Process for the annealing of double stranded oligonucleotides using crossflow filtration technology
Patent Information
- Authority / Receiving Office
- IL · IL
- Patent Type
- Applications
- Current Assignee / Owner
- F HOFFMANN LA ROCHE & CO AG
- Filing Date
- 2024-12-12
- Publication Date
- 2026-07-01
AI Technical Summary
Standard manufacturing processes for double-stranded oligonucleotides are resource-intensive and prone to errors due to multiple sampling operations and mixing small amounts of oligonucleotide solutions, leading to inaccuracies in concentration measurement and optimal ratio calculation for annealing.
The process involves using automated crossflow filtration equipment with a permeate line, retentate vessel, and membrane to continuously crossflow-filter solutions of single-strand oligonucleotides until target concentrations are reached and annealing is completed, as detected by UV signal changes.
This method streamlines the production of double-stranded oligonucleotides, reducing resource consumption and errors by allowing continuous processing and real-time monitoring of annealing completion, thereby achieving a more efficient and accurate production process.
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Abstract
Description
[0001] Process for the annealing of double stranded oligonucleotides using crossflow filtration technology
[0002] The invention relates to a novel process for the production of double stranded oligonucleotides by annealing a first single strand oligonucleotide with a second single strand oligonucleotide in an automated crossflow equipment.
[0003] Double stranded oligonucleotides can be used as therapeutic agents e.g. as small interfering RNA (siRNA). SiRNA is a type of RNA molecule that plays a crucial role in gene silencing or gene regulation. SiRNA is involved in the process of RNA interference (RNAi), where it helps in the degradation of specific messenger RNA (mRNA) molecules, preventing them from being translated into proteins. This targeted degradation of mRNA enables the selective suppression of gene expression, providing a powerful tool for studying gene function and potentially developing therapeutic interventions for various diseases. This was described, for example, in Anwar, S.; Mir, F.; Yokota, T. Enhancing the Effectiveness of Oligonucleotide Therapeutics Using Cell-Penetrating Peptide Conjugation, Chemical Modification, and Carrier-Based Delivery Strategies. Pharmaceutics 2023, 15 (4), 1130; or in Roberts, T. C.; Langer, R.; Wood, M. J. A. Advances in Oligonucleotide Drug Delivery. Nature Reviews Drug Discovery 2020. siRNAs have been widely studied for their therapeutic potential in various diseases, including genetic disorders, viral infections, and certain types of cancers. By targeting specific disease-causing genes or viral RNA, siRNAs can modulate gene expression, correct abnormal splicing patterns, inhibit protein production, or enhance degradation of target RNA. This targeted approach offers the potential for precise therapeutic interventions. Illustrative for siRNA therapeutics is e.g. Ozcan, G.; Ozpolat, B.; Coleman, R. L.; Sood, A. K.; Lopez -Berestein, G. Preclinical and Clinical Development of siRNA- Based Therapeutics. Advanced Drug Delivery Reviews 2015, 87, 108-119.
[0004] Standard manufacturing processes for double stranded oligonucleotides consist of separately synthesizing, purifying and desalting the individual single strands and combining them in the annealing step before ultimately isolating the final double stranded product as outlined in the Figure 1 and as described for instance in Seiffert, S.; Debelak, H.; Hadwiger, P.; Jahn-Hofmann, K.; Roehl, I.; Vomlocher, H.-P.; Noll, B. Characterization of Side Reactions during the Annealing of Small Interfering RNAs. Analytical Biochemistry 2011, 414 (1), 47-57. This widely applied process to perform the annealing involves an individual sampling of the purified and desalted single strand oligonucleotide solution 1 and 2 to determine the concentration of the respective oligonucleotide.
[0005] The concentration measurement involves a certain inaccuracy, which requires that the effective ratio for the annealing of the two single strand oligonucleotides have first to be calculated via a number of small scale annealing experiments.
[0006] Table 1 shows a test series to determine the optimal ratio for the bulk annealing of the single strand oligonucleotide 1 and 2
[0007] Table 1: Data of microtitration experiments used in a standard process to determine the ideal ratio of strands before bulk annealing. The experiment in Entry 5 of Table 1, provides the basis for the bulk annealing in a separate manufacturing operation.
[0008] This standard approach is resource intensive in terms of experimental, manufacturing and analytical work and is prone to errors due to multiple sampling operations and mixing small amounts of oligonucleotide solution. Accordingly, more efficient and foolproof processes are desirable.
[0009] Object of the present invention is therefore to provide a more efficient, streamlined and simplified process which does not comprise the described disadvantages of the known standard processes.
[0010] The object could be achieved with the novel process for producing double stranded oligonucleotides, wherein a) in an automated crossflow equipment, comprising a permeate line, a retentate vessel and a membrane, b) a solution of a first single strand oligonucleotide is fed into the retentate vessel and the retentate is continuously crossflow-filtered over the membrane until a target concentration is reached, thereafter; c) a solution of a second single strand oligonucleotide is fed into the retentate vessel and the retentate is continuously crossflow-filtered over the membrane under conditions which allow annealing until the completion of the annealing is detected.
[0011] The figures are explained as follows
[0012] Fig.1 shows the standard manufacturing process for the manufacturing of double stranded oligonucleotides which consists of separately synthesizing, purifying and desalting and combining them in the annealing step before ultimately isolating the final double stranded product.
[0013] Fig.2 shows the novel manufacturing process for the manufacturing of double stranded oligonucleotides which consists of separately synthesizing and purifying the individual single strands, but involving crossflow filtration for the subsequent desalting and the annealing.
[0014] Fig.3 shows the UV absorption data of a sensor which is placed in the permeate line over the course of an annealing experiment. The UV signal enables the determination of the completed annealing by an increase in the UV signal which is caused by small amounts of the second single strand passing through the membrane into the permeate line (highlighted in the figure with “Annealing completion”). Fig.4 shows the UV absorption data of a sensor which is placed in the retentate vessel over the course of an annealing experiment. The UV signal enables the determination of the completed annealing by an increase in the UV signal which is caused by excess single strand (highlighted in the figure with “Annealing completion”).
[0015] The following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention herein.
[0016] The term oligonucleotide as used herein is defined as it is generally understood by the skilled person as a molecule comprising two or more covalently linked nucleotides. For use as a therapeutically valuable oligonucleotide, oligonucleotides are typically synthesized as 10 to 40 nucleotides, preferably 10 to 25 nucleotides in length.
[0017] The oligonucleotides may consist of optionally modified DNA, RNA or LNA nucleoside monomers or combinations thereof.
[0018] The LNA nucleoside monomers are modified nucleosides which comprise a linker group or a bridge between C2’ and C4’ of the ribose sugar ring of a nucleotide. These nucleosides are also termed bridged nucleic acid or bicyclic nucleic acid (BNA) in the literature.
[0019] Optionally modified as used herein refers to nucleosides modified as compared to the equivalent DNA, RNA or LNA nucleoside by the introduction of one or more modifications of the sugar moiety or the nucleobase moiety. In a preferred embodiment the modified nucleoside comprises a modified sugar moiety, and may for example comprise one or more 2’ substituted nucleosides and / or one or more LNA nucleosides. The term modified nucleoside may also be used herein interchangeably with the term “nucleoside analogue” or modified “units” or modified “monomers”.
[0020] The DNA, RNA or LNA nucleosides are as a rule linked by a phosphodiester (P=O) and / or a phosphorothioate (P=S) intemucleoside linkage which covalently couples two nucleosides together.
[0021] Accordingly, in some oligonucleotides all intemucleoside linkages may consist of a phosphodiester (P=O), in other oligonucleotides all intemucleoside linkages may consist of a phosphorothioate (P=S) or in still other oligonucleotides the sequence of intemucleoside linkages vary and comprise both phosphodiester (P=O) and phosphorothioate (P=S) intemucleoside. The principles of the oligonucleotide manufacturing are well known in the art (see e.g. Oligonucleotide synthesis; Wikipedia, the free encyclopedia; https: / / en.wikipedi a. org / w / index.php?title=Oligonucleotide_synthesis&oldid=l 174276567 of November 8, 2023.
[0022] The term “double-stranded oligonucleotide” refers to two complementary or at least partially complementary strands of oligonucleotides, as defined before, which are bound together by hydrogen bonds. While double-stranded oligonucleotides involve the interaction of two complementary strands, their overall length and overlap may differ.
[0023] The term “Crossflow filtration” is a well-established separation technique used e.g. for desalting and concentration of liquid solutions based on particle size of the content. It involves the flow of the solution across a filtration membrane under pressure. Unlike conventional filtration, where the liquid flows perpendicular to the filtration medium, in crossflow filtration, the liquid flows parallel to the membrane surface while a portion of it passes through the membrane due to the applied pressure. This allows for continuous operation and is ideal for applications such as the desalting and concentration during oligonucleotides manufacturing (see e.g. Biotechnol. Prog. 2023;e3350).
[0024] Various types of membranes are suitable for the purpose of the crossflow filtration of oligonucleotides. Some common membrane materials used in crossflow filtration include:
[0025] • Polymeric membranes: These are typically made of materials such as poly sulfone, polyethersulfone, polyvinylidene fluoride (PVDF), or cellulose acetate. Polymeric membranes are widely used due to their versatility, cost-effectiveness, and compatibility with a wide range of solvents and pH levels.
[0026] • Ceramic membranes: These membranes are made of inorganic materials such as alumina, zirconia, or titania. Ceramic membranes are known for their high chemical and thermal stability, making them suitable for demanding applications that involve harsh conditions or high-temperature environments.
[0027] Preferred membranes are selected from regenerated cellulose and poly ethersulfone.
[0028] In a preferred embodiment the membrane has a molecular weight cut-off, which is dependent on the length of the oligonucleotides involved in the process, but preferably is between 1 kDa and 20 kDa, more preferably between 3 kDa and 15 kDa. Over all steps, the solutions are fed over the membrane in a manner that the loading in relation of the membrane area is between 100’000 OD / m2and 4’000’000 OD / m2, preferably between. 250’000 OD / m2and 2’500’000 OD / m2.
[0029] The process for producing double stranded oligonucleotides of the present invention essentially comprises a) in an automated crossflow equipment, comprising a permeate line, a retentate vessel and a membrane, b) feeding a solution of a first single strand oligonucleotide into the retentate vessel and the retentate is continuously crossflow-filtered over the membrane until a target concentration is reached, thereafter; c) feeding a solution of a second single strand oligonucleotide into the retentate vessel and the retentate is continuously crossflow-filtered over the membrane under conditions which allow annealing until the completion of the annealing is detected.
[0030] In a preferred embodiment subsequent to step c) in step d) a salt buffer solution is fed into to retentate vessel and the retentate is continuously crossflow-filtered over the membrane until excessive single strand oligonucleotides are removed, thereby maintaining the retentate volume constant and subsequent to step d) in step e) water is fed to retentate and the retentate is continuously crossflow-filtered over the membrane until remaining impurities are substantially reduced.
[0031] The retentate solution obtained subsequent to step e) contains the pure double strand oligonucleotide. Isolation of it can happen via lyophilization.
[0032] The solution of a first single strand oligonucleotide and of a second single strand oligonucleotide can be obtained directly from the oligonucleotide synthesis after an initial purification, for instance with HPLC (high performance liquid chromatography).
[0033] In a further preferred embodiment the pre-processing does not involve an extra desalting before the crossflow-filtration steps which enable the annealing of the single strand oligonucleotides. The concentration of the first single strand oligonucleotide in the feeding solution is as a rule in the range of 50 OD / mL to 1000 OD / mL, preferably from 125 OD / mL to 350 OD / mL.
[0034] The concentration of the second single strand oligonucleotide in the feeding solution is typically in the range of 50 OD / mL to 1000 OD / mL, preferably from 125 OD / mL to 350 OD / mL.
[0035] The solution in step b) is usually fed over the membrane with a transmembrane pressure between 0.5 and 3.0 bar, preferably between. 1.0 bar and 2.5 bar.
[0036] The target concentration to be reached in step b) is typically between 50 OD / mL to 1000 OD / mL, preferably between 300 OD / mL to 750 OD / mL.
[0037] The crossflow filtration in step b) is as a rule performed in such a manner that the target concentration corresponds to a concentration factor of 1 to 5, preferably 1.5 to 3.5. .
[0038] In step c) the solution is fed over the membrane with a transmembrane pressure between 0.5 and 3.0 bar, preferably between 1.0 bar and 2.5 bar, in in step d) the solution is fed over the membrane with a transmembrane pressure between 0.5 and 3.0 bar, preferably between 1.0 bar and 2.5 bar and in step e) the solution is fed over the membrane with a transmembrane pressure between 0.5 and 3.0 bar, preferably between 1.0 bar and 2.5 bar.
[0039] The completion of the annealing in step c) is detected by measuring changes of the UV signal in the permeate or in the retentate, whereby the UV signal turns up in the wavelength range of 230 to 300 nm, more preferably 250 to 280 nm .
[0040] In one implementation of the new process, the first single strand oligonucleotide is crossflow-filtered over the membrane with selected molecular weight cut off until a target concentration is reached. The second single strand oligonucleotide is then added until the UV sensor on the permeate line shows a change of the signal in the range mentioned above, indicating completion of the annealing.
[0041] In a more preferred implementation of the process the UV signal is measured with a suitable sensor in the retentate vessel. This sensor can be applied instead of the UV sensor in the permeate vessel, but more preferably it is applied in addition to the UV sensor in the permeate vessel. In this implementation of the new process, the first single strand oligonucleotide is crossflow-filtered over the membrane with selected molecular weight cut off until a target concentration is reached. The second single strand oligonucleotide is then added until the UV sensor on the retentate line detects a change in the UV signal. In this way, the excess of the second single strand oligonucleotide can be controlled on an even lower level as there is shorter delay compared to measurement of the UV signal in the permeate.
[0042] In a further preferred embodiment, UV measurement allows an automated management of the feed of the second single strand oligonucleotide in an optimal ratio, resulting in an annealing, which avoids unnecessary excess of one of the oligonucleotide strands. For instance feedback control stops the addition of the second single strand oligonucleotide once the UV signal reports the completion of the annealing.
[0043] Accordingly, in both implementations of the process, the approximate target ratio of the two strands can be determined based on a single point concentration measurement of the purified individual single strands. The accuracy of the measurement is not as critical as in the standard approach described in the introduction as the ratio is controlled during the addition process.
[0044] A heating-cooling cycle can be implemented to facilitate correct and complete annealing. Such a step may be advisable if, depending on the nature of the single strand oligonucleotides, the initial annealing has to be corrected by a heating step to disassemble the double strand and a subsequent careful cooling to assemble the strands in the desired manner. The heating-cooling cycle can be implemented in the crossflow-process.
[0045] As outlined above and in a preferred embodiment subsequent to step c) purification steps d) and e) follow to remove excess single strand oligonucleotides from the double strand solution by crossflow filtration and to reduce remaining impurities, such as salts and solvents, to a large extent.
[0046] In step d) salt buffers, i.e. aqueous solutions of salts are applied, which are capable to enhance permeation of the single strands through the membrane. Suitable salts with this capability contain anions with increasing kosmotropic strength (see ACS Omega 2020, 5, 12, 6229-6239). They can be selected from alkali halogenides, such as alkali chlorides or bromides, alkali sulfates, alkali hydrogen carbonates or alkali carbonates, preferably from alkali sulfates or alkali carbonates, more preferably from the respective sodium or potassium salts and even more preferably from sodium sulfate. . The concentration of salt in the aqueous buffer solution is between 0.05 M and 2.0 M, preferably between 0.1 M and 1.0 M.
[0047] Finally in step e) water is fed to retentate and the retentate is continuously crossflow-filtered over the membrane until remaining impurities are substantially reduced. These remaining impurities mainly consist of salts or solvents of the previous process steps.
[0048] As outlined above the isolation of the pure double strand oligonucleotide can happen via lyophilization, spray drying or by an isolation in the form of a concentrated solution. For the purpose of illustration the following single strand oligonucleotides have been applied.
[0049] OLIGO 1.1 : sense strand mGs.mAo.fCo.mAo.mAo.mAo.mAo.fAo.fUo.fCo.mCo.fUo.fCo.mAo.mCo.mAo.fAo.mU o.mAo.mAo.mGo.mCo.mAo.mGo.mCo.mCo.gGo.gAo.gAo.gAo.mGo.mGo.mCo.mUo.m Go.mC
[0050] OLIGO 1.2: anti-sense strand pUs.fUs.fAs.mUo.fUo.mGo.fUo.fGo.mAo.fGo.mGo.fAo.mUo.fUo.mUo.fUo.mUo.mGo.f Uo.mCs.mGs.mG
[0051] OLIGO 1.1 and OLIGO 1.2 have the following nucleobase sequence.1
[0052] SEQ ID No 1 : gacaaaaatc ctcacaataa gcagccgaaa ggctgc (OLIGO 1.1)
[0053] SEQ ID No 2: ttattgtgag gatttttgtc gg (OLIGO 1.2)
[0054] According to WIPO ST.26 format “f ’ represents U (uracil).
[0055] OLIGO 2.1: anti-sense strand
[0056] TGN.Lo.eAs.eTs.eEs.eEs.eEs.dAs.dEs.dGs.dEs.dEs.dEs.dEs.dTs.dGs.dTs.eEs.eEs.eAs.eG s.eE
[0057] OLIGO 2.2 : sense strand dGo.mCo.dTo.dGo.dGo.dAo.mCo.dAo.dGo.dGo.dGo.dGo.mCo.dGo.dTo.dGo.dGo.dGo.d Ao.dT
[0058] OLIGO 2.1 and OLIGO 2.2 have the following nucleobase sequence. SEQ ID No. 3: atcccacgcc cctgtccagc (OLIGO 2.1)
[0059] SEQ ID No. 4: atcccacgcc cctgtccagc (OLIGO 2.2)
[0060] OLIGO 3.1.: anti-sense strand ITs.lTs.lAs.dCs.lAs.dCs.dTs.dTs.dAs.dAs.dTs.dTs.dAs.dTs.dAs.dCs.dTs.lTs.lEs.lE
[0061] OLIGO 3.2 : sense strand dGo.dGo.dAo.dAo.dGo.dTo.dAo.dTo.dAo.dAo.dTo.dTo.dAo.dAo.dGo.dTo.dGo.dTo.dA o.dA
[0062] OLIGO 3.1 and OLIGO 3.2 have the following nucleobase sequence.
[0063] SEQ ID No. 5: ttacacttaa ttatacttcc (OLIGO 3.1)
[0064] SEQ ID No. 6: ggaagtataa ttaagtgtaa (OLIGO 3.2)
[0065] The following nomenclature rules apply for the sequence information above.
[0066] Sequence information is provided from 5'-end (left) to 3'-end (right). Each nucleotide is described by three-letters: First letter: Sugar (d = 2 ’-deoxyribose / DNA, e = 2'-O-(2-methoxyethyl) ribose / MOE, f = 2'-fluoro-2’deoxyribose, g = 2’-modified GalNAc ribose, 1 = 2’-O-CH2-4’-bridged ribose / LNA, m = 2'-O-methylribose , p = methylphosphonate-2’-O-methyl riboseisostere); Second letter: Nucleobase (A = adenine, C = cytosine, E = 5-methylcytosine, G = guanine, L = hexylaminolinker , T = thymine, U = uracil); Third letter: Backbone (o = phosphate, s = thiophosphate); Conjugate: TGN = Tris GalNAc cluster.
[0067] Examples:
[0068] Example 1: Permeate controlled annealing end point a1In an automated crossflow equipment, the prepared strand 1 solution (combined purification fractions or a desalted crude solution) was fed into the retentate vessel and concentrated by ultrafiltration utilizing a membrane with suitable molecular weight cut off (the same membrane was used for all following crossflow filtrations), until the target starting conditions were achieved. a2The prepared strand 2 solution (combined purification fractions or a desalted crude solution) was fed into the retentate vessel via the feed pump while the retentate was continuously crossflow filtered to maintain a constant volume level in the retentate vessel until the UV sensor in the permeate recognized a significant increase. a3The strand 2 solution was replaced with a salt buffer (e.g. Na2SC>4, NaCl, NaHCCh, NaBr, Nal) suitable to deplete the excess single strand while the double strand remains intact. The retentate was continuously crossflow filtered to maintain a constant volume level in the retentate vessel until fifteen volumes of buffer have passed the membrane. a4The salt buffer was replaced with water and the retentate was continuously crossflow filtered to maintain a constant volume level in the retentate vessel until fifteen volumes of water have passed the membrane.
[0069] The retentate was recovered and the retentate loop was washed twice with some water to collect the final product for lyophilisation and analysis.
[0070] Table 2: Data of annealing experiments using UV detection in permeate line. remaining excess of OLIGO 3.2
[0071] Example 2: Retentate controlled annealing end point a1In an automated crossflow equipment, the prepared strand 1 solution (combined purification fractions or a prefiltrated crude solution) was fed into the retentate and concentrated by ultrafiltration utilizing a membrane with suitable molecular weight cut off (the same membrane was used for all following crossflow filtrations), until the target starting conditions were achieved. a2The prepared strand 2 solution (combined purification fractions or a prefiltrated crude solution) was fed into the retentate via the feed pump while the retentate was continuously crossflow filtrated to maintain a constant volume level in the retentate until the UV sensor in the retentate recognized a significant slope change. a3The strand 2 solution was replaced with a salt buffer (e.g. Na2SO4, NaCl, NaHCCh, NaBr, Nal) suitable to deplete the excessive single strand while the double strand remains intact and the retentate was continuously crossflow filtrated to maintain a constant volume level in the retentate until fifteen volumes of buffer have passed the membrane. a4The salt buffer was replaced with water and the retentate was continuously crossflow filtrated to maintain a constant volume level in the retentate until fifteen volumes of water have passed the membrane.
[0072] The retentate was recovered and the retentate loop was washed twice with some water to collect the final product for lyophilisation and analysis.
[0073] Table 3: Data of annealing experiments using UV detection in retentate line. Example 3: Salt dependent permeation of single strands a1In an automated crossflow equipment, the prepared oligonucleotide solution (prefiltered crude solution) was fed into the retentate and concentrated by ultrafiltration utilizing a membrane with suitable molecular weight cut off (the same membrane was used for all following crossflow filtrations), until the target starting conditions were achieved. a2The prepared buffer (e.g. H2O, Na2SO4, NaCl, NaHCO3, Na2CO3NaBr, Nal) was fed into the retentate via the feed pump. During this process the retentate was continuously crossflow filtered to maintain a constant volume level in the retentate vessel. The process was continued until twenty to thirty volumes of buffer have passed or the oligonucleotide was completely gone to the permeate. A sample of the retentate solution was taken every two volumes for photometric optical density (OD / mL) evaluation.
[0074] For all experiments listed, the parameter ranges and conditions as specified in Table 4 were applied unless otherwise specified in the result data below:
[0075] Table 4: Parameters and conditions used for single strand permeation experiments. Table 5: Recovery data (%) from retentate of single strand permeation experiments of OLIGO 1.2 using regenerated cellulose membrane with 10 kDa molecular weight cut-off.
[0076] Table 5 continued
[0077] Table 6: Recovery data (%) from retentate of single strand permeation experiments of OLIGO 1.2 using poly ethersulfone membrane with 10 kDa molecular weight cut-off.
[0078]
[0079] Table 7: Recovery data (%) from retentate of single strand permeation experiments of OLIGO 2. 1 using regenerated cellulose membrane with 10 kDa molecular weight cut-off. Table 8: Recovery data (%) from retentate of single strand permeation experiments of OLIGO 2. 1 using regenerated cellulose membrane with 5 kDa molecular weight cut-off.
[0080] Table 9: Recovery data (%) from retentate of single strand permeation experiments of OLIGO 2. 1 using poly ethersulfone membrane with 10 kDa molecular weight cut-off.
[0081]
[0082] Table 10: Recovery data (%) from retentate of single strand permeation experiments of OLIGO 3. 1 using regenerated cellulose membrane with 5 kDa molecular weight cut-off.
[0083] Table 11: Recovery data (%) from retentate of single strand permeation experiments of OLIGO 3. 1 using polyethersulfone membrane with 10 kDa molecular weight cut-off.
Claims
Claims:
1. Process for producing double stranded oligonucleotides, wherein a) in an automated crossflow equipment, comprising a permeate line, a retentate vessel and a membrane, b) a solution of a first single strand oligonucleotide is fed into the retentate vessel and the retentate is continuously crossflow-filtered over the membrane until a target concentration is reached, thereafter; c) a solution of a second single strand oligonucleotide is fed into the retentate vessel and the retentate is continuously crossflow-filtered over the membrane under conditions which allow annealing until the completion of the annealing is detected.
2. Process of claim 1, wherein, subsequent to step c) in step d) a salt buffer solution is fed into to retentate vessel and the retentate is continuously crossflow-filtered over the membrane until excessive single strand oligonucleotides are removed, thereby maintaining the retentate volume constant and subsequent to step d) in step e) water is fed to retentate and the retentate is continuously crossflow-filtered over the membrane until remaining impurities are substantially reduced.
3. Process of claim 2, wherein subsequent to step e) the retentate solution is lyophilized to obtain the pure double strand oligonucleotide.
4. Process of any one of claims 1 to 3, wherein the solution of a first single strand oligonucleotide and of a second single strand oligonucleotide are obtained directly from the oligonucleotide synthesis after an initial purification.
5. Process of any one of claims 1 to 4, wherein the concentration of the first single strand oligonucleotide in the feeding solution is in the range of 50 OD / mL to 1000 OD / mL, preferably from 125 OD / mL to 350 OD / mL.
6. Process of any one of claims 1 to 5, wherein the concentration of the second single strand oligonucleotide in the feeding solution is in the range of 50 OD / mL to 1000 OD / mL, preferably from 125 OD / mL to 350 OD / mL.
7. Process of any one of claims 1 to 6, wherein the solution in step b) is fed over the membrane with a transmembrane pressure between 0.5 and 3.0 bar, preferably between.1.0 bar and 2.5 bar8. Process of claim 7, wherein the cross filtration in step b) is performed in such a manner that the target concentration corresponds to a concentration factor of 1 to 5, preferably 1.5 to 3.5.
9. Process of any one of claims 1 to 8, wherein the solution in step c) is fed over the membrane with a transmembrane pressure between 0.5 and 3.0 bar, preferably between 1.0 bar and 2.5 bar.
10. Process of any one of claims 2 to 8, wherein the solution in step d) is fed over the membrane with a transmembrane pressure between 0.5 and 3.0 bar, preferably between 1.0 bar and 2.5 bar.
11. Process of any one of claims 2 to 6, wherein the solution in step e) is fed over the membrane with a transmembrane pressure between 0.5 and 3.0 bar, preferably between.1.0 bar and 2.5 bar.
12. Process of any one of claims 1 to 11, wherein the solutions, over all steps, are fed over the membrane in a manner that the loading in relation of the membrane area is between 100’000 OD / m2and 4’000’000 OD / m2, preferably between. 250’000 OD / m2and 2’500’000 OD / m213. Process of any one of claims 1 to 12, wherein the membrane has a molecular weight cut-off, which is dependent on the length of the oligonucleotides involved in the process, but preferably is between 1 kDa and 20 kDa, more preferably between 3 kDa and 15 kDa.
14. Process of any one of claims 1 to 13, wherein target concentration to be reached in step b) is between 50 OD / mL to 1000 OD / mL, preferably from 300 OD / mL to 750 OD / mL.
15. Process of any one of claims 1 to 14, wherein the completion of the annealing in step c) is detected by measuring the UV signal in the permeate or in the retentate , which appears in the wavelength range of 230 - 300 nm, more preferably between 250 and 280 nm .
16. Process of any one of claims 2 to 15, wherein the salt buffer is an aqueous solution of a salt which is capable to enhance permeation of the single strand oligonucleotides.
17. Process of claim 16, wherein the salt is an alkali halogenide, alkali sulfate, alkali hydrogen carbonate or alkali carbonate, preferably an alkali sulfate or an alkali carbonate.
18. Process of claims 16 or 17 wherein the concentration of salt in the aqueous buffer solution is between 0.05 M and 2.0M, preferably between 0.1 M and 1.0 M.
19. Process of any one of claims 1 to 18, wherein the isolation of the pure double strand oligonucleotide happens via lyophilization, spray drying or by an isolation in the form of a concentrated solution.