Viscosity reducing excipients for highly concentrated nucleic acid compositions and combinations thereof

Abstract

The present invention relates to a liquid composition comprising a nucleic acid having a reduced viscosity. In addition, the present invention relates to a method of reducing the viscosity of a liquid composition comprising a nucleic acid.

Description

Technical Field

[0001] The present invention relates to a liquid composition comprising nucleic acids having a reduced viscosity. Furthermore, the present invention relates to a method for reducing the viscosity of a liquid composition comprising nucleic acids. Background Art

[0002] Although nucleic acids themselves have only been the subject of scientific research or as tools for genetic engineering work for decades, today they are used as therapeutically active substances or as important starting materials in the manufacturing process of new active therapeutics, including viruses, gene therapy products and mRNA or DNA vaccines. With the recent success of new treatments, reflected by an increasing number of medical approvals and positive results from clinical trials, the demand for improved formulations and more efficient production methods for the administration of nucleic acid drugs has increased.

[0003] Aqueous solutions of high molecular weight DNA have dynamic viscosities of hundreds and thousands of centipoise (cP) even at relatively low concentrations such as a few mg / ml (Creeth et al., J Chem Soc. 1947 September; 25: 1141-5; Butler et al., Transactions of the Faraday Society, Vol. 50, pp. 612–623195 1954). This makes it difficult to directly administer common doses of naked therapeutic nucleic acids (from a few milligrams in DNA vaccines to tens of milligrams in gene therapy) because the viscosity of injectable pharmaceutical preparations should not exceed about 50 cP (Srinivasan et al., Pharm Res 30, 1749-1757, 2013), and ideally should be less than half of this value. Nucleic acid delivery systems, such as viral delivery vectors, cationic liposomes and polycations, even with low viscosity, often have problems of toxicity, low in vivo stability and high cost (Dias and Lindman, DNA Interactions with Polymers and Surfactants, Wiley & Sons Inc. ISBN978-0-470-25818-7, 2008 Saraswat et al., Indian J. Pharm. Sci., 71, pp. 488-498 2009). Therefore, finding a method to reduce the viscosity of DNA solutions under drug-related conditions may have clinical uses for both the preparation of nucleic acid drugs and as independent treatments. (Elkin et al., Intern. J. of Pharmaceutics Vol. 494, No. 1, 15, pp. 66-72; 2015). Similarly, the rise of new technologies (such as RNA-based therapeutics and preventive vaccines) (particularly m-RNA).

[0004] Nucleic acids such as DNA, RNA, and especially m-RNA can only be concentrated to a few milligrams per milliliter at most. Above this concentration level, the viscosity of the solution will increase to a point where the set solution can no longer be injected into a patient or handled with standard filling and finishing equipment.

[0005] Nucleic acid therapy is usually administered parenterally, for example, by intravenous (iv), intramuscular (im) or subcutaneous (sc) routes. Because subcutaneous injection is likely to simplify patient administration (fast, small volume injection) and reduce treatment costs (shortening medical assistance), subcutaneous injection is particularly welcome for the delivery of nucleic acid therapy. To ensure patient compliance, it is desirable that the subcutaneous injection dosage form is isotonic and can be injected with a small volume (<2.0 ml for each injection site).

[0006] At the same time, nucleic acid therapies usually require doses ranging from a few milligrams to tens of milligrams. Therefore, the combination of high therapeutic doses and low injection volumes leads to the need for highly concentrated therapeutic nucleic acid preparations. However, nucleic acids, in addition to their potentially complex three-dimensional structures, also possess numerous functional groups. This makes their formulation difficult, especially when high concentrations are required.

[0007] One of the main problems is the high viscosity of solutions containing nucleic acids at concentrations above 1, 3 or 5 mg / ml. At such concentrations, nucleic acids tend to form highly viscous solutions, mainly due to unnatural self-association. Another problem that needs to be solved is the inability to increase the concentration of nucleic acids in solution to concentrations above 1, 3, 5 or 10 mg / ml. Typically, solutions containing nucleic acids cannot be processed by standard filling and final equipment or laboratory equipment. Another problem that needs to be solved is that high viscosity solutions containing nucleic acids hinder processing steps, including transfection of cell lines, polymerase chain reaction and incorporation of nucleic acids into viral vectors.

[0008] The rheological properties of biomacromolecule solutions such as nucleic acid preparations not only complicate their parenteral administration in medical applications, but the high viscosity of nucleic acid solutions also poses considerable challenges to their manufacturing itself, especially the intensification of the manufacturing process.

[0009] Many known manufacturing methods, particularly the manufacturing methods of relatively large amounts of nucleic acids (such as plasmid DNA or mRNA), include filtering procedures, chromatographic purification steps or mixing, pumping or filling operations to process product fluid streams. The performance of these steps is greatly affected by the rheological behavior of the fluid product stream to be processed. In the manufacturing process, highly viscous highly concentrated nucleic acid preparations present special difficulties to ultrafiltration and aseptic filtration. In addition, tangential flow filtration is generally used for buffer exchange and to increase nucleic acid concentration. However, due to the increased back pressure and shear stress shown by the viscous solution during injection and filtration, therapeutic nucleic acids may be unstable and / or the process time is extended. The increased shear stress often results in product loss. Both aspects have an adverse effect on process economy.

[0010] At the same time, high viscosity is unacceptable in terms of administration because it significantly limits the injectability of the nucleic acid.

[0011] In order to solve these problems and / or improve the stability of the solution, additives and excipients (such as EDTA and sodium chloride) are usually added to biopharmaceutical formulations at higher concentrations. However, the resulting solutions often cause pain due to high injection forces and resulting tissue damage. Some of these solutions may even no longer be administrable, resulting in a lack of treatment options for patients.

[0012] However, the formulation of nucleic acids (such as DNA or RNA, especially mRNA) requires careful selection of formulation additives and / or excipients to avoid denaturation and loss of biological activity. In addition, the excipients need to be pharmaceutically safe and physiologically compatible to avoid any undesirable side effects, such as allergic reactions.

[0013] Therefore, there is a strong need in the pharmaceutical industry for additional pharmaceutically acceptable, viscosity-reducing excipients, particularly as substitutes when standard solutions, as mentioned above, fail.

[0014] Therefore, the problem to be solved is to provide an excipient that can effectively reduce the viscosity of a nucleic acid solution. In addition, the problem to be solved is to provide an excipient combination that can effectively reduce the viscosity of a nucleic acid solution.

[0015] During the bioprocess, the solution must be pumped through the pipeline and the chromatographic column. Under high viscosity, the flow velocity through such a column is limited by the viscosity, which causes a longer processing time, a significant nucleic acid loss during the chromatography or may cause the complete non-processing of the nucleic acid solution. In addition, when entering a narrower column from a narrow tube through a connector, shearing forces may be generated. Shearing stress is a typical reason for nucleic acid denaturation and may gather and thereby reduce process yield. Obviously, the gathering that such shearing stress causes has an adverse effect on process economy. In addition, the gel bed in the chromatographic column may be damaged by high pressure.

[0016] In addition, some nucleic acids are formulated into high concentrations by tangential flow filtration (TFF). When the viscosity of the solution becomes critical, a gel-like layer may be formed near the film. In particular, the membrane flux is significantly reduced, resulting in an increase in processing time, which results in a significant increase in manufacturing costs. As previously mentioned, during TFF, shear stress may also occur, producing insoluble nucleic acid aggregates and reducing productivity.

[0017] Generally, it has been observed that high viscosity solutions develop a certain viscosity that makes it difficult to recover the complete solution from a container, pipe, or remove the entire material from a processing system. This material loss results in a significant reduction in product yield and has a significant adverse effect on process economics.

[0018] Furthermore, the problem to be solved is to provide an excipient combination which is able to effectively reduce the viscosity of nucleic acid solutions.

[0019] The high viscosity of nucleic acid solutions leads to numerous difficulties in bioprocessing. Since the additives known to date for reducing the viscosity of the corresponding nucleic acid solutions do not lead to a sufficient viscosity-lowering effect in many cases, it was an object of the present invention to find new possibilities by which the corresponding viscosity-lowering effect can be increased and the adverse effects on process economy can be reduced. Summary of the invention

[0020] This problem is solved by a liquid composition comprising a nucleic acid and at least one excipient that reduces viscosity, the excipient being selected from arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate. In a further embodiment, the liquid composition comprises a combination of two excipients and a nucleic acid, the excipient being selected from arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate. In a further embodiment, the liquid composition comprises a nucleic acid and a combination of a first excipient selected from arginine, phenylalanine, ornithine and meglumine and a second excipient selected from benzenesulfonic acid, pyridoxine and thiamine phosphate.

[0021] Likewise, 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 arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate to the liquid nucleic acid composition. In a further embodiment, a combination of two excipients selected from arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate is added to the liquid nucleic acid composition. In a further embodiment, a combination of a first excipient selected from arginine, phenylalanine, ornithine and meglumine and a second excipient selected from benzenesulfonic acid, pyridoxine and thiamine phosphate is added to the liquid nucleic acid composition.

[0022] The use of at least two excipients allows overcoming the problem that many viscosity reducing excipients used at relevant concentrations may have an adverse effect on nucleic acid stability by allowing the use of lower amounts of each individual excipient and taking advantage of the stabilizing effect of the second excipient. In addition, the use of at least two excipients produces a synergistic viscosity reduction.

[0023] Furthermore, the problem is solved by a freeze-dried nucleic acid formulation comprising a composition of a nucleic acid and at least one viscosity-reducing excipient selected from arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate. In a further embodiment, the composition comprises a combination of two excipients and a nucleic acid, the excipient being selected from arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate. In a further embodiment, the composition comprises a nucleic acid and a combination of a first excipient selected from arginine, phenylalanine, ornithine and meglumine and a second excipient selected from benzenesulfonic acid, pyridoxine and thiamine phosphate.

[0024] 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. In a further embodiment, the composition comprises a combination of two excipients and a nucleic acid, the excipient being selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate. In a further embodiment, 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.

[0025] Another subject of the present invention is a method for reducing the viscosity of a liquid nucleic acid composition in a biological process, comprising the step of adding at least one excipient selected from arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate to the liquid nucleic acid composition. In a further embodiment, a combination of two excipients selected from arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate is added to the liquid nucleic acid composition. In a further embodiment, a combination of a first excipient selected from arginine, phenylalanine, ornithine and meglumine and a second excipient selected from benzenesulfonic acid, pyridoxine and thiamine phosphate is added to the liquid nucleic acid composition. DETAILED DESCRIPTION

[0026] The present invention relates to a liquid composition comprising 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.

[0027] "Nucleic acid" is defined herein as a macromolecule composed of nucleotides. "Nucleotide" is the monomer structure of nucleic acid, which consists of three parts: a five-carbon sugar, a phosphate group, and a nitrogenous base. The two main categories of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). If the sugar is ribose, the polymer is RNA; if the sugar is the ribose derivative deoxyribose, the polymer is DNA. DNA has several forms, such as but not limited to plasmid DNA or genomic DNA. Similarly, RNA can exist in the form of messenger RNA (mRNA), transfer RNA (t-RNA), catalytic RNA, or structural RNA. DNA or RNA molecules can also be synthesized for different functions. In one embodiment, the nucleic acid is DNA or RNA. In one embodiment, the nucleic acid is DNA. In one embodiment, the nucleic acid is RNA.

[0028] Do not wish to be bound by mechanism, it is believed that the viscosity reduction effect of liquid composition according to the present invention is based on the interaction between excipient and nucleic acid residue or the sucrose / phosphate backbone of nucleic acid.Because all nucleic acids are made of the same nucleotide pool, the effect described herein is applicable to all types of nucleic acids.Therefore, liquid composition according to the present invention all has a beneficial effect on any type of nucleic acid, no matter how its sequence, size and structure.

[0029] The term "liquid composition comprising nucleic acids" as used herein refers to a liquid aqueous composition containing nucleic acids, preferably an aqueous solution containing nucleic acids. The composition may contain one or more nucleic acids at different concentrations. Typically, the concentration of one nucleic acid in the liquid composition is higher than 1, 3, 5 or 10 mg / ml.

[0030] In a preferred embodiment, the nucleic acid contained in the composition according to the invention is a therapeutic nucleic acid.

[0031] 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 may be administered for different purposes, such as replacing defective or abnormal proteins, enhancing existing pathways, providing new functions or activities, interfering with molecules or organisms, and delivering other compounds or proteins, such as radionuclides, cytotoxic drugs, or effector proteins. Therapeutic nucleic acids include naked DNA / RNA or modified DNA / RNA. Their sequences may be natural or engineered.

[0032] In a particularly preferred embodiment, the nucleic acid in the liquid composition according to the invention is naked DNA, in particular a therapeutic plasmid.

[0033] In another particularly preferred embodiment, the nucleic acid in the liquid composition according to the invention is RNA, such as mRNA.

[0034] In one embodiment, the nucleic acid is a biosimilar. A "biosimilar" is defined herein as a biopharmaceutical that is highly similar to another already approved biopharmaceutical. In a preferred embodiment, the biosimilar is RNA, such as mRNA.

[0035] In one embodiment, the liquid composition according to the invention comprises more than one type of nucleic acid.

[0036] As used herein, the term "excipient" refers to any compound known to reduce the viscosity of a composition comprising nucleic acid by at least 5% at appropriate concentrations compared to the same composition not comprising the excipient.

[0037] The liquid composition according to the invention comprises at least one excipient selected from:

[0038] Arginine (CAS Reg. No. 74-79-3)

[0039] Phenylalanine (CAS Reg. No. 63-91-2)

[0040] Ornithine (CAS Reg. No. 3184-13-2)

[0041] ·Meglumine (CAS registration number 6284-40-8)

[0042] Benzenesulfonic acid (CAS registration number 98-11-3)

[0043] Pyridoxine (vitamin B6, CAS registration number 65-23-6),

[0044] Thiamine phosphate (thiamine monophosphate, CAS Reg. No. 10023-48-0).

[0045] According to the present invention, excipients include salts or solvates of excipients. Preferred salts in the context of the present invention are physiologically acceptable salts of the compounds according to the invention. Salts which are not suitable for pharmaceutical use per se but which can be used, for example, for separation, purification or storage of the compounds according to the invention are also included.

[0046] Physiologically acceptable salts of the compounds according to the invention include salts with customary bases, such as, by way of example and preference, alkali metal salts (e.g. sodium and potassium salts), alkaline earth metal salts (e.g. calcium and magnesium salts) and ammonium salts derived from ammonia or organic amines having 1 to 16 C atoms (e.g., by way of example and preference, ethylamine, diethylamine, triethylamine, N,N-diisopropylethylamine, monoethanolamine, diethanolamine, triethanolamine, dimethylaminoethanol, diethylaminoethanol, procaine, dicyclohexylamine, dibenzylamine, N-methylpiperidine, N-methylmorpholine, arginine, lysine and 1,2-ethylenediamine).

[0047] Physiologically acceptable salts of the compounds according to the invention include salts of customary acids, such as, by way of example and preference, acetates, adipates, alginates, citrates, aspartates, benzoates, benzenesulfonates, bisulfates, butyrates, camphorates, camphorsulfonates, carbonates, digluconates, glycerophosphates, hemisulfates, heptanoates, hexanoates, formates, fumarates, hydrochlorides, hydrobromides, hydroiodides, 2-hydroxyethanesulfonates (isethionates), lactates, maleates, mesitylenesulfonates, methanesulfonates, naphthylsulfonates, nicotinates, 2-naphthalenesulfonates, oxalates, pamoates, pectinates, persulfates, 3-phenylpropionates, picrates, pivalates, propionates, succinates, sulfates, tartrates, trichloroacetates, trifluoroacetates, phosphates, glutamates, bicarbonates, p-toluenesulfonates and undecanoates.

[0048] For example, a physiologically acceptable salt may be a salt of ornithine, such as ornithine monohydrochloride.

[0049] Solvates in the context of the present invention are designated as those forms of the compounds according to the invention which form solid or liquid complexes by coordination with solvent molecules. Hydrates are a special form of solvates in which coordination occurs with water. Hydrates are preferred solvates in the context of the present invention.

[0050] According to the present invention, the excipient includes all enantiomers and stereoisomers as well as racemic mixtures of the excipient. For example, the excipient arginine includes L-arginine, D-arginine and a racemic mixture of L- and D-arginine.

[0051] Liquid composition according to the present invention comprises the excipient that is enough to reduce the amount of composition viscosity and / or stabilizing protein.For example, liquid composition according to the present invention can comprise at least one excipient of about 5mM to about 300mM, about 5mM to about 250mM or about 5mM to about 150mM.In exemplary embodiments, 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 300mM or larger.

[0052] In one aspect of the invention, these liquid compositions may further comprise additives for purposes other than reducing viscosity, such as stabilization, dissolution or preservation.

[0053] In another aspect of the invention, the liquid composition comprises more than one excipient. For example, the liquid compositions according to the invention may comprise two, three or four excipients, preferably they comprise two excipients.

[0054] The following mentions a combination of two excipients that is advantageous for the liquid composition comprising nucleic acid, the lyophilized preparation of the liquid composition comprising nucleic acid, the method for reducing the viscosity of the liquid composition comprising nucleic acid, and the kit comprising the composition comprising nucleic acid according to the present invention.

[0055] 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.

[0056] One embodiment of the present invention is a liquid composition comprising a first excipient selected from arginine, phenylalanine, ornithine and meglumine in combination with a second excipient selected from benzenesulfonic acid, pyridoxine and thiamine phosphate.

[0057] One embodiment of the present invention is a liquid composition comprising a first excipient selected from arginine, ornithine and meglumine in combination with a second excipient selected from benzenesulfonic acid, pyridoxine and thiamine phosphate.

[0058] 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, phenylalanine and ornithine, phenylalanine and meglumine, phenylalanine and benzenesulfonic acid, phenylalanine and pyridoxine, phenylalanine and thiamine phosphate, ornithine and meglumine, ornithine and benzenesulfonic acid, ornithine and pyridoxine, ornithine and thiamine phosphate, meglumine and benzenesulfonic acid, meglumine and pyridoxine, meglumine and thiamine phosphate, benzenesulfonic acid and pyridoxine, benzenesulfonic acid and thiamine phosphate, pyridoxine and thiamine phosphate.

[0059] Preferred combinations of the two excipients include arginine and benzenesulfonic acid, arginine and pyridoxine, arginine and thiamine phosphate, phenylalanine and benzenesulfonic acid, phenylalanine and pyridoxine, phenylalanine and thiamine phosphate, ornithine and benzenesulfonic acid, ornithine and pyridoxine, ornithine and thiamine phosphate, meglumine and benzenesulfonic acid, meglumine and pyridoxine, meglumine and thiamine phosphate.

[0060] More preferred combinations of the two excipients include arginine and benzenesulfonic acid, arginine and pyridoxine, arginine and thiamine phosphate, ornithine and benzenesulfonic acid, ornithine and pyridoxine, ornithine and thiamine phosphate, meglumine and benzenesulfonic acid, meglumine and pyridoxine, meglumine and thiamine phosphate.

[0061] In one embodiment, arginine and benzenesulfonic acid are combined. In another embodiment, arginine and pyridoxine are combined. In another embodiment, arginine and thiamine phosphate 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 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 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 are combined. In another embodiment, meglumine and pyridoxine are combined. In another embodiment, meglumine and thiamine phosphate are combined. In another embodiment, meglumine and pyridoxine are combined. In another embodiment, meglumine and thiamine phosphate are combined. In another embodiment, meglumine and pyridoxine are combined. In another embodiment, meglumine and thiamine phosphate are combined.

[0062] Fluid composition according to the present invention comprises a certain amount of combination of above-mentioned two excipients, and wherein the concentration of two kinds of excipients is enough to reduce the viscosity and / or the stable nucleic acid of fluid composition.For example, fluid composition according to the present invention can comprise every kind of excipient of about 5mM to about 300mM, about 5mM to about 250mM or about 5mM to about 150mM.In exemplary embodiments, the concentration of every kind of 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 300mM or higher.

[0063] In one embodiment, the liquid composition comprises a combination of two excipients, wherein the molar concentrations of the excipients may 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 specific embodiment, the molar concentrations of the two excipients are the same.

[0064] The excipients, excipient combinations, concentrations and molar ratios of excipients and excipient combinations, and nucleic acid concentrations disclosed above for liquid compositions comprising nucleic acids and at least one excipient should be equally applicable to all other embodiments extending to the invention, including lyophilized formulations of liquid compositions, methods for reducing the viscosity of liquid compositions, or use of the methods in biological processes.

[0065] In another preferred embodiment of the liquid composition, method of reducing viscosity or increasing stability, the excipient synergistically reduces viscosity and / or increases stability in the liquid composition comprising nucleic acid.

[0066] According to the present invention, a synergistic reduction in viscosity is given if the reduction in viscosity by the combination of two or more excipients is greater than the expected sum of the reductions in viscosity of each individual excipient. Preferably, a synergistic reduction in viscosity is given if the percentage reduction in viscosity by the combination of two or more excipients is greater than the expected sum of the percentage reductions in viscosity of each individual excipient.

[0067] In one embodiment, all combinations of two excipients mentioned in this application result in a synergistic reduction in the viscosity of the liquid composition comprising nucleic acids.

[0068] On the other hand, the present invention provides a lyophilized formulation of a liquid composition comprising a nucleic acid and at least one excipient selected from arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate. When reconstituted with an appropriate amount of diluent, the formulation exhibits a reduced viscosity relative to a control formulation of a composition that is otherwise identical but does not comprise at least one excipient. Therefore, the excipient is present in an amount that is effective to reduce viscosity when reconstituted with the diluent.

[0069] The lyophilized formulation according to the invention comprises a nucleic acid and at least one excipient according to the invention, which has been dried and is present as particles, e.g., in the form of a powder. In this context, the expression "powder" refers to a collection of substantially dry particles, i.e., a moisture content of at least less than about 10 wt. %, 6 wt. %, 4 wt. % or less.

[0070] As defined herein, "viscosity" refers to the resistance of a substance (usually a liquid) to flow. Viscosity is related to the concept of shear; it can be understood as the effect of different layers of fluid exerting shear forces on each other or on other surfaces as they move relative to each other. There are several ways to express viscosity. The unit of viscosity is Ns / m 2 , called Pascal-seconds (Pas). Viscosity can be "kinematic" or "absolute". Kinematic viscosity is a measure of the rate at which momentum is transferred through a fluid. It is measured in stokes (St). Kinematic viscosity is a measure of a fluid's resistance to flow under the influence of gravity. When two fluids of equal volume and different viscosities are placed in identical capillary viscometers 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 the other takes 400 seconds, then on the kinematic viscosity scale the second fluid is said to be twice as viscous as the first fluid. The dimension of kinematic viscosity is length 2 / time. Usually, kinematic viscosity is expressed in centistokes (cSt). The SI unit of kinematic viscosity is mm 2 / s, which is equal 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). The SI unit of absolute viscosity is milliPascal-second (mPas), where 1 cP = 1 mPas.

[0071] Viscosity can be measured at a given shear rate or multiple shear rates using, for example, a viscometer. "Extrapolated zero shear" viscosity can be determined by creating a best fit line of four highest shear points in a graph of absolute viscosity versus shear rate and linearly extrapolating the viscosity back to zero shear. Alternatively, for Newtonian fluids, viscosity can be determined by taking the average viscosity value at multiple shear rates. Viscosity can also be measured at a single or multiple shear rates (also referred to as flow rate) using a microfluidic viscometer, where the absolute viscosity is derived from the pressure change when the liquid flows through the channel. Viscosity is equal to the ratio of shear stress to shear rate. In some embodiments, the viscosity measured with a microfluidic viscometer can be directly compared to the extrapolated zero shear viscosity, such as those extrapolated from the viscosity measured at multiple shear rates using a cone-plate viscometer. According to the present invention, when at least one of the above methods exhibits a stabilizing effect, the viscosity of the liquid composition decreases. Preferably, the viscosity is measured using a microfluidic viscometer at 20°C. More preferably, the viscosity is measured using a RheoSense mVROC microfluidic viscometer at 20°C. Most preferably, the viscosity is measured using a RheoSense mVROC microfluidic viscometer at 20°C and using a 250 μl syringe, a shear rate of 1500s-1 or 1000s-1 and a volume of 60-80 μl.

[0072] Those of ordinary skill in the art are familiar with viscosity measurements using microfluidic viscometers. RheoSensemVROC microfluidic viscometer (mVROC) can be used TM Technology) is used as a microfluidic viscometer, especially the parameters described above. Detailed specifications, methods and settings can be found in 901003.5.1-mVROC_User's_Manual.

[0073] In this article, "shear rate" refers to the rate of change of velocity of one layer of fluid passing through the adjacent layer. The velocity gradient is the rate of change of velocity with distance from the plate. This simple example shows a uniform velocity gradient with a shear rate of (v1-v2) / h, with units of (cm / sec) / (cm)=1 / sec. Therefore, the shear rate units are the reciprocal of seconds, or, more generally, the reciprocal of time. For microfluidic viscometers, changes in pressure and flow rate are related to the shear rate. "Shear rate" refers to the speed at which a material deforms. When measuring with a cone and plate viscometer and a cone spindle, shear rates are usually measured in the range of about 0.5s -1 To about 200s -1A formulation containing a nucleic acid and an excipient, wherein the cone and plate viscometer and cone rotor are appropriately selected by a person skilled in the art to accurately measure the viscosity within the viscosity range of the sample of interest (i.e., a 20 cP sample is most accurately measured on a CPE40 cone rotor fixed on a DV2T viscometer (Brookfield); when measured using a microfluidic viscometer, the shear rate range is generally greater than about 20 s -1 to about 3,000s -1 A preparation containing a nucleic acid and an excipient.

[0074] For the classical "Newtonian" fluids commonly used herein, viscosity is substantially independent of shear rate. However, for "non-Newtonian fluids", viscosity decreases or increases with increasing shear rate, e.g., the fluid is "shear-thinning" or "shear-thickening", respectively. In the case of concentrated (i.e., high concentration) liquid compositions comprising nucleic acids, this may manifest as pseudoplastic shear-thinning behavior, i.e., viscosity decreases with shear rate.

[0075] In one embodiment, the liquid composition of the invention exhibits a decrease in viscosity of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% compared to the same composition not comprising at least one excipient.

[0076] In one embodiment, the liquid composition of the invention exhibits a decrease in viscosity of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% compared to the same composition not comprising the combination of the two excipients.

[0077] Liquid compositions according to the present invention may additionally comprise a pharmaceutically acceptable diluent, solvent, carrier, adhesive, binder, preservative, solubilizer, surfactant, penetration enhancer, stabilizer, emulsifier or bioavailability enhancer. The technician knows how to select suitable additives for a safe and well-tolerated liquid composition comprising nucleic acid.

[0078] The present invention also provides a liquid composition according to the present invention, however the molecular weight of the nucleic acid is 100 base pairs or more, preferably 50 to 50,000 base pairs.

[0079] In a preferred embodiment, 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 and more preferably at least 10 mg / ml. In another preferred embodiment, 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.

[0080] The present invention also provides a liquid composition according to the present invention, which also comprises a buffer having a concentration of 10mM to 50mM. The buffer may be a suitable tris(hydroxymethyl)aminomethane salt and provides a pH of about 7. The buffer may be supplemented with other ingredients, 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) may be used instead of acetic acid.

[0081] The present invention also provides a liquid composition according to the present invention, however the viscosity is between 1 mPas and 60 mPas, preferably between 1 mPas and 50 mPas, more preferably between 1 mPas and 30 mPas, most preferably between 1 mPas and 20 mPas. Preferably, the viscosity is measured using a microfluidic viscometer at 20°C. More preferably, the viscosity is measured using a RheoSense mVROC microfluidic viscometer at 20°C. Most preferably, the viscosity is measured using a RheoSense mVROC microfluidic viscometer at 20°C, using a 250 μl syringe, 1500 s -1 or 1000s -1 The viscosity was measured at a shear rate of 200 μl and a volume of 60-80 μl.

[0082] The present invention also relates to a kit comprising a lyophilized formulation according to the present invention (optionally in a container), instructions for its reconstruction and administration, optionally with a vial of a sterile diluent, and optionally with a syringe or other administration device. Exemplary containers include vials, test tubes, bottles, single-chamber or multi-chamber pre-filled syringes or barrels, and 96-well plates containing ready-to-use lyophilized formulations or spray-dried formulations in wells. Exemplary administration devices include syringes with or without needles, infusion pumps, jet injectors, pen devices, percutaneous injectors or other needle-free injectors.

[0083] The invention further relates to a method for reducing the viscosity of a liquid composition comprising nucleic acid, for the purpose of making all of the above-mentioned liquid compositions. All embodiments regarding the excipients, combinations and concentrations of nucleic acids, concentrations and molecular weights of nucleic acids, pH, buffers and buffer concentrations mentioned above for the liquid composition are also applicable to the method for reducing the viscosity of a composition comprising nucleic acid.

[0084] The present invention also provides a method for reducing the viscosity of a liquid composition containing nucleic acids, which comprises the step of adding at least one excipient to the liquid nucleic acid composition, wherein the excipient is selected from arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate or a salt or solvate thereof.

[0085] The present invention also provides a method for reducing the viscosity of a liquid composition containing nucleic acids, which comprises the step of adding a combination of two excipients to the liquid nucleic acid composition, wherein the excipients are selected from arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate or their salts or solvates.

[0086] The present invention also provides a method for reducing the viscosity of a liquid composition containing nucleic acids, comprising the step of adding a first excipient selected from arginine, phenylalanine, ornithine and meglumine in combination with a second excipient selected from benzenesulfonic acid, pyridoxine and thiamine phosphate.

[0087] All embodiments of the liquid composition comprising nucleic acid according to the present invention described above are also applicable to methods for reducing the viscosity of the liquid composition comprising nucleic acid. This includes all embodiments regarding the nature and concentration of nucleic acid, the combination of excipients, the concentration and ratio of excipients, the additional ingredients of the liquid composition, the viscosity reduction of the liquid composition or the stability of the nucleic acid.

[0088] Likewise, the present invention relates to a method for increasing the stability of a liquid composition comprising nucleic acids, which comprises the steps mentioned in the method for reducing the viscosity of a liquid composition comprising nucleic acids.

[0089] The present invention also provides a method for preventing self-association of nucleic acids in a liquid composition containing nucleic acids, which comprises the steps mentioned in the method for reducing the viscosity of the liquid composition containing nucleic acids.

[0090] In the method according to the invention, excipients can be added to the composition comprising nucleic acid in any manner known to the technician. When adding more than one excipient, the excipients can be pre-mixed and then added to the liquid composition comprising nucleic acid. Equally, excipients can be added to the liquid composition comprising nucleic acid respectively.

[0091] In a preferred embodiment of the present invention, the liquid composition is a pharmaceutical composition. The present invention also relates to a pharmaceutical composition as described above, comprising a therapeutic nucleic acid for treating a disease.

[0092] In particular, the pharmaceutical liquid composition as described above comprising a therapeutic nucleic acid is suitable for treating 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.

[0093] The present invention also relates to a method of treatment, wherein the treatment comprises administering a pharmaceutical composition comprising a therapeutic nucleic acid as described above.

[0094] In one aspect, the method of treatment is a method of treating cancer. That is, the liquid composition according to the present invention can be used to treat 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.

[0095] Furthermore, it has been found that excipients and excipient combinations as described above are beneficial in biological processes.

[0096] In the bioprocesses referred to here, the addition of these excipients, which have been found to act as viscosity-reducing additives, leads to improved process economy, since on the one hand the yield of intact nucleic acids can be increased and on the other hand the duration of the process can be shortened.

[0097] In the technical method, when the solution passes through a filter or medium (such as a gel bed, a packed bed or fluidized bed of porous or non-porous particles, a membrane or a monolithic material) under the force applied by the back pressure (for example, during chromatographic purification), the disclosed excipient has a beneficial effect. During the elution of nucleic acids, the back pressure of the chromatography material will increase sharply, and if it is completely blocked, it will cause a potential risk of process failure, and this event may especially occur on high-capacity chromatography materials operated at high loads. Although the filtration step is mainly used in downstream processes, excipients may also be beneficial in upstream processes. When the concentration of nucleic acids rises to the level at which it causes viscosity, with the negative effects of pressure limitation and shear force, when the solution passes through a pipe or filter to remove cell material and debris, the present invention will obviously have a beneficial effect. In order to measure the process efficiency in tangential flow filtration, most of the field flow passes tangentially through the filter surface, rather than through the filter, compared with the previously used methods, a laboratory scale TFF system is used. Although the filtration principle is different from the method described above, the filtration efficiency here also depends on the resistance of the membrane (also kept constant here) and the viscosity of the solution (which is changed by the present invention). Filtration methods are typical unit operations used to exchange formulation buffers or to bring the concentration of biomolecules to a desired level. The stirred cell used in this article is representative of a dead-end filter, where the feed is passed through a filter material that retains larger molecules on top of the material, releasing the filtrate at the other end of the device. When in TFF mode, the stirred cell uses an agitator to create turbulence of the feed solution on the membrane surface by introducing sufficient cross-flow velocity of the feed solution to reduce concentration polarization and membrane surface clogging.

[0098] The common method of exchanging buffer and concentrating nucleic acid is tangential flow filtration, when compared with the method used before, most of the field flow tangentially passes through the filter surface, rather than through the filter. As when using a stirred cell in tangential flow filtration, by making the described smaller molecules pass through a suitable filter material, macromolecules are separated from smaller molecules. Compared with a stirred cell representing a form of dead-end filtration, in tangential flow filtration, the flow geometry of the feed is different to avoid forming filter cakes and allow continuous processes. When using a stirred cell, the formation of filter cakes is also prevented by using a stirring device. Therefore, although the filter geometry is different, the stirred cell is very similar to the tangential flow filtration device. The efficiency of these two methods depends critically on membrane resistance. In addition, it is known that high viscosity reduces the flux rate that can be used, thereby increasing processing time, resulting in higher production costs. Therefore, it is expected that the reduced viscosity allows a more effective filtering process, while the shear force remains low, producing higher nucleic acid concentrations in the filtrate. This point is highlighted in the work of Hung et al., who state: "During the production of concentrated monoclonal antibody formulations by tangential flow ultrafiltration (TFF), high viscosity and aggregation often lead to extensive membrane fouling, flux decay, and low product yields" (Journal of Membrane Science, Vol. 508, June 15, 2016, pp. 113-126).

[0099] Excipients selected from arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate or salts or solvates thereof are suitable for improving the economics of the bioprocess as described above, especially according to the combination of the liquid composition containing nucleic acid in various ratios to improve the economics of the bioprocess as described above.

[0100] Therefore, another aspect of the present invention is to provide a method for reducing the viscosity of a composition containing nucleic acids in a biological process, which comprises the step of combining the composition containing nucleic acids with at least one excipient selected from arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate or a salt or solvate thereof.

[0101] In another aspect, the composition comprising the nucleic acid is combined with a combination of two excipients selected from arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine, and thiamine phosphate or a salt or solvate thereof.

[0102] In another aspect, the composition comprising the nucleic acid is combined with a combination of a first excipient selected from arginine, phenylalanine, ornithine and meglumine and a second excipient selected from benzenesulfonic acid, pyridoxine and thiamine phosphate or a salt or solvate thereof.

[0103] According to the present invention, all the parameters mentioned above; such as the combination of excipients, the concentration of excipients, the concentration of nucleic acids, the ratio of excipients, other components of the composition, pH value, viscosity reduction, specifications of nucleic acids also apply to the method for reducing the viscosity of a composition containing nucleic acids in a bioprocess.

[0104] Another aspect of the present invention is to provide a method for reducing the viscosity of a composition comprising nucleic acids in the above-mentioned biological process, wherein the permeate flux of the composition comprising nucleic acids in the filtration step is increased compared to the same composition comprising nucleic acids without at least one excipient selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate, or compared to the same composition comprising nucleic acids without a combination of two excipients selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate, or compared to the same composition comprising nucleic acids without 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.

[0105] Another aspect of the invention is the use of the method in the above-mentioned bioprocess, wherein the permeate flux of the composition comprising nucleic acids in the filtration step is increased compared to the same composition comprising nucleic acids without at least one excipient selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate, or compared to the same composition comprising nucleic acids without a combination of two excipients selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate, or compared to the same composition comprising nucleic acids without 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.

[0106] An increase in permeate flux refers to a percentage increase of at least 2%, preferably at least 5%, more preferably at least 10%, and most preferably from 10% to 100%.

[0107] Another aspect of the present invention is to provide a method for reducing the viscosity of a composition comprising nucleic acids in the above-mentioned bioprocess, wherein the volume reduction in the filter and the recovery of nucleic acids after buffer exchange are increased compared to the same composition comprising nucleic acids without at least one excipient selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate, or compared to the same composition comprising nucleic acids without a combination of two excipients selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate, or compared to the same composition comprising nucleic acids without 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.

[0108] An increase in nucleic acid recovery after volume reduction and buffer exchange in the filter refers to an increase in the percentage of nucleic acid recovery of at least 1%, preferably at least 2%, more preferably at least 5%, and most preferably between 5% and 20%.

[0109] Another aspect of the present invention is to provide a method for reducing the viscosity of a composition comprising nucleic acids in the above-mentioned biological process, wherein the process time of a filtration step, preferably a filtration step for concentrating the nucleic acids, is reduced compared to the same composition comprising nucleic acids without at least one excipient selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate, or compared to the same composition comprising nucleic acids without a combination of two excipients selected from the group consisting of arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate, or compared to the same composition comprising nucleic acids without 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.

[0110] A reduction in the process time of the filtration step means a percentage reduction of at least 5%, preferably at least 10%, more preferably at least 25%, most preferably between 25% and 100%.

[0111] In a particular embodiment of the invention, the filtration step is tangential flow filtration (TFF).

[0112] The term "bioprocess" refers to the therapeutic cell manufacturing process, which can be divided into upstream and downstream processes. The upstream process is defined as the entire process before the nucleic acid is isolated from the cellular compound. The upstream process includes early cell isolation and culture, cell banking and cell culture expansion until 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 purity and quality requirements. Some types of cells need to be destroyed when entering the downstream process. Still other cells may secrete the target nucleic acid into the culture medium, which needs to be removed by filtration. Further downstream processing is usually divided into main parts: purification part and polishing part. The bioprocess can be a batch process or a semi-continuous or continuous process.

[0113] The term "permeate flux" refers to the volume that passes through a given filter in a specific period of time, usually on the order of minutes.

[0114] The term "filtration step" refers to a process step in which a liquid is passed through a material having a defined pore size that allows separation of materials based on their size. For some filters, the pore size is defined in nanometers. However, for other filters, the pore size is not directly defined, but the weight of the molecules to be retained is given. The filter materials can be placed in such a way that they block the cross section of the filtering device (dead-end filtration). However, the filter materials can be placed in such a way that the solution to be filtered flows tangentially over the surface of the material, such as tangential flow filtration. The filter material can be a membrane, a glass filter, a metal filter or a resin. The resin can be held in a chromatographic column. The resin can be a cation exchange resin or an anion exchange resin, an affinity resin, such as protein A or glutathione resin, or a hydrophobic or hydrophilic resin.

[0115] The term "nucleic acid recovery" after buffer exchange and volume reduction refers to the fraction of nucleic acid recovered after a process step.

[0116] The term "tangential flow filtration" or "TFF" refers to a filtration method in which a solution is passed tangentially through a defined filter. The pressure generated by the solution flow rate, viscosity, temperature, and other factors forces materials smaller than the filter pores to exit the solution through the filter.

[0117] Example

[0118] 1. Effect of excipients on the viscosity reduction of DNA in TE buffer (pH 7.4)

[0119] Buffer preparation

[0120] Dilute the concentrated nuclease-free TE buffer by adding nuclease-free water to make 1x TE buffer. If necessary, adjust the pH to pH 7.4 using HCI and NaOH.

[0121] Sample preparation

[0122] Prepare 150 mM excipient solutions of ornithine (Orn), arginine (Arg), meglumine (Meg), benzenesulfonic acid (BSacid), pyridoxine (Pyr) and thiamine monophosphate (TMP) in TE-buffer, pH 7.4, respectively. Likewise, prepare 125 mM excipient solution of phenylalanine (Phe) in TE-buffer, pH 7.4. If necessary, adjust pH with HCI or NaOH.

[0123] The original buffer was exchanged for a buffer containing the relevant excipients using a centrifugal filter (Amicon, 30 kDA MWCO) and the volume of the solution was reduced to prepare a concentrated pDNA solution containing the desired excipients. The pDNA was then diluted to 8760 μg / mL and 7000 μg / mL, respectively.

[0124] DNA concentration measurement

[0125] DNA concentration was determined using absorption spectroscopy according to the Lambert-Beer law. For absorption spectroscopy, a NanoDrop TM The absorbance at 260 nm was measured by OneC (Thermo Fisher Scientific) at 260 nm. The layer thickness was adjusted by the device to give the best resolution of sample concentration.

[0126] Viscosity measurement

[0127] The mVROC™ Technology (Rheo Sense, San Ramon, California, USA) was used for viscosity measurement. A 250 μl syringe and 1500 s -1 The shear rate was measured at 1.5 % and 2.5 % respectively. The volume used was 80 μl. All samples were measured three times.

[0128] Figure 1 and 2 The viscosities of 4 kb pDNA and 14 kb pDNA solutions without (control) and with different excipients are shown.

[0129] 2. Effect of excipient combinations on the viscosity reduction of DNA in TE buffer (pH 7.4)

[0130] Buffer preparation

[0131] See Example 1.

[0132] Sample preparation

[0133] Prepare excipient solutions of 75 mM ornithine, arginine, phenylalanine or meglumine in TE-buffer pH 7.4 and supplement with 75 mM benzenesulfonic acid, pyridoxine or thiamine monophosphate, respectively. If necessary, adjust the pH with HCl or NaOH.

[0134] The original buffer was exchanged for a buffer containing the relevant excipients using a centrifugal filter (Amicon, 30 kDA MWCO) and the volume of the solution was reduced to prepare a concentrated pDNA solution containing the desired excipients. The pDNA was then diluted to 9170 μg / mL and 7000 μg / mL, respectively.

[0135] DNA concentration measurement

[0136] DNA concentration was determined using absorption spectroscopy according to the Lambert-Beer law. For absorption spectroscopy, a NanoDrop TM The absorbance at 260 nm was measured by OneC (Thermo Fisher Scientific) at 260 nm.

[0137] The layer thickness is adjusted by the device to obtain the best resolution of the sample concentration.

[0138] Viscosity measurement

[0139] The mVROC™ Technology (Rheo Sense, San Ramon, California, USA) was used for viscosity measurement. A 250 μl syringe and 1500 s -1 The shear rate was measured at 1.5 % and 2.5 % respectively. The volume used was 80 μl. All samples were measured three times.

[0140] Figure 3 and 4 The viscosities of 4 kb pDNA and 14 kb pDNA solutions without (control) and with different excipients are shown.

[0141] 3. Effect of excipients on the viscosity reduction of RNA in TE buffer (pH 7.0)

[0142] Buffer preparation

[0143] Dilute the concentrated nuclease-free TE buffer by adding nuclease-free water to make 1x TE buffer. If necessary, adjust the pH to pH 7.0 using HCI and NaOH.

[0144] Sample preparation

[0145] Prepare 150 mM excipient solutions of ornithine, arginine, benzenesulfonic acid, pyridoxine, thiamine monophosphate, respectively, in TE-buffer pH 7.0. Likewise, prepare 125 mM excipient solution of phenylalanine in TE-buffer, pH 7.4. If necessary, adjust pH with HCl or NaOH.

[0146] The original buffer was exchanged for a buffer containing the relevant excipients using a centrifugal filter (Amicon, 30 kDA MWCO) and the volume of the solution was reduced to prepare a concentrated mRNA solution containing the desired excipients. The mRNA was then diluted to 8800 μg / mL, 5680 μg / mL and 7530 μg / mL, respectively.

[0147] mRNA concentration measurement

[0148] mRNA concentration was determined using fluorescence spectroscopy. For fluorescence spectroscopy, an assay (Quant-IT TM RNA XR Assay Kit, Thermo Fisher Scientific). The fluorophore was excited at 644 nm and the Fluorescence was measured at 673 nm using a multimode plate reader (Tecan). The assay was performed and interpreted according to the manufacturer's instructions.

[0149] Viscosity measurement

[0150] The mVROC™ Technology (Rheo Sense, San Ramon, California, USA) was used for viscosity measurement. A 250 μl syringe and 1500 s -1 The shear rate was measured at 1.5 % and 2.5 % respectively. The volume used was 80 μl. All samples were measured three times.

[0151] Figures 5 to 7 The viscosity of 2000 bp, 4000 bp and 6000 bp mRNA solutions without (w / o) and with different excipients is shown.

[0152] 4. Effect of excipient combination on viscosity reduction of RNA in TE buffer (pH 7.0)

[0153] Buffer preparation

[0154] Dilute the concentrated nuclease-free TE buffer by adding nuclease-free water to make 1x TE buffer. If necessary, adjust the pH to pH 7.0 using HCI and NaOH.

[0155] Sample preparation

[0156] Prepare excipient solutions of 75 mM ornithine, arginine, phenylalanine in TE-buffer pH 7.0 and supplement with 75 mM benzenesulfonic acid, pyridoxine or thiamine monophosphate, respectively. If necessary, adjust the pH with HCl or NaOH.

[0157] The original buffer was exchanged for a buffer containing the relevant excipients using a centrifugal filter (Amicon, 30 kDA MWCO) and the volume of the solution was reduced to prepare a concentrated mRNA solution containing the desired excipients. The mRNA was then diluted to 8800 μg / mL, 5680 μg / mL and 7530 μg / mL, respectively.

[0158] mRNA concentration measurement

[0159] mRNA concentration was determined using fluorescence spectroscopy. For fluorescence spectroscopy, an assay (Quant-IT TM RNA XR Assay Kit, Thermo Fisher Scientific). The fluorophore was excited at 644 nm and the Fluorescence was measured at 673 nm using a multimode plate reader (Tecan). The assay was performed and interpreted according to the manufacturer's instructions.

[0160] Viscosity measurement

[0161] mVROC™ Technology (Rheo Sense, San Ramon, California, USA) was used for viscosity measurements.

[0162] Use a 250 μl syringe and 1500 s -1 The shear rate was measured at 1.5 % and 2.5 % respectively. The volume used was 80 μl. All samples were measured three times.

[0163] Figures 8 to 10 The viscosity of 2000 bp, 4000 bp and 6000 bp mRNA solutions without (control) and with different excipients is shown.

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.

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.

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

4. The liquid composition according to any one of claims 1 to 3, having a reduced viscosity compared to the same composition not comprising the at least one excipient.

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

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

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

8. The liquid composition according to any one of claims 1 to 7, wherein the size of the nucleic acid is 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 nucleic acids, comprising the step of adding at least one excipient to the liquid nucleic acid composition, wherein the excipient is selected from arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate or a salt or solvate thereof.

12. The method of claim 11, wherein a combination of two excipients is added to the liquid nucleic acid composition, the excipients being selected from arginine, phenylalanine, ornithine, meglumine, benzenesulfonic acid, pyridoxine and thiamine phosphate or a salt or solvate thereof.

13. The method according to claim 11 or 12, wherein a combination of a first excipient selected from arginine, phenylalanine, ornithine and meglumine or a salt or solvate thereof and a second excipient selected from benzenesulfonic acid, pyridoxine and thiamine phosphate or a salt or solvate 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 any one of claims 11 or 14 in a bioprocess.

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