Excipients for reducing the viscosity of highly concentrated nucleic acid compositions and combinations thereof

Abstract

The present invention relates to a liquid composition containing a nucleic acid with reduced viscosity. Further, the present invention relates to a method for reducing the viscosity of a liquid composition containing a nucleic acid.

Description

Technical Field

[0001] Technical Field The present invention relates to a liquid composition containing a nucleic acid with reduced viscosity. Further, the present invention relates to a method for reducing the viscosity of a liquid composition containing a nucleic acid.

[0002] Background For decades, nucleic acids themselves have been exclusively the subject of scientific investigation or used as tools in genetic engineering research. Today, however, they are being used as therapeutic agents or serve as important starting materials in the manufacturing processes of novel active therapeutic agents, including viruses, gene therapy products, and mRNA or DNA vaccines. With the recent success of new treatment approaches, reflecting the increasing number of positive results from medical approvals and clinical trials, there is a growing need for improved formulations and more efficient production methods for the administration of nucleic acid pharmaceuticals.

[0003] An aqueous solution of high molecular weight DNA possesses a dynamic viscosity reaching several hundreds to thousands of centipoises (cP) even at a relatively low concentration of several mg / ml (Creeth et al., J Chem Soc. 1947 Sep; 25:1141-5.; Butler et al., Transactions of the Faraday Society, Volume 50, Pages 612 - 623195 1954). Since the viscosity of injectable pharmaceutical formulations should not exceed about 50 cP (Srinivasan et al., Pharm Res 30, 1749-1757, 2013) and ideally should be less than half of that, this makes it difficult to directly administer naked therapeutic nucleic acids at typical doses ranging from milligrams in DNA vaccines to tens of milligrams in gene therapy. Nucleic acid delivery systems such as viral delivery vectors, cationic liposomes, and polycations are often plagued by toxicity, low in vivo stability, and high cost even when the viscosity is low (Dias and Lindman, DNA Interactions with Polymers and Surfactants, Wiley & Sons Inc. ISBN 978-0-470-25818-7,; 2008, Saraswat et al., Indian J. Pharm. Sci., 71, pp. 488-498 2009). Therefore, finding a way to reduce the viscosity of DNA solutions under pharmaceutically appropriate conditions could be clinically useful both for the formulation of nucleic acid pharmaceuticals and as a standalone therapeutic agent (Elkin et al. Intern. J. of Pharmaceutics Vol. 494, Iss. 1, 15, Pages 66-72; 2015). Similarly, the emergence of new technologies such as RNA-based treatments and prophylactic vaccines (especially m-RNA).

[0004] Nucleic acids such as DNA, RNA, especially mRNA, can only be concentrated up to several milligrams per milliliter. Beyond that concentration level, the viscosity of the solution increases to the extent that the set solution can no longer be injected into the patient or handled by standard filling and finishing equipment.

[0005] Nucleic acid therapeutics are typically administered parenterally via, for example, intravenous (iv), intramuscular (im), or subcutaneous (sc) routes. Subcutaneous injection is particularly popular for the delivery of nucleic acid therapeutics because it can simplify administration to the patient (a small injection in a short time) and reduce treatment costs (more abbreviated medical assistance). To ensure patient compliance, the dosage form for subcutaneous injection is isotonic and desirably can be injected in a small amount (less than 2.0 ml per injection site).

[0006] At the same time, nucleic acid therapy usually requires a dosage of several milligrams to several tens of milligrams. Therefore, the combination of a high therapeutic dose and a low injection volume leads to the need for a highly concentrated formulation of the therapeutic nucleic acid. However, nucleic acids possess a number of functional groups in addition to a potentially complex three-dimensional structure. This makes formulation difficult, especially when high concentrations are required.

[0007] One of the main problems is that solutions containing nucleic acids at concentrations exceeding 1, 3, or 5 mg / ml have a high viscosity. At such concentrations, mainly due to non-natural self-association, nucleic acids tend to form highly viscous solutions. Another problem to be solved is that the concentration of nucleic acids in the solution cannot be increased to concentrations exceeding 1, 3, 5, or 10 mg / ml. In many cases, nucleic acid-containing solutions cannot be handled by standard filling and finishing equipment or experimental equipment. Yet another problem to be solved is that nucleic acid-containing solutions with high viscosity interfere with processing steps including transfection of cell lines, polymerase chain reaction, and incorporation of nucleic acids into viral vectors.

[0008] The rheological properties of biopolymer solutions such as nucleic acid formulations not only complicate parenteral administration for medical use, but the high viscosity of nucleic acid solutions also presents significant challenges for manufacturing itself, particularly for enhancing the manufacturing process.

[0009] Many known manufacturing methods, particularly many known manufacturing methods for relatively large amounts of nucleic acids such as plasmid DNA and mRNA, involve filtration procedures, chromatographic purification steps, or mixing, pump through, or filling operations for processing the flow of the product fluid. The performance of these steps is greatly affected by the rheological behavior of the product fluid flow being processed. In the manufacturing process, highly concentrated nucleic acid formulations with high viscosities present particular difficulties for ultrafiltration and sterile filtration. Tangential flow filtration is also often used for buffer exchange and increasing nucleic acid concentration. However, viscous solutions exhibit an increase in backpressure and shear stress during injection and filtration, resulting in the potential instability of therapeutic nucleic acids and / or longer processing times. The increase in said shear stress often results in product loss. Both aspects have an adverse impact on the economics of the process.

[0010] At the same time, high viscosity significantly limits the injectability of nucleic acids and is unacceptable with regard to administration.

[0011] To address these issues and / or improve the stability of the solution, additives and excipients such as EDTA and sodium chloride are typically added to biopharmaceutical formulations at high concentrations. However, the resulting solutions often cause pain due to high injection forces and resulting tissue damage. Some of these solutions are no longer administrable, resulting in a lack of treatment options for patients.

[0012] However, in formulating nucleic acids such as DNA or RNA, particularly mRNA, it is necessary to carefully select formulation additives and / or excipients in order to avoid denaturation and loss of biological activity. Also, excipients need to be pharmaceutically safe and physiologically compatible in order to avoid any undesirable side effects such as allergic reactions.

[0013] As a result, there is a strong need in the pharmaceutical industry for pharmaceutically acceptable additional excipients that reduce viscosity, particularly as an alternative when standard solutions such as those described above fail.

[0014] Accordingly, the problem to be solved is to provide excipients that can effectively reduce the viscosity of nucleic acid solutions. Further, the problem to be solved is to provide combinations of excipients that can effectively reduce the viscosity of nucleic acid solutions.

[0015] During the bioprocess, the solution needs to be pumped through tubes and chromatography columns. At high viscosities, the flow rate through such columns is limited by the viscosity, which leads to longer processing times, significant loss of nucleic acid during chromatography, or the potential for incomplete processing of the nucleic acid solution. Further, shear forces can occur when passing through connectors from thin tubes into thinner columns. Shear stress is a typical reason for nucleic acids to denature and potentially aggregate, thereby reducing the yield of the process. Clearly, such aggregation induced by shear stress has an adverse effect on the economics of the process. Additionally, the gel bed in the chromatography column can be damaged by high pressure.

[0016] In addition, some nucleic acids are formulated at high concentrations through tangential flow filtration (TFF). When the viscosity of the solution becomes significant, a gel-like layer may form near the membrane. In particular, the flux of the membrane significantly reduces productivity, increases the processing time, and as a result, the manufacturing cost becomes significantly high. As discussed previously, shear stress also occurs during TFF, which can produce insoluble nucleic acid aggregates and reduce the yield.

[0017] Generally, it has been observed that a highly viscous solution generates a certain degree of adhesiveness, making it difficult to completely recover the solution from the container and out of the tube, or to remove the entire substance from the processing system. Such loss of the substance leads to a significant reduction in the productivity of the product, along with an obvious adverse effect on the economics of the process.

[0018] Furthermore, the problem to be solved is to provide a combination of excipients that can effectively reduce the viscosity of the nucleic acid solution. A high viscosity of the nucleic acid solution causes many difficulties in bioprocessing. Known additives that have been used to reduce the viscosity of the corresponding nucleic acid solution often do not lead to a sufficient viscosity reduction effect. Therefore, it is an object of the present invention to find new possibilities to improve the corresponding viscosity reduction effect and reduce the adverse impact on the economics of the process. SUMMARY OF THE INVENTION

[0019] Summary of the Invention The problem is solved by a liquid composition comprising a nucleic acid and at least one viscosity-reducing excipient selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine, and thiamine phosphate. In a further aspect, the liquid composition comprises a nucleic acid and a combination of two excipients selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine, and thiamine phosphate. In a further aspect, the liquid composition comprises a nucleic acid and a combination of a first excipient selected from the group consisting of arginine, phenylalanine, ornithine, and meglumine and a second excipient selected from the group consisting of benzenesulfonic acid, pyridoxine, and thiamine phosphate.

[0020] Similarly, the problem is solved by a method for reducing the viscosity of a liquid nucleic acid composition, the method comprising the step of adding at least one excipient selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine, and thiamine phosphate to the liquid nucleic acid composition. In a further aspect, a combination of two excipients selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine, and thiamine phosphate is added to the liquid nucleic acid composition. In a further aspect, a combination of a first excipient selected from the group consisting of arginine, phenylalanine, ornithine, and meglumine and a second excipient selected from the group consisting of benzenesulfonic acid, pyridoxine, and thiamine phosphate is added to the liquid nucleic acid composition.

[0021] By using at least two excipients, it is possible to lower the amount of each individual excipient used and to overcome the problem that many viscosity-reducing excipients used at appropriate concentrations can have an adverse effect on the stability of the nucleic acid by taking advantage of the stabilizing effect of the second excipient. In addition, using at least two excipients leads to a synergistic reduction in viscosity.

[0022] Furthermore, the problem is solved by a lyophilized nucleic acid preparation of a composition comprising a nucleic acid and at least one viscosity-reducing excipient selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine, and thiamine phosphate ester. In a further aspect, the composition comprises a nucleic acid and a combination of two excipients selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine, and thiamine phosphate ester. In a further aspect, the composition comprises a nucleic acid and a combination of a first excipient selected from the group consisting of arginine, phenylalanine, ornithine, and meglumine and a second excipient selected from the group consisting of benzenesulfonic acid, pyridoxine, and thiamine phosphate ester.

[0023] Furthermore, the problem is solved by a kit comprising a composition comprising a nucleic acid and at least one first excipient selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine, and thiamine phosphate ester. In a further aspect, the composition comprises a nucleic acid and a combination of two excipients selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine, and thiamine phosphate ester. In a further aspect, the composition comprises a nucleic acid and a combination of a first excipient selected from the group consisting of arginine, phenylalanine, ornithine, and meglumine and a second excipient selected from the group consisting of benzenesulfonic acid, pyridoxine, and thiamine phosphate ester.

[0024] A further subject of the present invention is a method for reducing the viscosity of a liquid nucleic acid composition in a bioprocess, comprising the step of adding at least one excipient selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine, and thiamine phosphate ester to the liquid nucleic acid composition. In a further aspect, a combination of two excipients selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine, and thiamine phosphate ester is added to the liquid nucleic acid composition. In a further aspect, a combination of a first excipient selected from the group consisting of arginine, phenylalanine, ornithine, and meglumine and a second excipient selected from the group consisting of benzenesulfonic acid, pyridoxine, and thiamine phosphate ester is added to the liquid nucleic acid composition.

Brief Description of the Drawings

[0025] [Fig. 1] Figures 1 and 2 show the viscosities of 4 kb pDNA and 14 kb pDNA solutions without excipients (control) and with various excipients. [Fig. 2] Figures 1 and 2 show the viscosities of 4 kb pDNA and 14 kb pDNA solutions without excipients (control) and with various excipients.

[0026] [Fig. 3] Figures 3 and 4 show the viscosities of 4 kb pDNA and 14 kb pDNA solutions without excipients (control) and with various excipients. [Fig. 4] Figures 3 and 4 show the viscosities of 4 kb pDNA and 14 kb pDNA solutions without excipients (control) and with various excipients.

[0027] [Fig. 5]Figures 5 to 7 show the viscosities of mRNA solutions of 2000 bp, 4000 bp, and 6000 bp without (w / o) and with various excipients. [Fig. 6] Figures 5 to 7 show the viscosities of mRNA solutions of 2000 bp, 4000 bp, and 6000 bp without (w / o) and with various excipients. [Fig. 7] Figures 5 to 7 show the viscosities of mRNA solutions of 2000 bp, 4000 bp, and 6000 bp without (w / o) and with various excipients.

[0028] [Fig. 8] Figures 8 to 10 show the viscosities of mRNA solutions of 2000 bp, 4000 bp, and 6000 bp without (control) and with various excipients. [Fig. 9] Figures 8 to 10 show the viscosities of mRNA solutions of 2000 bp, 4000 bp, and 6000 bp without (control) and with various excipients. [Fig. 10] Figures 8 to 10 show the viscosities of mRNA solutions of 2000 bp, 4000 bp, and 6000 bp without (control) and with various excipients. Mode for Carrying Out the Invention

[0029] Detailed Description of the Invention The present invention is directed to a liquid composition comprising a nucleic acid and at least one viscosity-reducing excipient selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine, and thiamine phosphate ester.

[0030] As used herein, "nucleic acid" is defined as a polymer composed of nucleotides. A "nucleotide" is the monomeric structure of a nucleic acid that contains three components: a five-carbon sugar, a phosphate group, and a nitrogenous base. The two main classes of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). When the sugar is ribose, the polymer is RNA; when the sugar is the ribose derivative deoxyribose, the polymer is DNA. For example, although there are several forms of DNA, it is not limited to plasmid DNA or genomic DNA. Similarly, RNA can be found in the form of messenger RNA (mRNA), transfer RNA (t-RNA), catalytic RNA, or structural RNA. DNA or RNA molecules can also be synthetically engineered for various functions. In one aspect, the nucleic acid is DNA or RNA. In one aspect, the nucleic acid is DNA. In one aspect, the nucleic acid is RNA.

[0031] Although not wishing to be bound by theory, the viscosity-reducing effect of the liquid compositions according to the invention is believed to be based on the interaction between the excipient and the nucleic acid residues or the sucrose / phosphate backbone of the nucleic acid. Since all nucleic acids are made from the same nucleotide pool, the effects described herein are applicable to all types of nucleic acids. Thus, the liquid compositions according to the invention have an advantageous effect on all types of nucleic acids, regardless of their sequence, size, and structure.

[0032] The term "liquid composition containing a nucleic acid" as used herein refers to a liquid aqueous nucleic acid-containing composition, preferably an aqueous nucleic acid-containing solution. The composition can contain one or more nucleic acids at various concentrations. Usually, the concentration of one nucleic acid in the liquid composition exceeds 1, 3, 5, or 10 mg / ml.

[0033] In a preferred aspect, the nucleic acid contained in the composition according to the invention is a therapeutic nucleic acid. As used herein, the term "therapeutic nucleic acid" refers to any nucleic acid administered to a subject for the purpose of treating or preventing a disease or medical condition. In particular, the subject may be a mammal or a human. Therapeutic proteins can be administered for various purposes such as replacement of a deficient or abnormal protein, enhancement of existing pathways, provision of new functions or activities, interference with molecules or organisms, and delivery of other compounds or proteins such as radionuclides, cytotoxic drugs, or effector proteins. Therapeutic nucleic acids encompass naked DNA / RNA or modified DNA / RNA. Their sequences can be natural or engineered.

[0034] In a particularly preferred embodiment, the nucleic acid in the liquid composition according to the invention is naked DNA, in particular a therapeutic plasmid. In an even more preferred embodiment, the nucleic acid in the liquid composition according to the invention is RNA such as mRNA.

[0035] In one embodiment, the nucleic acid is a biosimilar. As used herein, "biosimilar" is defined as a biological pharmaceutical that is very similar to another biological pharmaceutical that has already been approved. In a preferred embodiment, the biosimilar is RNA such as mRNA. In one embodiment, the liquid composition according to the invention contains more than one type of nucleic acid.

[0036] As used herein, the term "excipient" refers to any compound at a suitable concentration known to reduce the viscosity of a composition containing a nucleic acid by at least 5% compared to the same composition without the excipient.

[0037] The liquid composition according to the invention contains at least one excipient selected from the group consisting of: · Arginine (CAS registration number 74-79-3) · Phenylalanine (CAS registration number 63-91-2) · Ornithine (CAS registration number 3184-13-2) · Meglumine (CAS registration number 6284-40-8) · Benzenesulfonic acid (CAS registration number 98-11-3) · Pyridoxine (vitamin B6, CAS registration number 65-23-6), · Thiamine monophosphate (thiamine monophosphate, CAS registration number 10023-48-0).

[0038] According to the present invention, the excipient includes salts or solvates of the excipient. Preferred salts in the context of the present invention are physiologically acceptable salts of the compounds according to the present invention. Also included are salts which are not per se suitable for pharmaceutical use but which can be used, for example, for the isolation, purification or storage of the compounds according to the present invention.

[0039] Examples of physiologically acceptable salts of the compounds according to the present invention include, preferably, alkali metal salts (for example, sodium salts and potassium salts), alkaline earth metal salts (for example, calcium salts and magnesium salts), and ammonium salts derived from ammonia or organic amines having 1 to 16 carbon atoms (for example, preferably, ethylamine, diethylamine, triethylamine, N,N-diisopropylethylamine, monoethanolamine, diethanolamine, triethanolamine, dimethylaminoethanol, diethylaminoethanol, procaine, dicyclohexylamine, dibenzylamine, N-methylpiperidine, N-methylmorpholine, arginine, lysine, and 1,2-ethylenediamine) and other salts of conventional bases are included.

[0040] Examples of physiologically acceptable salts of the compounds according to the present invention include, preferably, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, carbonate, digluconate, glycerophosphate, hemisulfonate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethionate), lactate, maleate, mesitylenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, sulfate, tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, paratoluenesulfonate, and undecanoate, and salts of other conventional acids are included. By way of example, a physiologically acceptable salt can be a salt of ornithine, for example, ornithine monohydrochloride.

[0041] Solvates in the context of the present invention are designated as those forms of the compounds according to the present invention that form complexes in the solid or liquid state by coordination with solvent molecules. Hydrates are a specific form of solvates in which the coordination occurs with water. In the context of the present invention, hydrates are preferred solvates.

[0042] According to the present invention, excipients include all enantiomers and stereoisomers of the excipients, as well as racemic mixtures. By way of example, the excipient arginine includes L-arginine, D-arginine, and a racemic mixture of L-arginine and D-arginine.

[0043] The liquid composition of the present invention contains an excipient in an amount sufficient to reduce the viscosity of the composition and / or to stabilize the protein. For example, the liquid composition according to the present invention may contain at least one excipient from about 5 mM to about 300 mM, from about 5 mM to about 250 mM or from about 5 mM to about 150 mM. In an exemplary embodiment, the concentration of at least one excipient is 1, 5, 10, 12, 13, 15, 20, 25, 30, 35, 50, 75, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 250 or 300 mM or more.

[0044] In one aspect of the present invention, these liquid compositions may further contain additives used for purposes other than viscosity reduction, such as for stabilization, solubilization or preservation. In another aspect of the present invention, the liquid composition contains more than one excipient. For example, the liquid composition according to the present invention may contain two, three, or four excipients, preferably containing two excipients.

[0045] Hereinafter, a combination of two excipients advantageous for a liquid composition containing a nucleic acid as described in the present invention, a lyophilized preparation of the liquid composition containing a nucleic acid, a method for reducing the viscosity of the liquid composition containing a nucleic acid, and a kit containing a composition containing a nucleic acid will be described.

[0046] One embodiment of the present invention is a liquid composition comprising a combination of two excipients selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate.

[0047] One embodiment of the present invention is a liquid composition comprising a combination of a first excipient selected from the group consisting of arginine, phenylalanine, ornithine and meglumine and a second excipient selected from the group consisting of benzenesulfonic acid, pyridoxine and thiamine phosphate.

[0048] One aspect of the present invention is a liquid composition comprising a combination of a first excipient selected from the group consisting of arginine, ornithine, and meglumine, and a second excipient selected from the group consisting of benzenesulfonic acid, pyridoxine, and thiamine phosphate ester.

[0049] The combinations of the two excipients include arginine and phenylalanine, arginine and ornithine, arginine and meglumine, arginine and benzenesulfonic acid, arginine and pyridoxine, arginine and thiamine phosphate ester, phenylalanine and ornithine, phenylalanine and meglumine, phenylalanine and benzenesulfonic acid, phenylalanine and pyridoxine, phenylalanine and thiamine phosphate ester, ornithine and meglumine, ornithine and benzenesulfonic acid, ornithine and pyridoxine, ornithine and thiamine phosphate ester, meglumine and benzenesulfonic acid, meglumine and pyridoxine, meglumine and thiamine phosphate ester, benzenesulfonic acid and pyridoxine, benzenesulfonic acid and thiamine phosphate ester, pyridoxine and thiamine phosphate ester.

[0050] Preferred combinations of the two excipients include arginine and benzenesulfonic acid, arginine and pyridoxine, arginine and thiamine phosphate ester, phenylalanine and benzenesulfonic acid, phenylalanine and pyridoxine, phenylalanine and thiamine phosphate ester, ornithine and benzenesulfonic acid, ornithine and pyridoxine, ornithine and thiamine phosphate ester, meglumine and benzenesulfonic acid, meglumine and pyridoxine, meglumine and thiamine phosphate ester.

[0051] More preferred combinations of the two excipients include arginine and benzenesulfonic acid, arginine and pyridoxine, arginine and thiamine phosphate ester, ornithine and benzenesulfonic acid, ornithine and pyridoxine, ornithine and thiamine phosphate ester, meglumine and benzenesulfonic acid, meglumine and pyridoxine, and meglumine and thiamine phosphate ester.

[0052] In one embodiment, arginine and benzenesulfonic acid are combined. In another embodiment, arginine and pyridoxine are combined. In another embodiment, arginine and thiamine phosphate ester are combined. In another embodiment, phenylalanine and benzenesulfonic acid are combined. In another embodiment, phenylalanine and pyridoxine are combined. In another embodiment, phenylalanine and thiamine phosphate ester are combined. In another embodiment, ornithine and benzenesulfonic acid are combined. In another embodiment, ornithine and pyridoxine are combined. In another embodiment, ornithine and thiamine phosphate ester are combined. In another embodiment, meglumine and benzenesulfonic acid are combined. In another embodiment, meglumine and pyridoxine are combined. In another embodiment, meglumine and thiamine phosphate ester are combined. In another embodiment, meglumine and pyridoxine are combined. In another embodiment, meglumine and thiamine phosphate ester are combined. In another embodiment, meglumine and pyridoxine are combined. In another embodiment, meglumine and thiamine phosphate ester are combined.

[0053] The liquid composition according to the present invention contains the amounts of the combination of the two excipients as described above, where the concentrations of both excipients are sufficient to reduce the viscosity of the liquid composition and / or are sufficient to stabilize the nucleic acid. For example, the liquid composition according to the present invention may contain each excipient at about 5 mM to about 300 mM, about 5 mM to about 250 mM, or about 5 mM to about 150 mM. In an exemplary embodiment, the concentration of each excipient is 1, 5, 10, 12, 13, 15, 20, 25, 30, 35, 50, 75, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 250, or 300 mM or more.

[0054] In one aspect, the liquid composition contains a combination of two excipients, where the molar concentrations of the excipients can be the same or different. The molar ratio of the first and second excipients is between 1:100 and 100:1, preferably between 1:10 and 10:1, more preferably between 1:5 and 5:1, and most preferably between 1:2 and 2:1. In a particular embodiment, the molar concentrations of the two excipients are the same.

[0055] For a liquid composition containing a nucleic acid and at least one excipient, the excipients, combinations of excipients, concentrations and molar ratios of excipients and combinations of excipients, and concentration of the nucleic acid as disclosed above should apply to all other aspects of the present invention to the same extent, including the lyophilized formulation of the liquid composition, the method of reducing the viscosity of the liquid composition, or the use of the method in a bioprocess.

[0056] In a further preferred aspect of the liquid composition, the method of reducing viscosity or the method of increasing stability, the excipient synergistically reduces the viscosity and / or increases the stability in the liquid composition containing the nucleic acid.

[0057] According to the present invention, a synergistic viscosity reduction is provided when the viscosity reduction by a combination of two or more excipients is greater than the expected sum of the viscosity reductions of the individual excipients. A synergistic viscosity reduction is preferably provided when the percentage of viscosity reduction by a combination of two or more excipients is greater than the expected sum of the viscosity reduction rates of the individual excipients.

[0058] In one aspect, all combinations of two excipients described in this application result in a synergistic reduction in the viscosity of the liquid composition containing the nucleic acid.

[0059] In another aspect, the present invention provides a lyophilized formulation of a liquid composition comprising a nucleic acid and at least one excipient selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine, and thiamine phosphate ester. When reconstituted with a suitable amount of diluent, the formulation exhibits a reduced viscosity compared to a control formulation having the same composition otherwise but lacking at least one excipient. Thus, when reconstituted with a diluent, the excipient is present in an amount effective to reduce the viscosity.

[0060] The lyophilized formulation according to the present invention includes a nucleic acid and at least one excipient according to the present invention present as dried particles, for example, particles in powder form. In this context, the expression "powder" refers to an essentially dry collection of particles, i.e., having a moisture content of less than at least about 10 wt%, less than 6 wt%, less than 4 wt%, or lower.

[0061] As defined herein, "viscosity" refers to the resistance to flow of a substance (typically a liquid). Viscosity is related to the concept of shear force; it can be understood that shear force is generated because the actions of the various layers of a fluid move relative to each other or relative to other surfaces as they move in opposite directions to each other. There are several ways to express viscosity. The unit of viscosity is NS / m 2and is known as Pascal seconds (Pas). Viscosity can be either "kinematic viscosity" or "absolute viscosity". Kinematic viscosity is a measure of the rate at which momentum moves through a fluid. The unit of measurement is the Stokes (St). Kinematic viscosity is a measure of the resistance flow of a fluid under the influence of gravity. When two fluids of equal volume but different viscosities are placed in the same capillary viscometer and allowed to flow under gravity, the more viscous fluid takes longer to flow through the capillary than the less viscous fluid. For example, if one fluid takes 200 seconds (s) to complete its flow and another fluid takes 400 s, the second fluid is said to be twice as viscous as the first fluid on the kinematic viscosity scale. The dimension of kinematic viscosity is length 2 / time. Generally, kinematic viscosity is expressed in centistokes (cSt). The SI unit of kinematic viscosity is mm 2 / s, which is equivalent to 1 cSt. "Absolute viscosity", sometimes called "dynamic viscosity" or "simple viscosity", is the product of kinematic viscosity and fluid density. Absolute viscosity is expressed in centipoise (cP) units. The SI unit of absolute viscosity is millipascal seconds (mPa), and 1 cP = 1 mPa.

[0062] Viscosity may be measured, for example, using a viscometer at a given shear rate or multiple shear rates. The "extrapolated zero shear" viscosity can be determined by creating an optimal fit line for the four highest shear points of a plot of absolute viscosity versus shear rate and extrapolating linearly to return the viscosity to zero shear. Alternatively, for Newtonian fluids, viscosity can be determined by averaging the viscosity values at multiple shear rates. Viscosity can also be measured using a microfluidic viscometer at a single or multiple shear rates (also called flow rates), where the absolute viscosity is derived from the change in pressure as the liquid flows through the flow channel. Viscosity is equivalent to shear stress with respect to shear rate. The viscosity measured with a microfluidic viscometer can, in some embodiments, be directly compared to the extrapolated zero shear viscosity, for example, those extrapolated from viscosities measured at multiple shear rates using a cone-plate viscometer. According to the present invention, the viscosity of the liquid composition is reduced when at least one of the above methods exhibits a stabilizing effect. Preferably, the viscosity is measured at 20 °C using a microfluidic viscometer. More preferably, the viscosity is measured at 20 °C using a RheoSense mVROC microfluidic viscometer. Most preferably, the viscosity is measured at 20 °C using a RheoSense mVRoc microfluidic viscometer and a 250 μl syringe at a shear rate of 1500 s-1 or 1000 s-1 and a volume of 60 - 80 μl.

[0063] One skilled in the art is proficient in viscosity measurements using a microfluidic viscometer. As the microfluidic viscometer, a RheoSense mVRoc microfluidic viscometer (mVROC (trademark) Technology), particularly one using the above parameters, can be used. Detailed specifications, methods, and settings can be found in 901003.5.1 - mVROC_User’s_Manual.

[0064] As used herein, "shear rate" refers to the rate of change of the velocity at which one layer of a fluid passes through an adjacent layer. The velocity gradient is the rate of change of velocity with respect to the distance from the plate. This simple case shows a uniform velocity gradient having a shear rate of (v1 - v2) / h in units of (cm / sec) / (cm) = 1 / sec. Therefore, the unit of shear rate is reciprocal seconds, or generally reciprocal hours. For a microfluidic viscometer, changes in pressure and flow rate are related to the shear rate. "Shear rate" becomes the rate at which the material deforms. When measured using a spindle appropriately selected by one of ordinary skill in the art to accurately measure viscosity in the viscosity range of a cone-plate viscometer and the sample of interest, a formulation containing a nucleic acid and an excipient typically has a shear rate in the range of about 0.5 s -1 to about 200 s -1 (i.e., a 20 cP sample is most accurately measured against a CPE40 spindle attached to a DV2T viscometer (Brookfield)); when measured using a microfluidic viscometer, about 20 s -1 to about 3,000 s -1 .

[0065] In the case of classical "Newtonian" fluids commonly used herein, viscosity is essentially independent of shear rate. However, in the case of "non-Newtonian fluids", viscosity decreases or increases with an increase in shear rate, for example, the fluid becomes "shear thinning" or "shear thickening", respectively. In the case of a concentrated (i.e., high-concentration) liquid composition containing a nucleic acid, this may appear as pseudoplastic shear thinning behavior, i.e., a decrease in viscosity with increasing shear rate.

[0066] In one aspect, the liquid composition of the present invention exhibits a viscosity reduction of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% compared to the same composition without at least one excipient.

[0067] In one aspect, the liquid composition of the present invention exhibits a viscosity reduction of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% compared to the same composition that does not contain a combination of two excipients.

[0068] The liquid composition according to the present invention may further contain a pharmaceutically acceptable diluent, solvent, carrier, adhesive, binder, preservative, solubilizer, surfactant, penetration enhancer, stabilizer, emulsifier or bioavailability enhancer. Those skilled in the art know how to select additives suitable for a liquid composition containing a safe and highly tolerable nucleic acid.

[0069] The present invention further provides a liquid composition according to the present invention, wherein the nucleic acid has a molecular weight of 100 base pairs or more, preferably 50 to 5000 base pairs.

[0070] In a preferred aspect, the nucleic acid concentration in the liquid composition according to the present invention is at least 1 mg / ml, at least 3 mg / ml, at least 5 mg / ml, preferably at least 7.5 mg / ml, more preferably at least 10 mg / ml. In another preferred aspect, the nucleic acid concentration is between 1 mg / ml and 50 mg / ml, or between 3 mg / ml and 50 mg / ml, or between 3 mg / ml and 30 mg / ml, or between 3 mg / ml and 20 mg / ml or between 1 mg / ml and 10 mg / ml.

[0071] The present invention further provides a liquid composition according to the present invention that further contains a buffer at a concentration of 10 mM to 50 mM. The buffer can be a suitable tris(hydroxymethyl)aminomethane salt and provides a pH of about 7. The buffer can be supplemented with other components such as ethylenediaminetetraacetic acid (TE buffer), ethylenediaminetetraacetic acid and sodium chloride (TSE buffer), or ethylenediaminetetraacetic acid and acetic acid (TAE buffer). Other acids such as boric acid or phosphoric acid can be used instead of acetic acid.

[0072] The present invention further provides a liquid composition according to the present invention, wherein the viscosity is between 1 MPa and 60 MPa, preferably between 1 MPa and 50 MPa, more preferably between 1 MPa and 30 MPa, and most preferably between 1 MPa and 20 MPa. Preferably, the viscosity is measured at 20 °C using a microfluidic viscometer. More preferably, the viscosity is measured at 20 °C using a RheoSense mVROC microfluidic viscometer. Most preferably, the viscosity is measured at 20 °C using a RheoSense mVRoc microfluidic viscometer, a 250 μl syringe, a shear rate of 1500 s-1 or 1000 s-1, and a volume of 60-80 μl.

[0073] The present invention is also directed to a kit comprising a lyophilized formulation according to the present invention, optionally contained in a container, together with instructions for its reconstitution and administration, optionally together with a vial of sterile diluent, and optionally together with a syringe or other administration device. Exemplary containers include vials, tubes, bottles, single-chamber or multi-chamber prefilled syringes, or cartridges, etc., and also include 96-well plates containing freeze-dried or spray-dried formulations that are immediately usable in wells. Exemplary administration devices include syringes with or without needles, infusion pumps, jet injectors, pen devices, transdermal syringes, or other needleless injectors.

[0074] The present invention is also directed to a method for reducing the viscosity of a liquid composition containing nucleic acid for the purpose of manufacturing all of the above liquid compositions. All aspects regarding the combination and concentration of excipients, nucleic acid, nucleic acid concentration and molecular weight, pH, buffer and buffer concentration described for the above liquid compositions are also applicable to the method for reducing the viscosity of a composition containing nucleic acid.

[0075] The present invention further provides a method for reducing the viscosity of a liquid composition containing nucleic acid, which comprises the step of adding at least one excipient selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate ester or salts or solvates thereof to the liquid nucleic acid composition.

[0076] The present invention further provides a method for reducing the viscosity of a liquid composition containing nucleic acid, which comprises the step of adding a combination of two excipients selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate ester or salts or solvates thereof to the liquid nucleic acid composition.

[0077] The present invention further provides a method for reducing the viscosity of a liquid composition containing nucleic acid, which comprises the step of adding a combination of a first excipient selected from the group consisting of arginine, phenylalanine, ornithine and meglumine and a second excipient selected from the group consisting of benzenesulfonic acid, pyridoxine and thiamine phosphate ester.

[0078] All of the above-described aspects for the liquid composition containing nucleic acid according to the present invention are also applicable to the method for reducing the viscosity of the liquid composition containing nucleic acid. This includes all aspects related to the properties and concentration of the nucleic acid, the combination of excipients, the concentration and ratio of the excipients, additional components of the liquid composition, the reduction of the viscosity of the liquid composition, or the stability of the nucleic acid.

[0079] Similarly, the present invention is directed to a method for increasing the stability of a liquid composition containing nucleic acid, which comprises the steps described above for the method for reducing the viscosity of the liquid composition containing nucleic acid.

[0080] The present invention further provides a method for preventing self-association of nucleic acid in a liquid composition containing nucleic acid, which comprises the steps as described for the method for reducing the viscosity of the liquid composition containing nucleic acid.

[0081] In the method according to the invention, the excipient may be added to the composition containing the nucleic acid by any method known to those skilled in the art. When adding more than one excipient, the excipients may be premixed and subsequently added to the liquid composition containing the nucleic acid. Similarly, the excipients may be added separately to the liquid composition containing the nucleic acid.

[0082] In a preferred embodiment of the invention, the liquid composition is a pharmaceutical composition. The invention is also directed to the above pharmaceutical composition containing a therapeutic nucleic acid for the treatment of diseases.

[0083] In particular, the above-described pharmaceutical liquid composition containing a therapeutic nucleic acid is suitable for the treatment of cancer, rheumatoid arthritis, Crohn's disease, ulcerative colitis, ankylosing spondylitis, psoriatic arthritis, psoriasis, hypercholesterolemia, mixed dyslipidemia, homozygous familial hypercholesterolemia, myocardial infarction, peripheral arterial disease or immunodeficiency disorders.

[0084] The invention is also suitable for a treatment method, where the treatment comprises administration of the above-described pharmaceutical composition containing a therapeutic nucleic acid.

[0085] In one aspect, the treatment method is a method for treating cancer. That is, the liquid composition according to the invention is useful for the treatment of cancer, rheumatoid arthritis, Crohn's disease, ulcerative colitis, ankylosing spondylitis, psoriatic arthritis, psoriasis, hypercholesterolemia, mixed dyslipidemia, homozygous familial hypercholesterolemia, myocardial infarction, peripheral arterial disease or immunodeficiency disorders.

[0086] Furthermore, it has been found that the above-described excipients and combinations of excipients are beneficial in the bioprocess.

[0087] In the bioprocess as meant herein, the addition of these excipients, which have been found to serve as additives for reducing viscosity, can on the one hand improve the yield of intact nucleic acid and on the other hand reduce the duration of the process, leading to an improvement in the economy of the process.

[0088] For example, in a technical approach where, during chromatographic purification, a solution passes through a filter or medium such as a gel bed, porous or non-porous particles in a packed or fluidized bed, a membrane (membare) or a monolith, due to the force applied by the back-end pressure, the disclosed excipients have beneficial effects. The backpressure of the chromatographic material can increase rapidly during the elution of nucleic acids, which can cause a potential risk of process obstacles to an event that can occur in a completely blocked case, especially in a large-capacity chromatographic material operated at a high load. Although the filtration step is mainly used in downstream processes, the excipients can also be beneficial in upstream processes. When the concentration of nucleic acids rises to a level that causes viscosity, there are described negative effects of pressure limits and shear forces when passing the solution through a tube or filter to remove cell materials and debris, but the present invention will clearly have beneficial effects. In contrast to previously used methods, a laboratory-scale TFF system was used to measure the process efficiency of tangential flow filtration, where most of the field flow crosses the surface of the filter tangentially rather than passing through the filter. The principle of filtration is different from the previously described methods, but here too, the filtration efficiency depends on the resistance of the membrane (which remains constant here) and the viscosity of the solution modified by the present invention. The filtration method is a typical unit operation used to exchange formulation buffers or adjust the concentration of biomolecules to a desired level. The stirred cell used herein is a representative example of a dead-end filter, where the feed passes through a filter medium that retains large molecules at the top of the material and releases the filtrate at the other end of the device. In TFF mode, concentration polarization and membrane surface fouling are reduced by introducing a sufficient cross-flow velocity of the feed solution, while the stirred cell uses a stirrer to generate a turbulent flow of the feed solution on the membrane surface.

[0089] A method commonly used for buffer exchange and nucleic acid concentration is tangential flow filtration, where, in contrast to methods used heretofore, most of the field flow moves tangentially across the surface of the filter rather than passing through the filter. Similar to when a stirred cell is used for tangential flow filtration, large molecules are separated from small molecules by passing the small molecules through a suitable filter material. In contrast to a stirred cell, which represents one form of dead-end filtration, in tangential flow filtration, the flow profile of the feed is different to avoid formation of a filter cake and enable a continuous process. When using a stirred cell, formation of a filter cake is similarly prevented by using a stirrer. Thus, a stirred cell closely resembles a tangential flow filtration device, regardless of differences in filter shape. The efficiency of both methods depends critically on the membrane resistance. It is also known that high viscosities result in reduced flux velocities that can be used, increased processing times, and higher production costs. Therefore, reducing viscosity is expected to enable a more efficient filtration process while keeping shear forces low and result in higher nucleic acid concentrations in the filtrate. This is emphasized in the study by Hung et al, which presents "During production of concentrated monoclonal antibody formulations by tangential flow ultrafiltration (TFF), high viscosities and aggregation often cause extensive membrane fouling, flux decay and low product yields" (Journal of Member Science Volume 508, 15 June 2016, Pages 113-126).

[0090] Excipients selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine, and thiamine phosphate ester or salts or solvates thereof are suitable for improving the economy of the above-described bioprocess. In particular, combinations in various ratios according to the composition of the liquid containing nucleic acid improve the economy of the bioprocess as described.

[0091] Accordingly, another aspect of the present invention provides a method for reducing the viscosity of a composition containing nucleic acid in a bioprocess, which includes the step of combining a composition containing nucleic acid with at least one excipient selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate ester or salts or solvates thereof.

[0092] In another aspect, the composition containing nucleic acid is combined with a combination of two excipients selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate ester, or salts or solvates thereof.

[0093] In another aspect, the composition containing nucleic acid is combined with a combination of a first excipient selected from the group consisting of arginine, phenylalanine, ornithine and meglumine and a second excipient selected from the group consisting of benzenesulfonic acid, pyridoxine and thiamine phosphate ester or salts or solvates thereof.

[0094] According to the present invention, all the above-described parameters, for example, the combination of excipients, the concentration of excipients, the concentration of nucleic acid, the ratio of excipients, further components of the composition, pH value, viscosity reduction, and specifications of nucleic acid, are also applicable to the method for reducing the viscosity of a composition containing nucleic acid in a bioprocess.

[0095] Another aspect of the present invention is to provide a method for reducing the viscosity of a composition containing a nucleic acid in the bioprocess as described above, where the nucleic acid is compared with the same composition not containing at least one excipient selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine, and thiamine phosphate ester, or with the same composition not containing a combination of two excipients selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine, and thiamine phosphate ester, or with the same composition not containing a combination of a first excipient selected from the group consisting of arginine, phenylalanine, ornithine, and meglumine and a second excipient selected from the group consisting of benzenesulfonic acid, pyridoxine, and thiamine phosphate ester, and the permeate flux of the composition containing the nucleic acid in the filtration step is increased.

[0096] Another aspect of the present invention is the use of the method in the bioprocess as described above, where the nucleic acid is compared with the same composition not containing at least one excipient selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine, and thiamine phosphate ester, or with the same composition not containing a combination of two excipients selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine, and thiamine phosphate ester, or with the same composition not containing a combination of a first excipient selected from the group consisting of arginine, phenylalanine, ornithine, and meglumine and a second excipient selected from the group consisting of benzenesulfonic acid, pyridoxine, and thiamine phosphate ester, and the permeate flux of the composition containing the nucleic acid in the filtration step is increased.

[0097] The increase in permeate flux means an increase in percentage of at least 2%, preferably at least 5%, more preferably at least 10%, and most preferably 10% to 100%.

[0098] Another aspect of the present invention is to provide a method for reducing the viscosity of a composition containing a nucleic acid in the bioprocess as described above, wherein arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate ester Compared with the same composition containing a nucleic acid that does not contain at least one excipient selected from the group consisting of, or compared with the same composition containing a nucleic acid that does not contain a combination of two excipients selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate ester, or a first excipient selected from the group consisting of arginine, phenylalanine, ornithine and meglumine and a second excipient selected from the group consisting of benzenesulfonic acid, pyridoxine and thiamine phosphate ester Compared with the same composition containing a nucleic acid that does not contain the combination of, the recovery of the nucleic acid after buffer exchange and reduction of the volume in the filter is increased.

[0099] The increase in the recovery of the nucleic acid after buffer exchange and reduction of the volume in the filter means that the recovery of the nucleic acid increases by at least 1%, preferably at least 2%, more preferably at least 5%, and most preferably 5% to 20% in percentage.

[0100] Another aspect of the present invention is to provide a method for reducing the viscosity of a composition containing nucleic acid in the bioprocess as described above, where, compared with the same composition containing nucleic acid that does not contain at least one excipient selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate ester, or compared with the same composition containing nucleic acid that does not contain a combination of two excipients selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate ester, or compared with the same composition containing nucleic acid that does not contain a combination of a first excipient selected from the group consisting of arginine, phenylalanine, ornithine and meglumine and a second excipient selected from the group consisting of benzenesulfonic acid, pyridoxine and thiamine phosphate ester, the processing time for the filtration step, preferably the filtration step in which the nucleic acid is concentrated, is reduced.

[0101] The reduction in the processing time for the filtration step means a reduction in percentage of at least 5%, preferably at least 10%, more preferably at least 25%, most preferably 25% to 100%.

[0102] In a particular embodiment of the present invention, the filtration step is tangential flow filtration (TFF).

[0103] The term "bioprocess" refers to the manufacturing process of therapeutic cells, which can be divided into an upstream process and a downstream process. The upstream process is defined as the entire process before separating nucleic acids from cell compounds. The upstream process includes initial cell isolation and culture, cell banking, and cell culture expansion from the initial harvest to the final harvest. The downstream part of the bioprocess refers to the part where the target nucleic acid is purified from the upstream feed and processed to meet the requirements of purity and quality. Some types of cells need to be disrupted when entering the downstream process. Additionally, other cells may secrete the target nucleic acid into the medium and need to be removed through filtration. Furthermore, the downstream process is usually divided into the main sections of a purification section and a polishing section. The bioprocess can be a batch process, or a semi - continuous or continuous process.

[0104] The term "permeate flux" refers to the volume passing through a defined filter within a specific time (typically on the order of minutes).

[0105] The term "filtration step" refers to a process step in which a liquid is passed through a material having a defined pore size and the material is separated based on its size. For some filters, the pore size is defined in nanometers. However, for other filters, the pore size is not directly defined and the weight of the molecules to be retained is given. The filter medium can be arranged to block the cross - section of the filtration device (dead - end filtration). However, the filter medium can be arranged such that the solution to be filtered flows tangentially across the surface of the material, for example, it can be tangential flow filtration. The filter medium can be a membrane, a glass filter, a metal filter, or a resin. The resin can be held in a chromatography column. The resin can be a cationic or anionic exchange resin, an affinity resin such as protein A or glutathione resin, or a hydrophobic or hydrophilic resin.

[0106] The term "nucleic acid recovery" after buffer exchange and volume reduction refers to the fraction of nucleic acid recovered after a process step.

[0107] The term "tangential flow filtration" or "TFF" refers to a filtration method in which a solution passes tangentially over a defined filter. Substances smaller than the pores of the filter are pushed out of the solution through the filter by the pressure resulting from the flow rate, viscosity, temperature, and other factors of the solution.

[0108] Example 1. Viscosity-reducing effect of excipients on deoxyribonucleic acid in TE buffer, pH 7.4 Preparation of buffer The concentrated nuclease-free TE buffer was diluted by adding nuclease-free water to yield 1x TE buffer. The pH was adjusted to pH 7.4 using HCl and NaOH as needed.

[0109] Preparation of samples Excipient solutions of 150 mM ornithine (Orn), arginine (Arg), meglumine (Meg), benzenesulfonic acid (BSacid), pyridoxine (Pyr), and thiamine monophosphate (TMP) were each prepared in TE buffer pH 7.4. Similarly, an excipient solution of 125 mM phenylalanine (Phe) was prepared in TE buffer at pH 7.4. The pH was adjusted using HCl or NaOH as needed. A centrifugal filter (Amicon, 30 kDa MWCO) was used to prepare a concentrated pDNA solution containing the desired excipient, and the original buffer was exchanged with a buffer containing the relevant excipient to reduce the volume of the solution. Subsequently, the pDNA was diluted to 8760 μg / mL and 7000 μg / mL, respectively.

[0110] Measurement of DNA concentration The DNA concentration was determined using absorption spectroscopy applying Lambert-Beer's law. The absorbance at 260 nm was measured at 260 nm using NanoDrop™ OneC (Thermo Fisher Scientific) in absorption spectroscopy. The layer thickness was adjusted with the device so as to obtain an optimal resolution for the concentration of the sample.

[0111] Viscosity measurement For viscosity measurement, mVROCTM Technology (Rheo Sense, San Ramon, California USA) was used. Measurements were carried out using a 250 μl syringe and a shear rate of 1500 s -1 . A volume of 80 μl was used. All samples were measured three times. Figures 1 and 2 show the viscosities of 4 kb pDNA and 14 kb pDNA solutions without excipient (control) and with various excipients.

[0112] 2. Viscosity reducing effect of combinations of excipients on deoxyribonucleic acid in TE buffer, pH 7.4 Buffer preparation See Example 1.

[0113] Sample preparation Excipient solutions of 75 mM ornithine, arginine, phenylalanine, or meglumine were prepared in TE buffer pH 7.4 and supplemented with 75 mM benzenesulfonic acid, pyridoxine, or thiamine monophosphate, respectively. The pH was adjusted using HCl or NaOH as required. Concentrated pDNA solutions containing the desired excipient were prepared using a centrifugal filter (Amicon, 30 kDa MWCO), the original buffer was exchanged with a buffer containing the relevant excipient, and the volume of the solution was reduced. Thereafter, the pDNA was diluted to 9170 μg / mL and 7000 μg / mL, respectively.

[0114] Measurement of DNA concentration The DNA concentration was determined using absorption spectroscopy applying Lambert-Beer's law. The absorbance at 260 nm was measured at 260 nm using a NanoDrop™ OneC (Thermo Fisher Scientific) in absorption spectroscopy. The thickness of the layer was adjusted with the device so as to obtain the optimal resolution for the concentration of the sample.

[0115] Viscosity measurement For viscosity measurement, mVROCTM Technology (Rheo Sense, San Ramon, California USA) was used. The measurement was carried out using a 250 μl syringe and a shear rate of 1500 s-1. A volume of 80 μl was used. All samples were measured three times. Figures 3 and 4 show the viscosities of 4 kb pDNA and 14 kb pDNA solutions without excipient (control) and with various excipients.

[0116] 3. Viscosity reduction effect of excipients on ribonucleic acid in 3. TE buffer, pH 7.0 Buffer preparation The concentrated nuclease-free TE buffer was diluted by adding nuclease-free water to yield 1x TE buffer. The pH was adjusted to pH 7.0 using HCl and NaOH as necessary.

[0117] Sample preparation Excipient solutions of 150 mM ornithine, arginine, benzenesulfonic acid, pyridoxine, thiamine monophosphate were each prepared in TE buffer pH 7.0. Similarly, an excipient solution of 125 mM phenylalanine was prepared in TE buffer (pH 7.4). The pH was adjusted using HCl or NaOH as necessary. A centrifugal filter (Amicon, 30 kDa MWCO) was used to prepare a concentrated mRNA solution containing the desired excipient, and the original buffer was exchanged with a buffer containing the relevant excipient to reduce the volume of the solution. Subsequently, the mRNA was diluted to 8800 μg / mL, 5680 μg / mL, and 7530 μg / mL, respectively.

[0118] Measurement of mRNA Concentration The mRNA concentration was measured using fluorescence spectroscopy. For fluorescence spectroscopy, an assay (Quant-IT™ RNA XR Assay Kit, Thermo Fisher Scientific) was utilized. A Spark® multimode plate reader (Tecan) was used to excite the fluorophore at 644 nm and measure the fluorescence at 673 nm. The assay was performed and interpreted according to the manufacturer's instructions.

[0119] Viscosity Measurement For viscosity measurement, mVROCTM Technology (Rheo Sense, San Ramon, California USA) was used. The measurement was carried out using a 250 μl syringe and a shear rate of 1500 s-1. A volume of 80 μl was used. All samples were measured three times. Figures 5 - 7 show the viscosities of 2000 bp, 4000 bp, and 6000 bp mRNA solutions without excipient (w / o) and with various excipients.

[0120] 4. Viscosity Reduction Effect of Combinations of Excipients on Ribonucleic Acid in TE Buffer, pH 7.0 Preparation of Buffer Concentrated nuclease-free TE buffer was diluted by adding nuclease-free water to yield 1xTE buffer. The pH was adjusted to pH 7.0 using HCl and NaOH as needed.

[0121] Preparation of Samples A vehicle solution of 75 mM ornithine, arginine, and phenylalanine was prepared in TE buffer pH 7.0 and supplemented with 75 mM benzenesulfonic acid, pyridoxine, or thiamine monophosphate, respectively. The pH was adjusted using HCl or NaOH as needed. A centrifugal filter (Amicon, 30 kDa MWCO) was used to prepare a concentrated mRNA solution containing the desired vehicle and the original buffer was exchanged with a buffer containing the relevant vehicle to reduce the volume of the solution. The mRNA was then diluted to 8800 μg / mL, 5680 μg / mL, and 7530 μg / mL, respectively.

[0122] Measurement of mRNA concentration The mRNA concentration was measured using fluorescence spectroscopy. For fluorescence spectroscopy, an assay (Quant-IT™ RNA XR Assay Kit, Thermo Fisher Scientific) was utilized. A Spark® multimode plate reader (Tecan) was used to excite the fluorophore at 644 nm and measure the fluorescence at 673 nm. The assay was performed and interpreted according to the manufacturer's instructions.

[0123] Viscosity measurement For viscosity measurement, mVROCTM Technology (Rheo Sense, San Ramon, California USA) was used. Measurements were performed using a 250 μl syringe and a shear rate of 1500 s-1. A volume of 80 μl was used. All samples were measured three times. Figures 8 - 10 show the viscosities of 2000 bp, 4000 bp, and 6000 bp mRNA solutions in the absence of vehicle (control) and in the presence of various vehicles.

Claims

1. A liquid composition comprising a nucleic acid and at least one excipient selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine, and thiamine phosphate ester.

2. The liquid composition according to claim 1, comprising a combination of two excipients selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine, and thiamine phosphate ester.

3. The liquid composition according to claim 1 or 2, comprising a combination of a first excipient selected from the group consisting of arginine, phenylalanine, ornithine, and meglumine and a second excipient selected from the group consisting of benzenesulfonic acid, pyridoxine, and thiamine phosphate ester.

4. The liquid composition according to any one of claims 1 to 3, having a reduced viscosity as compared to the same composition without at least one excipient.

5. The liquid composition according to any one of claims 1 to 4, wherein the concentration of the nucleic acid is between 1 and 50 mg / ml.

6. The liquid composition according to any one of claims 1 to 5, wherein the concentration of each of the at least one excipient is between 5 mM and 300 mM.

7. The liquid composition according to any one of claims 1 to 6, having a viscosity between 1 MPa and 60 MPa at 20 °C measured using a microfluidic viscometer.

8. The liquid composition according to any one of claims 1 to 7, wherein the nucleic acid has a size of 100 base pairs or more.

9. The liquid composition according to any one of claims 1 to 8, wherein the nucleic acid is DNA or RNA.

10. A lyophilized preparation of the liquid composition according to any one of claims 1 to 9.

11. A method for reducing the viscosity of a liquid composition containing a nucleic acid, the method comprising adding at least one excipient selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine, and thiamine phosphate ester or salts or solvates thereof to the liquid nucleic acid composition.

12. The method according to claim 11, wherein a combination of two excipients selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate ester or salts or solvates thereof is added to the liquid nucleic acid composition.

13. The method according to claim 11 or 12, wherein a combination of a first excipient selected from the group consisting of arginine, phenylalanine, ornithine, and meglumine or salts or solvates thereof and a second excipient selected from the group consisting of benzenesulfonic acid, pyridoxine and thiamine phosphate ester or salts or solvates thereof is added to the liquid nucleic acid composition.

14. The method according to any one of claims 11 to 13, wherein the nucleic acid is DNA or RNA.

15. Use of the method according to claim 11 or 14 in a bioprocess.

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