sup.2 membrane. mRNA in WFI, 0.1 M TEAA, 13% acetonitrile and 0.2 M NaCl was concentrated from 0.1 g/l to 5 g/l and FLUX rates were determined. Diafiltration against 10 diafiltration volumes (dv) of 0.2 M NaCl solution and 10 dv of WFI was performed.
(96) Results:
(97) The overall time for concentration and diafiltration of 380 mg mRNA was very similar: NovaSet: 2.8 h, Hydrosart: 2.68 h. Respective FLUX rates for the diafiltration step are shown in
Example 12—TFF of Linearization Reaction with Higher Membrane Load
(98) For concentration and diafiltration of the linearization reaction we tested TFF membranes made from different material, with a MWCO of 100 kDa and membrane area of 200 cm.sup.2 from different suppliers (The PES-based membranes Sartocon Slice 200 from Sartorius and the NovaSet-LS ProStream (Low Binding mPES) from NovaSep and the cellulose-based membrane Sartocon Slice 200, Hydrosart from Sartorius) with a high membrane load (5.6 and 6 g plasmid DNA/m.sup.2). The parameters selected for TFF of the transcription reaction were applied in this step as well, as they showed good results before. Therefore dp and TMP=1 bar (P1=1.5 bar, P2=0.5 bar and P3=0 bar) were selected to concentrate pDNA (P1452.8 120 mg) from 0.2 g/l to approximately 1.5 g/l and afterwards for diafiltration against 10 diafiltration volumes WFI.
(99) Results:
(100) All tested membranes showed similar results. During concentration of the linearization reaction (
Example 13—Complete Process
(101) 13.1—Linearization of pDNA Linearization of the plasmid DNA P1141 was conducted according to Example 2.
(102) 13.2—Concentration and Diafiltration of Linearized pDNA Using TFF
(103) For tangential flow filtration of the linearization reaction, a Vivaflow50 filter cassette (PES membrane, MWCO 100 kDa, Sartorius) was used. Before assembling the diafiltration setup, all product contacting components (tubes, feed tank etc.) were thoroughly washed with ethanol and water. Next, the setup was washed with ultra-pure water. Then, the setup was chemically sanitized with 500 mM NaOH solution. Subsequently, the setup was washed with WFI until a pH of 7 was measured in the retentate. Then, the feed tank was filled with 150 ml linearization reaction according to Example 2.
(104) In a first concentration step, approximately 100 ml of restriction reaction was filtered, to obtain 50 ml retentate with a higher pDNA concentration. Next, a vacuum was applied to the feed tank, and the feed tube was connected to a WFI bottle. Then, the diafiltration procedure with 10 volumes (500 ml in total) WFI was performed. Subsequently, the retentate was concentrated as much as possible. After concentration, the retentate was collected in a sterile 50 ml reaction tube. The retentate was analyzed on a DNA agarose gel (see
(105) Results
(106) Photometrical Determination of the DNA Concentration after Concentration and Diafiltration:
(107) The DNA concentration of the different DNA samples was determined by measuring the absorption at 260 nm:
(108) TABLE-US-00008 TABLE 8 Plasmid concentration Volume for Concentration agarose gel [g/l] [μl] Permeate from concentration step n/a 10 Permeate from diafiltration step n/a 10 Retentate from diafiltration step 0.10 3 Linearized plasmid 0.17 1.8 Plasmid control 0.10 3
(109) The dsDNA concentration in the retentate was photometrically determined to be 1.05 g/l in a final volume of 22 ml. The diafiltration of the linearized pDNA using TFF yielded 96.6% of input DNA.
(110) Agarose Gel Electrophoresis:
(111) Only a negligible amount of plasmid DNA is visible in the permeate of the concentration step and of the diafiltration step.
(112) 13.3—RNA In Vitro Transcription
(113) 800 ml of in vitro transcription mix as described in Example 4 was incubated for 3 h at 37° C. Next, CaCl.sub.2) and DNase I was added and subsequently incubated for 2 h at 37° C.
(114) 13.4—Diafiltration of the Transcription Reaction
(115) The TFF system Sartoflow 200 with two PES membranes (Sartorius, 200 cm.sup.2, 100 kDa) was used to exchange the buffer of three aliquots (400 ml each) of the transcription reaction to WFI (process parameters indicated in Table 9). First, container and tubes were cleaned with ultrapure water, ethanol and WFI. Then the setup was assembled and the filter cassette was mounted on the Sartocon holder according to the manufacturer's instructions. The whole system was cleaned with ultra-pure water. Subsequently, the system was chemically sanitized by a 1 hour wash with 1 M NaOH. Then, the setup was washed with WFI until a pH of 7 was measured in the retentate and the system was equilibrated with 500 ml WFI. After that, 400 ml transcription reaction (see 13.3) were added to the retentate reservoir, pressures (dp and TMP) were set to 1 bar, and the diafiltration procedure was started against 10 diafiltration volumes (DFV) water for injection (WFI). After diafiltration by TFF the RNA concentration was measured photometrically.
(116) TABLE-US-00009 TABLE 9 TFF parameters used for diafiltration of the transcription reaction Parameters Amount of RNA [mg] 2100-2200 Volume of RNA-solution [ml] 410-430 membrane Sartorius, PES, 100 kDa Number of membranes 2 pressures [bar] P1 = 1.5 P2 = 0.5 P3 = 0 TMP und dp = 1 bar Diafiltration volume WFI [1] 4.1-4.3 Membrane load [mg RNA/cm.sup.2] 5-5.6
(117) Results:
(118) The final concentration of the RNA after diafiltration was 5 g/l and the total recovery rate of the RNA after buffer exchange via diafiltration was 98%.
(119) Example 13.5—RP-HPLC Purification of the Conditioned Transcription Reaction
(120) The RNA solution obtained from Example 13.4 was diluted to 100 mM TEAA and a concentration of 1 g/l by addition of 1 M triethylammonium acetate (TEAA) and WFI. The RNA was step-wise purified according to Example 5. The HPLC fractions were collected, the product-containing fractions were pooled and divided in three pools I to III.
(121) Moreover, fractions were analyzed for RNA content (UV260/280) and every fraction was analyzed for RNA integrity.
(122) Results:
(123) TABLE-US-00010 TABLE 10 Determination of RNA concentration and RNA integrity after RP-HPLC purification RNA RNA Pools Volume conc. amount integrity Pool I Ca. 7.2 L 0.12 g/l Ca. 840 mg 97.8% Pool II Ca. 7.3 L 0.12 g/l Ca. 840 mg 97.7% Pool III Ca. 6.2 L 0.12 g/l Ca. 744 mg 95.7%
(124) Compared to the starting material (TFF conditioned RNA), the integrity could be increased by >10% (86.5% RNA integrity before RP-HPLC) by separating aborted RNA species.
(125) Example 13.6—Concentration and Diafiltration of the RP-HPLC Pool Via TFF
(126) The three RP-HPLC purified RNA pools were processed separately using an additional concentration and diafiltration step with TFF to further separate the RNA from impurities (e.g. spermidine contaminations) and to exchange the solvent.
(127) First, every pool was concentrated from 0.12 g/l to approximately 5 g/l using TFF. 5M NaCl solution was added to the retentate to get a final concentration of 0.2 M NaCl. Then, a diafiltration was performed against 0.2 M NaCl (10 DFV) to remove spermidine impurities. Next, the diafiltration solution was exchanged to WFI (10 DFV). The process parameters are shown in Table 11. After TFF RNA concentration was determined by measuring the absorption at 260 nm. For agarose gel electrophoresis the indicated amounts of RNA were used for gel electrophoresis.
(128) TABLE-US-00011 TABLE 11 Parameters used for TFF of the RP-HPLC pools Pool 1 Pool 2 Pool 3 Pressures P1 = 2 P1 = 2 P1 = 2 used for P2 = 0.5 P2 = 1 P2 = 1 concentration P3 = 0 P3 = 0 P3 = 0 [bar] TMP = 1.2 TMP = 1.5 TMP = 1.5 dp = 1.5 dp = 1.0 dp = 1.0 Pressures P1 = 2 P1 = 2 P1 = 2 used for P2 = 1 P2 = 1 P2 = 1 diafiltration P3 = 0 P3 = 0 P3 = 0 [bar] TMP = 1.5 TMP = 1.5 TMP = 1.5 dp = 1.0 dp = 1.0 dp = 1.0 TFF Hydrosart- Hydrosart- Hydrosart- membrane Membrane Membrane Membrane 200 cm.sup.2; 100 kDa 200 cm.sup.2; 100 kDa 200 cm.sup.2; 100 kDa Membrane 2.1 mg RNA/cm.sup.2 2.1 mg RNA/cm.sup.2 1.9 mg RNA/cm.sup.2 load start volume 7.2 L 7.3 L 6.2 L
(129) Results:
(130) The yield after the diafiltration was spectrometrically determined to be 94.44%, 94.3% and 90.5% respectively.
(131) Agarose Gel Electrophoresis:
(132) Samples of the TFF diafiltration procedure after RP-HPLC purification were analyzed. Respective samples were analyzed using agarose gel electrophoresis (see
(133) TABLE-US-00012 TABLE 12 Loading scheme of permeate and retentate samples taken during TFF of the RP-HPLC pools. RNA conc. Volume used for agarose Lane Sample [μg/μl] gel electrophoresis [μl] 1 RNA marker 8 2 RP-HPLC 0.12 9 Pool I 3 RP-HPLC 0.12 9 Pool II 4 RP-HPLC 0.12 9 Pool III 5 TFF permeate n.a. 9 6 TFF permeate n.a. 9 (40× concentrated) 7 TFF retentate 5.00 1 Pool I 8 TFF retentate 5.00 1 Pool II 9 TFF retentate 4.84 1 Pool III 10 Final product 4.84 1 11 Control 5.00 1 12 Control 1.00 1 13 empty 14 RNA marker 8
(134) The TFF permeate samples did not contain detectable RNA levels (even though samples that had been concentrated 40×).
(135) The TFF retentate samples and the final TFF conditioned RNA pool contained RNA of integrity of about 100%, all of those with a band size of 2476 bases, which was in accordance to the theoretically expected size.
(136) Example 13.7—Determination of Protein Content Using a BCA Assay
(137) To determine the protein content in the samples, the BCA-test was used. The total protein concentration contained in a sample was measured photometrically via absorption at 562 nm compared to a protein standard (bovine serum albumin, BSA). The test was performed using a commercially available BCA kit, according to the manufacturer's instructions.
(138) To produce a 20 μg/ml bovine serum albumin (BSA) solution stock solution, 20 μl BSA solution [1 mg/ml] was mixed with 980 μl water for injection. This BSA stock solution was used to generate a standard curve using BSA solutions of different concentrations (50 μl each, diluted in WFI): 2.5 μg/ml; 5 μg/ml; 10 μg/ml; 15 μg/ml; 20 μg/ml
(139) The protein content was determined in samples from the TFF after linearization reaction (Example 13.2), from the transcription reaction (Example 13.3), before RP-HPLC (Example 13.4) and after RP-HPLC and TFF against 0.2 M NaCl (Examples 13.5 and 13.6).
(140) Results:
(141) The measurements were performed in a standard photometer. Results are displayed in Table 13.
(142) TABLE-US-00013 TABLE 13 Determined protein concentrations A562 RNA/DNA nm (AU) protein conc. protein/RNA Sample average Dilution [μg/ml] [mg/ml] [μg/mg] 1 TFF retentate after 0.717 20 324.2 1.05 308.8 linearization 2 transcription reaction 0.404 1000 6823.4 5.07 1345.8 3 TFF retentate before RP- 0.596 10 122.9 4.30 28.6 HPLC 4 TFF retentate before RP- 0.632 10 136 5,.2 26.7 HPLC 5 TFF retentate before RP- 0.625 10 131.2 5.22 25.1 HPLC 6 TFF retentate after RP- 0.397 2 13.2 5.03 2.6 HPLC Pool I 7 TFF retentate after RP- 0.352 2 10.7 5.00 2.1 HPLC Pool II 8 TFF retentate after RP- 0.455 2 16.6 4.84 3.4 HPLC Pool III
(143) Values of the BCA-assay are shown. 1: TFF retentate of linearization reaction; 2: Transcription reaction; 3-5: TFF retentates of transcription reactions before RP-HPLC; 6-8: TFF retentates of transcription reactions after RP-HPLC
(144) A step-wise depletion of the protein content per RNA over the whole RNA purification procedure could be observed.
(145) SDS-PAGE:
(146) SDS-PAGE was used to determine the protein content in the different samples. The indicated amounts of samples were used for SDS-PAGE. The results are shown in
(147) TABLE-US-00014 TABLE 14 Samples used for SDS PAGE RNA/DNA Volume Lane Sample conc. [mg/ml] [μl] 1 Protein Marker 2 TFF retentate after 1.05 9.5 linearization 3 transcription reaction 5.07 2.0 4 TFF retentate before 4.30 2.3 RP-HPLC 5 TFF retentate before 5,.2 2.0 RP-HPLC 6 TFF retentate before 5.22 1.9 RP-HPLC 7 TFF retentate after 5.03 2.0 RP-HPLC Pool I 8 TFF retentate after 5.00 2.0 RP-HPLC Pool II 9 TFF retentate after 4.84 2.1 RP-HPLC Pool III 10 Final Product 4.84 2.1 11 control 5.00 2.0 12 Protein Marker
(148) No protein bands were detectable in samples after RP-HPLC purification.
(149) Example 13.8—Determination of Spermidine Concentration
(150) Spermidine concentration was measured in the TFF retentates before RP-HPLC (Example 13.4) and after RP-HPLC purification and TFF against 0.2 M NaCl (Example 13.5 and 13.6) according to Example 6.3.
(151) Results:
(152) TABLE-US-00015 TABLE 15 Spermidine concentrations in RNA samples RNA conc. Spermidin/RNA Sample [g/l] [ng/mg] TFF retentate before 0.22 39964.50 RP-HPLC (1:20) TFF retentate before 0.26 39039.21 RP-HPLC (1:20) TFF retentate before 0.26 37748.26 RP-HPLC (1:20) RP-HPLC-Pool I 0.12 1834.98 RP-HPLC-Pool II 0.12 30.08 RP-HPLC-Pool III 0.12 110.90 TFF retentate after 5.03 1.00 RP-Pool I TFF retentate after 5.00 3.08 RP-Pool II TFF retentate after 4.84 1.48 RP-Pool III
(153) Results:
(154) Spermidine was detectable in samples of the TFF retentates before RP-HPLC purification. In the samples purified by RP-HPLC and TFF using 0.2 M NaCl only a very low amount of spermidine was detectable (see Table 15).
(155) Example 13.9—Determination of Organic Solvents
(156) The concentration of acetonitrile (ACN) and TEAA was determined according to Example 6.4.
(157) Results:
(158) The final sample after RP-HPLC purification and TFF contained less than 40 ppm ACN and less than 2 ppm TEAA.
Example 14—Overview of the Process and Key Process Parameters
(159) In the following, a further example of the inventive method is illustrated, providing process parameters of the method for each of the individual steps including concentration of the linearization reaction and diafiltration of linearized plasmid DNA, diafiltration of RNA in vitro transcription reaction and concentration and diafiltration of an RP-HPLC RNA pool.
(160) 14.1—Concentration of the Linearization Reaction and Diafiltration of Linearized Plasmid DNA Using TFF:
(161) Linearization of the plasmid DNA was performed as described in Example 2. For tangential flow filtration of the linearization reaction (conducted according to Example 2), a Hydrosart filter cassette (cellulose based membrane, MWCO 100 kDa, Sartorius) was used. The plasmid DNA concentration procedure as well as the diafiltration procedure was performed as explained above (see Example 13). The result of the concentration of the linearization mix is provided in
(162) TABLE-US-00016 TABLE 16 TFF process parameters of plasmid DNA concentration and diafiltration Process parameter Concentration Initial pDNA concentration 0.2 of the [g/l] linearization Pressures used for concentration P1 = 1.5 reaction of the pDNA linearization P2 = 0.5 mix [bar] P3 = 0 TMP = 1 dp = 1 Obtained pDNA concentration 1.0 or 1.5 [g/l] Diafiltration of Pressures used for diafiltration P1 = 1.5 the linearized of linearized pDNA [bar] P2 = 0.5 pDNA in WFI P3 = 0 TMP = 1 dp = 1 DFV 10 DF buffer WFI General TFF membrane cassettes Hydrosart parameters membrane cassette; 200 cm.sup.2; 100 kDa or NovaSet-LS ProStream (Low Binding mPES), 100 kDa Membrane load 0.1-0.6 mg DNA/cm.sup.2 Feed flowrate [l/h/m.sup.2] 750-900 Permeate flux rate [l/h/m.sup.2] 30-100
(163) 14.2—Diafiltration of the RNA IVT Reaction by TFF:
(164) RNA in vitro transcription was performed as described in Example 4. For tangential flow filtration of the RNA IVT reaction (conducted according to Example 4), a Hydrosart filter cassette (cellulose based membrane, MWCO 100 kDa, Sartorius) was used. The conditioning of the RNA IVT reaction was performed as explained above (see Example 13). The result of the diafiltration of the RNA IVT reaction is provided in
(165) TABLE-US-00017 TABLE 17 TFF Process parameters of the RNA IVT reaction diafiltration Process parameter Pressures used for diafiltration of P1 = 1.5 RNA IVT reaction [bar] P2 = 0.5 P3 = 0 TMP = 1-1.5 dp = 1 DFV 10 DF buffer WFI TFF membrane cassettes Hydrosart membrane cassette; 200 cm.sup.2; 100 kDa or NovaSet-LS ProStream (Low Binding mPES), 100 kDa Membrane load 2.5-6.5 mg RNA/cm.sup.2 Feed flowrate [l/h/m.sup.2] 300-1050 Permeate flux rate [l/h/m.sup.2] 20-120
(166) 14.3—Concentration and Diafiltration of the RP-HPLC RNA Pool
(167) The in vitro transcribed RNA was purified by RP-HPLC as described in Example 5. For tangential flow filtration of the RP-HPLC RNA pool, a Hydrosart filter cassette (cellulose based membrane, MWCO 100 kDa, Sartorius) was used. The concentration of the RP-HPLC RNA pool was performed as explained above (see Example 13). The result of the concentration of the RP-HPLC RNA is provided in
(168) TABLE-US-00018 TABLE 18 TFF process parameters of the RP-HPLC RNA concentration and diafiltration Process parameter Concentration Initial RNA concentration 0.1 of the [g/l] RP-HPLC RNA Pressures used for P1 = 1.5 pool concentration of the pDNA P2 = 0.5 linearization mix [bar] P3 = 0 TMP = 1 dp = 1 Obtained RNA 5 +/− 0.25 concentration [g/l] Diafiltration of Pressures used for P1 = 1.5 the RP-HPLC diafiltration of RNA P2 = 0.5 RNA [bar] P3 = 0 pool in NaCl TMP = 1-1.5 buffer dp = 1 DFV 10 DF buffer 0.2M NaCL Diafiltration of Pressures used for P1 = 1.5 the RP-HPLC diafiltration of RNA P2 = 0.5 RNA [bar] P3 = 0 pool in WFI TMP = 1-1.5 dp = 1 DFV 10 DF buffer WFI General TFF membrane cassettes Hydrosart membrane parameters cassette; 200 cm.sup.2; 100 kDa or NovaSet-LS ProStream (Low Binding mPES), 100 kDa Membrane load 2 mg-2.5 mg RNA/cm.sup.2 Feed flowrate [l/h/m.sup.2] 900-1500 Permeate flux rate [l/h/m.sup.2] 25-140 Temperature [° C.] 17° C. or <17° C.
(169) Embodiment List:
(170) 1. A method for producing and purifying RNA, comprising the steps of
(171) A) providing DNA encoding the RNA;
(172) B) transcription of the DNA to yield a solution comprising transcribed RNA; and
(173) C) conditioning and/or purifying of the solution comprising transcribed RNA by one or more steps of tangential flow filtration (TFF).
(174) 2. The method according to item 1, wherein in step A) plasmid DNA is provided as DNA encoding the RNA and the method comprises subsequently to step A) the steps:
(175) A1) linearization of the plasmid DNA in a linearization reaction;
(176) A2) optionally termination of the linearization reaction; and
(177) A3) conditioning and/or purifying of the linearization reaction comprising linearized plasmid DNA by one or more steps of TFF.
(178) 3. The method according to item 1 or 2, wherein step C) comprises at least one diafiltration step and/or at least one concentration step using TFF.
(179) 4. The method according to item 3, wherein the at least one diafiltration step using TFF in step C) comprises diafiltration with an aqueous salt solution.
(180) 5. The method according to item 4, wherein the aqueous salt solution is a NaCl solution, preferably an aqueous solution comprising from about 0.1 M NaCl to about 1 M NaCl, more preferably a solution comprising from about 0.2 to about 0.5 M NaCl.
(181) 6. The method according to item 3, wherein the at least one diafiltration step using TFF of step C) comprises diafiltration with water.
(182) 7. The method according to any one of items 1 to 6, wherein the method does not comprise a step of phenol/chloroform extraction and/or DNA and/or RNA precipitation.
(183) 8. The method according to any one of items 1 to 7, wherein the method does not comprise a step of using a TFF hollow fiber membrane.
(184) 9. The method according to any one of items 1 to 8, wherein the at least one or more steps of TFF comprises using a TFF membrane with a molecular weight cutoff of 500 kDa, preferably of ≤200 kDa and most preferably of 100 kDa.
(185) 10. The method according to any one of items 1 to 9, wherein the at least one or more steps of TFF comprises using a TFF membrane comprising at least one of polyethersulfone (PES), modified polyethersulfone (mPES), a cellulose derivative membrane or combinations thereof.
(186) 11. The method according to any one of items 1 to 10, wherein the at least one or more steps of TFF comprises using a TFF membrane comprising a cellulose derivative membrane with a molecular weight cutoff of about 100 kDa.
(187) 12. The method according to any one of items 1 to 11, wherein the at least one or more steps of TFF comprises using a TFF membrane cassette.
(188) 13. The method according to any one of items 1 to 12, wherein the method comprises in step C) at least one further purification method before or after the one or more steps of TFF.
(189) 14. The method according to any of items 1 to 13, wherein the method comprises in step C) the steps:
(190) C1) optionally termination of transcription;
(191) C2) conditioning and/or purifying of the solution comprising the transcribed RNA by one or more steps of TFF;
(192) C3) purifying the RNA by any further purification method; and
(193) C4) conditioning and/or optionally purifying of the solution comprising the transcribed RNA obtained after step C3) by one or more steps of TFF.
(194) 15. The method according to item 14, wherein step C2) comprises at least one step of diafiltration using TFF with water and/or diafiltration with an aqueous salt solution, preferably an aqueous NaCl solution and more preferably with an aqueous solution comprising from about 0.1 M NaCl to about 1 M NaCl, more preferably a solution comprising from about 0.2 to about 0.5 M NaCl.
(195) 16. The method according to item 14 or 15, wherein step C4) comprises at least one first diafiltration step using TFF.
(196) 17. The method according to item 16, wherein step C4) comprises at least one second diafiltration step using TFF.
(197) 18. The method according to any one of items 16 or 17, wherein the at least one first diafiltration step using TFF in step C4) comprises diafiltration with an aqueous salt solution, preferably an aqueous NaCl solution and more preferably with an aqueous solution comprising from about 0.1 M NaCl to about 1 M NaCl, more preferably a solution comprising from about 0.2 to about 0.5 M NaCl.
(198) 19. The method according to item 17 or 18, wherein the second diafiltration step using TFF of step C4) comprises diafiltration with water.
(199) 20. The method according to any one of items 13 to 19, wherein the at least one further purification method is performed by means of high performance liquid chromatography (HPLC) or low normal pressure liquid chromatography methods.
(200) 21. The method according to any one of items 13 to 20, wherein the at least one further purification method is a reversed phase chromatography method.
(201) 22. The method according to any one of items 1 to 21, wherein all steps of TFF are performed with the same TFF membrane.
(202) 23. The method according to any one of items 1 to 22, wherein the transcribed RNA is selected from the group consisting of mRNA, viral RNA, retroviral RNA and replicon RNA, small interfering RNA (siRNA), antisense RNA, CRISPR 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) or whole-cell RNA and preferably is mRNA.
(203) 24. The method according to any one of items 1 to 23, wherein the transcription of DNA in step B) is performed as in vitro transcription.