Compositions comprising rnase inhibitors

Abstract

The present invention relates to a lyophilization-ready biocompatible solution and a lyophilisate comprising active RNase Inh(-SH)n, a kit comprising the solution and / or lyophilisate of the invention and a method for producing the solution and / or lyophilisate of the invention. The present invention further relates to the use of the solution, kit and / or lyophilisate disclosed herein for inhibiting RNase.

Description

Technical Field

[0001] This invention relates to lyophilized, biocompatible solutions and lyophilized products containing active RNase Inh(-SH)n, kits containing the solutions and / or lyophilized products of this invention, and methods for producing the solutions and / or lyophilized products of this invention. The invention further relates to the use of the solutions, kits, and / or lyophilized products disclosed herein for inhibiting RNases. Background Technology

[0002] RNases are ubiquitous, and when working with RNA, for example, in the reverse transcription of RNA to produce cDNA via RT-PCR, it is necessary to prevent these RNases from degrading RNA.

[0003] RNase inhibitors are proteins that work in conjunction with RNA to inhibit RNases. Most RNase inhibitors irreversibly inhibit RNases and thus protect RNA from RNase degradation. One possibility for improving storage stability and / or reducing storage costs, as well as certain requirements for specialized compositions, is lyophilization, also known as freeze-drying. Storage in lyophilized form, i.e., powder form, increases stability. However, lyophilization itself stresses the protein and can lead to loss of activity through degradation and / or precipitation. A common cryoprotectant in aqueous compositions is glycerol, which is able to prevent damage to proteins during freezing. However, glycerol interferes with any lyophilization process due to its low glass transition temperature. To provide an aqueous composition containing a lyophilizable RNase inhibitor, a lyophilization-ready composition containing the RNase inhibitor needs to be stabilized during the lyophilization process. Furthermore, it is desirable that the RNase inhibitor remain active after reconstitution of the lyophilized material obtainable from the desired composition, so that the reconstituted RNase inhibitor can again protect RNA from degradation.

[0004] RNase inhibitors typically contain reduced cysteine ​​residues, and therefore require reducing agents during production and storage to maintain their activity and prevent cysteine ​​oxidation. Maintaining the thiol group (–SH) of the cysteine ​​residue in its reduced state increases the stability of the RNase inhibitor. Dithiothreitol (DTT) is commonly used as a standard reducing agent in biochemistry because it is inexpensive and, compared to glutathione, does not form mixed disulfide species. Furthermore, DTT is considered more convenient because its odor is not as strong as other reducing agents, particularly β-mercaptoethanol.

[0005] WO 2022 / 114981 A1 discloses a composition having a recombinant human RNase inhibitor, reportedly suitable for its lyophilized and freeze-dried formulations. The disclosed composition does not contain glycerol, but instead contains a buffer, a salt, and DTT as a reducing agent.

[0006] Given the desire to provide improved solutions for the production of freeze-dried formulations containing RNase inhibitors, known technical solutions to date only offer compositions with drawbacks. Among these, the insufficient stability of the RNase inhibitor activity in the freeze-dried formulation is particularly significant.

[0007] Therefore, there remains a need to provide compositions, such as solutions or lyophilized products, containing RNase inhibitors with increased activity stability, especially when stored for longer periods and / or exposed to stressors, such as elevated temperatures and / or lyophilization. Summary of the Invention

[0008] In a first aspect, the present invention relates to a lyophilized, biocompatible solution comprising...

[0009] (i) An active RNase inhibitor containing at least one cysteine ​​residue having a reduced thiol group, and

[0010] (ii) Tris(2-carboxyethyl)phosphine (TCEP).

[0011] The inventors were surprised to discover that, generally, the stability of RNase inhibitor activity in lyophilized-ready biocompatible solutions can be increased by including tris(2-carboxyethyl)phosphine (TCEP) in the solution. In particular, this applies to solutions intended for lyophilization. As illustrated in the examples, the stability of RNase inhibitor activity exposed to different stressors is higher in compositions containing TCEP than in compositions containing DTT. The latter is a biochemically standard reducing agent known to those skilled in the art.

[0012] In a second aspect, the present invention relates to a freeze-dried ready biocompatible solution comprising activeRNaseInh(-SH)n, a reducing agent, a buffer, a monovalent salt, trehalose, a nonionic surfactant, and water, wherein the reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

[0013] In a third aspect, the present invention relates to lyophilized products containing activeRNaseInh(-SH)n and TCEP.

[0014] In a fourth aspect, the present invention relates to a kit comprising, in a container, at least one lyophilized ready biocompatible solution of the first or second aspect, or at least one lyophilized product of the third aspect.

[0015] In a fifth aspect, the present invention relates to a method for producing a lyophilized product containing active RNaseInh(-SH)n, wherein the method comprises the following steps: Lyophilized lyophilized biocompatible solutions containing active RNase Inh(-SH)n and TCEP, particularly lyophilized biocompatible solutions of the first or second aspect.

[0016] In a sixth aspect, the present invention relates to a method for producing a solution comprising active RNase Inh(-SH)n and TCEP, wherein the method comprises the following steps: To reconstitute the lyophilized product obtained by the third aspect or, in particular, by the fifth aspect.

[0017] In a seventh aspect, the present invention relates to a solution that can be obtained by, in particular by, the method of the sixth aspect, wherein, compared with the same solution before lyophilization, the RNase inhibitory activity of the solution is at least 90% after lyophilization and storage of the lyophilized product for up to 3 days.

[0018] In an eighth aspect, the present invention relates to a solution that can be obtained by, in particular by, the method of the sixth aspect, wherein the RNase inhibitory activity of the solution is at least 75% after the lyophilized product is stored at 35°C for 3 weeks, compared to the same solution before lyophilization.

[0019] In a ninth aspect, the present invention relates to a solution that can be obtained by, in particular by, the method of the sixth aspect, wherein, compared with a second solution containing the same amount of the same components after being stored at -20°C for 7 months, the RNase inhibitory activity of the solution is at least 77% after being lyophilized and the lyophilized product is stored for at most 3 days and the solution is stored for 6 months.

[0020] In a tenth aspect, the present invention relates to a solution that can be obtained by, in particular by, the method of the sixth aspect, wherein, compared with a second solution containing the same amount of the same components after being stored at -20°C for 7 months, the RNase inhibitory activity of the solution is at least 90% after being stored at 35°C for 3 weeks and subsequently at -20°C for 5 months.

[0021] In the eleventh aspect, the present invention relates to the use of the lyophilized biocompatible solution of the first or second aspect, the solution of the seventh, eighth, ninth or tenth aspect, the lyophilized product of the third aspect or the kit of the fourth aspect for inhibiting RNase.

[0022] In a twelfth aspect, the present invention relates to the use of lyophilized biocompatible solutions for producing lyophilized products (particularly the third aspect) containing activeRNaseInh(-SH)n, wherein the lyophilized biocompatible solution contains activeRNaseInh(-SH)n and tris(2-carboxyethyl)phosphine (TCEP). Attached Figure Description

[0023] Figure 1 shows photographs of two solutions containing RNase inhibitors, formulation S1 (left) and formulation S2 (right), which were obtained by reconstituted previously stressed lyophilized material, with turbidity visible in formulation S2. Detailed Implementation

[0024] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific embodiments and examples described herein, as these embodiments and examples can vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which will be limited only by the appended claims. Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0025] This specification incorporates numerous references throughout. Each of these references (including all patents, patent applications, scientific publications, manufacturer's instructions, user manuals, etc.), whether cited above or below, is incorporated herein by reference in its entirety. In the event of any conflict between the definitions or teachings of such incorporated references and those cited in this specification, the definitions and teachings in this specification shall prevail.

[0026] Elements of the invention will now be described. These elements are listed along with specific embodiments; however, it should be understood that they can be combined in any manner and in any number to create other embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention solely to the explicitly described embodiments. This description should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, unless the context otherwise requires, any arrangement and combination of the elements described herein should be considered as disclosed in the specification of this application.

[0027] The following definitions and embodiments apply in their entirety to this disclosure, and are particularly applicable to all aspects and embodiments of the invention.

[0028] As used in this specification and the appended claims, unless otherwise expressly stated, the singular forms “a,” “an,” and “the” include the plural referents.

[0029] The word “comprise” and its variants such as “comprises” and “comprising” should be understood to imply the inclusion of the stated integer or step or group of integers or steps, but not to exclude any other integer or step or group of integers or steps.

[0030] The use of alternatives (e.g., "or") should be understood to mean any one, both, or any combination of alternatives.

[0031] The term “and / or” should be understood to mean either or both of the alternatives.

[0032] Percentages, concentrations, amounts, and other numerical data may be expressed or presented in a “range” format throughout this document. It should be understood that this range format is used solely for convenience and brevity, and therefore should be flexibly interpreted to include not only the values ​​explicitly listed as range limits, but also all individual values ​​or subranges covered by the range, as if each value and subrange were explicitly listed. For example, the numerical range “4% to 20%” should be interpreted to include not only the explicitly listed values ​​of 4% to 20%, but also the individual values ​​and subranges within the indicated range. Therefore, this numerical range includes individual values ​​such as 4, 5, 6, 7, 8, 9, 10, … 18, 19, 20% and subranges such as 4% to 10%, 5% to 15%, 10% to 20%, etc. The same principle applies to ranges referencing minimum or maximum values. Furthermore, this interpretation applies regardless of the breadth of the range or characteristic.

[0033] As used herein and unless otherwise stated, the term “about” should be understood to be used synonymously with the term “approximately”. Demonstratively and unless otherwise stated, the use of the term “about” when combined with a specified numerical value or range indicates a value or range slightly greater than or slightly less than the specified value, within ±15% of the specified value, within ±10% of the specified value, within ±5% of the specified value, or conveniently within ±2% of the specified value. Therefore, such values ​​are covered by the scope of the claims that reference the terms “about” or “approximately”.

[0034] As used herein, the term "solution" refers to a composition comprising one or more solvents, each of which is in a liquid state under ambient conditions, and one or more solutes, each of which is in a solid or gaseous state under ambient conditions in the absence of a solvent. Examples of solvents include water, ethanol, glycerol, DMSO (dimethyl sulfoxide), or THF (tetrahydrofuran).

[0035] A "biocompatible" solution is a solution comprising a homogeneous mixture of water and one or more solutes, each present at a specific concentration, wherein all the compounds in the solution together are suited to provide conditions that support and / or preserve and / or maintain the structural integrity, activity, and / or function of a given biomolecule (such as a polypeptide or nucleic acid). Such conditions vary depending on the specific biomolecule in question. Specifically, active polypeptide compounds, such as, but not limited to, enzymes and compounds involved in intramolecular interactions (e.g., receptors, ligands, activators, inhibitors, etc.), may require, depending on the enzyme, a favorable buffered pH, one or more salts, and often other additives in a biocompatible solution. Examples of additives are surfactants and compounds that maintain a certain redox potential in the solution.

[0036] "Freeze-drying ready" solutions are aqueous solutions that can be freeze-dried to obtain a solid cake. Specifically, it refers to solutions that are substantially free of or contain at most 0.5% (w / v), typically at most 0.1% (w / v) of freeze-drying inhibitory solvents, or substantially free of any of the various freeze-drying inhibitory solvents or at most 0.5% (w / v) of each. "Freeze-drying inhibitory solvent" should be understood to mean a solvent that is significantly different from water with respect to one or both of its boiling point and vapor pressure. In embodiments, under ambient conditions, the boiling point of the freeze-drying inhibitory solvent is 0.01% to 10% higher than the boiling point of water; in specific embodiments, it is 0.1% to 5% higher than the boiling point of water; and in more specific embodiments, it is 0.1% to 1% higher than the boiling point of water. In another embodiment, under ambient conditions, the vapor pressure of the lyophilization inhibiting solvent is 0.01% to 10% lower than that of water; in a specific embodiment, it is 0.1% to 5% lower than that of water; and in a more specific embodiment, it is 0.1% to 1% lower than that of water. Examples of lyophilization inhibiting solvents include glycerol and other polyols, or some ionic liquids. The lyophilization-ready solution is particularly a glycerol-free solution.

[0037] The term "glycerol-free" refers to the nature of a composition (e.g., a solution or lyophilized product) and means that the composition does not contain at most 0.5% (w / v), typically at most 0.1% (w / v) glycerol. Glycerol is commonly used as a cryoprotectant to increase the stability of proteins (such as enzymes) during storage. However, it can also interfere with lyophilization. Therefore, it is recommended to avoid the use of glycerol in compositions intended for lyophilization.

[0038] "Freeze-dried products" are powders with low water content. Typically, the water content of freeze-dried products is at most 15% (w / w), at most 10% (w / w), at most 5% (w / w), at most 2% (w / w), or at most 1% (w / w).

[0039] As used in this disclosure, "% (w / v)" refers to weight-volume percentage, which is a unit of concentration expressed as a percentage of the amount of solute (e.g., in grams (g)) as a percentage of the total volume of the solution (e.g., in milliliters (ml)).

[0040] As used in this disclosure, "% (v / v)" refers to volume percentage, which is a unit of concentration expressed as a percentage of the total volume of a solution (e.g., in milliliters (ml)) of a particular solute.

[0041] As used in this disclosure, "% (w / w)" means weight percentage, which is a unit of measurement of the amount of a particular substance (e.g., in grams (g)) as a percentage of the total weight (e.g., in grams (g)) of a mixture (which may be a powder or a solution).

[0042] The terms "lyophilising" or "lyophilization" refer to the process of freezing a substance by freezing it and then reducing the ambient pressure (e.g., below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or lower), causing the freezing medium in the substance to sublimate directly from the solid phase to the gas phase. Therefore, the terms "lyophilizing" and "lyophilization" are used interchangeably herein.

[0043] The terms “reconstitution” or “resolution” refer to adding a solvent, such as water, to a dried product, such as a lyophilized product, to restore it to a liquid state, such as its original liquid state.

[0044] In the context of this disclosure, the term "recombinant" means "prepared by genetic engineering." In some embodiments, the "recombinant protein" in the context of this disclosure is not naturally occurring.

[0045] As used herein, the terms “room temperature” and “ambient temperature” are used interchangeably and refer to temperatures ranging from at least about 15°C, for example from about 15°C to about 35°C, from about 15°C to about 30°C, from about 15°C to about 25°C, or from about 17°C to about 22°C. Such temperatures will include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, and 22°C.

[0046] As used herein, the term “environmental conditions” refers to conditions having an ambient temperature and an atmospheric pressure of approximately 10¹³ ± 50 hPa.

[0047] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, peptides or nucleic acids that exist in organisms (including viruses), can be isolated from natural sources, and have not been intentionally modified in a laboratory are naturally occurring. The term "found in nature" means "existing in nature," including known objects as well as objects that have not yet been discovered and / or are isolated from nature but may be discovered in the future and / or are isolated from natural sources. The term "natural" refers to naturally occurring objects.

[0048] According to the present invention, the term "peptide" refers to a molecule comprising about two or more, about three or more, about four or more, about six or more, about eight or more, about ten or more, about thirteen or more, about sixteen or more, about 20 or more, and up to about 50, about 100, or about 150 amino acids linked together by peptide bonds. The term "polypeptide" refers to a large peptide, particularly a peptide having at least about 151 amino acids. Both "peptide" and "polypeptide" are protein molecules. Therefore, the terms "peptide," "protein," and "polypeptide" are generally used as synonyms herein.

[0049] For the purposes of this invention, a “variant” of an amino acid sequence (peptide or polypeptide) may include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term “variant” includes all mutants, splicing variants, post-translational modification variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those that are naturally occurring. The term “variant” includes, in particular, fragments of an amino acid sequence. An amino acid insertion variant includes the insertion of one or more amino acids into a specific amino acid sequence. In the case of a variant with an inserted amino acid sequence, one or more amino acid residues are inserted into a specific site in the amino acid sequence, although random insertion and appropriate screening of the resulting product are also possible. An amino acid addition variant includes the fusion of one or more amino acid terms at the amino and / or carboxyl ends, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. An amino acid deletion variant is characterized by the removal of one or more amino acids from the sequence, such as the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Deletions can occur at any location in a protein. Amino acid deletion variants containing deletions at the N-terminus and / or C-terminus of a protein are also called N-terminal and / or C-terminal truncation variants. Amino acid substitution variants are characterized by the removal of at least one residue from the sequence and the insertion of another residue in its position. Modifications at non-conserved positions in the amino acid sequence between homologous peptides or polypeptides and / or substitution of amino acids with other amino acids having similar properties are preferred. In some embodiments, amino acid changes in peptide and polypeptide variants are conserved amino acid changes, i.e., substitutions of similar charged or uncharged amino acids. Conserved amino acid changes involve substitutions of one of the relevant amino acid families in their side chains. Naturally occurring amino acids are generally classified into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and non-polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. In some embodiments, conserved amino acid substitutions include substitutions within the group consisting of: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0050] "Sequence similarity" refers to the percentage of identical or conserved amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of identical amino acids between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of identical nucleotides between the sequences. Alignments used to determine sequence similarity, such as sequence identity, can be performed using tools known in the art, such as optimal sequence alignment, for example using Align, with standard settings, preferably EMBOSS::Needle, matrix: Blosum62, vacancy open 10.0, vacancy extension 0.5.

[0051] The terms “%identical” and “%compatibility” or similar terms are intended to specifically refer to the percentage of identical nucleotides or amino acids in the best alignment between sequences to be compared. This percentage is purely statistical, and the differences between two sequences may, but are not necessarily, randomly distributed across the entire length of the sequences being compared. Comparison of two sequences is typically performed by comparing the sequences relative to fragments or a “comparison window” after best alignment in order to identify local regions of the corresponding sequences. The optimal alignment for comparison can be performed manually or by means of algorithms, such as Smith and Waterman's local homology algorithm, 1981, Ads App. Math. 2, 482; Needleman and Wunsch's local homology algorithm, 1970, J. Mol. Biol. 48, 443; Pearson and Lipman's similarity search algorithm, 1988, Proc. Natl Acad. Sci. USA 88, 2444; or by means of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, the percentage identity of the two sequences is determined using the BLASTN or BLASTP algorithm (as available on the National Center for Biotechnology Information (NCBI) website). In some embodiments, the algorithm parameters for the BLASTN algorithm on the NCBI website include: (i) an expected threshold set to 10; (ii) a word size set to 28; (iii) a maximum match within the query range set to 0; (iv) match / no-match scores set to 1, -2; (v) a gap cost set to linear; and (vi) a filter for the low-complexity region being used. In some embodiments, the algorithm parameters for the BLASTP algorithm on the NCBI website include: (i) an expected threshold set to 10; (ii) a word size set to 3; (iii) a maximum match within the query range set to 0; (iv) a matrix set to BLOSUM62; (v) a gap cost set to exist: 11, expand: 1; and (vi) conditional combination score matrix adjustment.

[0052] Percentage identity is obtained by determining the number of identical positions corresponding to the sequences to be compared, dividing that number by the number of positions being compared (e.g., the number of positions in the reference sequence), and then multiplying the result by 100.

[0053] The amino acid sequence variants described herein can be readily prepared by those skilled in the art, for example, through recombinant DNA manipulation. Operations for preparing DNA sequences having substituted, added, inserted, or deleted peptides or polypeptides are described in detail in Molecular Cloning: e.g., A Laboratory Manual, 4th Edition, MR Green and J. Sambrook et al. (1989), eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012. Furthermore, the peptides, polypeptides, and amino acid variants described herein can be readily prepared using known peptide synthesis techniques, such as, for example, solid-phase synthesis and similar methods. In some embodiments, fragments or variants of amino acid sequences (peptides or polypeptides) are “functional fragments” or “functional variants.” The term “functional fragment” or “functional variant” of an amino acid sequence refers to any fragment or variant exhibiting one or more functional properties that are identical or similar to those functional properties of the amino acid sequence from which it is derived; that is, it is functionally equivalent. Regarding the sequence of a binder (such as an antibody), a specific function is one or more binding activities exhibited by the amino acid sequence from which the fragment or variant is derived. As used herein, the terms "functional fragment" or "functional variant" specifically refer to a variant molecule or sequence comprising an amino acid sequence altered by one or more amino acids compared to the amino acid sequence of a parent molecule or sequence, and which still performs one or more functions of the parent molecule or sequence, such as binding to a target molecule. In some embodiments, modifications to the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced, but still significantly present; for example, the function of the functional fragment or functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence. However, in other embodiments, the function of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0054] According to various embodiments of this disclosure, nucleic acids encoding peptides or polypeptides are taken up or introduced into cells, i.e., transfected or transduced into cells that may be present in vitro or in a subject, thereby resulting in the expression of said peptide or polypeptide. Cells may, for example, express the encoded peptide or polypeptide intracellularly (e.g., in the cytoplasm and / or in the nucleus), may secrete the encoded peptide or polypeptide, and / or may express it on a surface. According to this disclosure, terms such as “expressing nucleic acid” and “encoding nucleic acid” or similar terms are used interchangeably herein, and with respect to a particular peptide or polypeptide, refers to the nucleic acid, if present in a suitable environment, such as intracellularly, that can be expressed to produce said peptide or polypeptide.

[0055] The term “expression” as used in this article includes transcription and / or translation of a specific nucleotide sequence.

[0056] In the context of this disclosure, the term "transcription" refers to the process in which the genetic code in a DNA sequence is transcribed into RNA (specifically mRNA). Subsequently, the RNA can be translated into peptides or polypeptides.

[0057] Regarding RNA, the terms "expression" or "translation" refer to a process within the cell's ribosomes through which the mRNA chain directs the assembly of amino acid sequences to produce peptides or polypeptides.

[0058] The term “free of DTT and / or glutathione” refers to the nature of a composition (e.g., a solution or lyophilized product) and means that the composition does not contain up to 0.5 mM, typically up to 0.1 mM, typically up to 0.01 mM of DTT and / or glutathione.

[0059] The "active" RNase inhibitor according to the invention is an RNase inhibitor capable of inhibiting RNA degradation in solution. The amount of activity can be measured in units. In particular, an RNase inhibitor containing at least one cysteine ​​in its polypeptide chain and in which the cysteine ​​is in its free form (i.e., not part of a disulfide bridge) is active when the free cysteine ​​is reduced to its thiol form. The term "activeRNaseInh(-SH)n" refers to an active RNase inhibitor having n cysteines, wherein the cysteine ​​is reduced and n is one or more cysteines.

[0060] The term "unit" or "international unit" (IU), in relation to RNase inhibitors, refers to the amount of RNase inhibitor required to inhibit an RNase. One unit of RNase inhibitor is the amount required to inhibit 50% of the activity of 5 ng of RNase A (see Blackburn P., Ribonuclease Inhibitor from Human Placenta: Rapid Purification and Assay, J Biol Chem, Vol. 254, No. 24, pp. 12484-12487, 1979). The activity of an RNase inhibitor, and thus the number of units contained in solution, can be determined by measuring the inhibition of 2',3'-cyclocytidine monophosphate hydrolysis, as described, for example, by Peter Blackburn (see Blackburn P., Ribonucase Inhibitor from Human Placenta: Rapid Purification and Assay, J Biol Chem, Vol. 254, No. 24, pp. 12484-12487, 1979).

[0061] The term "activity stability" refers to the property of a solution or lyophilized product to at least partially prevent the loss of active RNase inhibitor within that solution or lyophilized product for a certain period of time. The extent to which a solution or lyophilized product must prevent the loss of active RNase inhibitor activity to stabilize its activity depends on the conditions to which the solution is exposed. For example, a solution containing 100 units of RNase inhibitor is considered stable when the activity of the RNase inhibitor in an equal volume of solution is, for example, 80 units, obtained by reconstituted a lyophilized product containing 100 units of the solution and storing the lyophilized product under ambient conditions for 3 weeks.

[0062] Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0063] In a first aspect, the present invention relates to a lyophilized, biocompatible solution comprising...

[0064] (i) An active RNase inhibitor containing at least one cysteine ​​residue having a reduced thiol group, and

[0065] (ii) Tris(2-carboxyethyl)phosphine (TCEP).

[0066] In some embodiments of the invention, the lyophilized-ready biocompatible solution is an aqueous solution. In some embodiments of the invention, the lyophilized-ready biocompatible aqueous solution contains water and up to 10% (v / v), up to 5% (v / v), 1% (v / v), or up to 0.5% (v / v) of other solvents.

[0067] In some embodiments of the present invention, a lyophilization-ready biocompatible solution is used to produce lyophilized products. In some embodiments of the present invention, a lyophilization-ready biocompatible solution is used to produce lyophilized products containing active RNase Inh(-SH)n and TCEP.

[0068] In some embodiments of the present invention, the RNase inhibitory activity of the lyophilized biocompatible solution is at least 90% after the solution is stored at 35°C for 3 weeks, compared to the same solution before storage.

[0069] In some embodiments of the present invention, RNase inhibitory activity is measured by measuring RNase inhibitory activity according to methods known to those skilled in the art, particularly according to the method of Blackburn P., Ribonucase Inhibitor from Human Placenta: Rapid Purification and Assay, J Biol Chem, Vol. 254, No. 24, pp. 12484-12487, 1979.

[0070] In some embodiments of the invention, the pH of the lyophilized, biocompatible solution is 5.0 to 9.0 at 4°C, 7.0 to 8.0 at 4°C, or 7.6 at 4°C.

[0071] In some embodiments of the present invention, the freeze-dried biocompatible solution is free of dithiothreitol (DTT) and / or glutathione.

[0072] In some embodiments of the present invention, the concentration of TCEP is 1 to 20 mM, 5 to 10 mM, or 5 mM.

[0073] In some embodiments of the invention, the freeze-dried ready biocompatible solution further comprises a buffer.

[0074] In some embodiments of the invention, the buffer is selected from the group consisting of: TES (2-{[1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl]amino}ethane-1-sulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), Tris (tris(hydroxymethyl)aminomethane), phosphate esters, and combinations thereof. In some embodiments of the invention, the buffer is HEPES. In some embodiments of the invention, the concentration of the buffer is 5 to 100 mM, 10 to 50 mM, or 20 mM.

[0075] In some embodiments of the invention, the freeze-dried ready biocompatible solution further comprises salt.

[0076] In some embodiments of the invention, the freeze-dried ready biocompatible solution further comprises a monovalent salt.

[0077] In some embodiments of the present invention, the monovalent salt is NaCl and / or KCl. In some embodiments of the present invention, the monovalent salt is NaCl or KCl. In some embodiments of the present invention, the monovalent salt is NaCl. In some embodiments of the present invention, the monovalent salt is KCl. In some embodiments of the present invention, the concentration of the monovalent salt is 1 to 500 mM, 5 to 300 mM, 5 to 250 mM, 50 to 150 mM, or 100 mM. In some embodiments of the present invention, the concentration of the monovalent salt is 100 mM.

[0078] In some embodiments of the invention, the lyophilized, biocompatible solution further comprises trehalose. In some embodiments of the invention, the concentration of trehalose is 10% to 40% (w / v), 15% to 35% (w / v), or 20% to 30% (w / v). Trehalose, compared to other carbohydrates, particularly sucrose, can further help increase the stability of the activity of the RNase inhibitor in the compositions of the invention.

[0079] In some embodiments of the invention, the lyophilized, biocompatible solution further comprises a nonionic surfactant. In some embodiments of the invention, the nonionic surfactant is selected from the group consisting of: Triton X-100, polysorbate 20, polysorbate 60, polysorbate 80, polydextrose, and combinations thereof. In some embodiments of the invention, the nonionic surfactant is polysorbate 20. In some embodiments of the invention, the concentration of the nonionic surfactant is 0.005% to 0.5% (v / v), 0.005% to 0.2% (v / v), 0.01% to 0.1% (v / v), or 0.05% to 0.1% (v / v). In some embodiments of the invention, the concentration of the nonionic surfactant is 0.05% to 0.1% (v / v).

[0080] In some embodiments of the present invention, the freeze-dried ready biocompatible solution comprises active RNase Inh(-SH)n, tris(2-carboxyethyl)phosphine (TCEP), a buffer, salt, trehalose, and water.

[0081] In some embodiments of the present invention, the freeze-dried ready biocompatible solution comprises active RNase Inh(-SH)n, tris(2-carboxyethyl)phosphine (TCEP), a buffer, a salt, trehalose, a nonionic surfactant, and water.

[0082] In some embodiments of the present invention, the freeze-dried ready biocompatible solution comprises active RNaseInh(-SH)n, tris(2-carboxyethyl)phosphine (TCEP), a buffer, a monovalent salt, trehalose, a nonionic surfactant, and water.

[0083] In some embodiments of the present invention, the freeze-dried ready biocompatible solution comprises TCEP, HEPES, KCl, trehalose, and polysorbate 20.

[0084] In some embodiments of the present invention, the lyophilized ready biocompatible solution comprises 1 to 20 mM TCEP, 5 to 100 mM buffer, 5 to 300 mM monovalent salt, 10% to 40% (w / v) trehalose and 0.005% to 0.5% (v / v) nonionic surfactant.

[0085] In some embodiments of the present invention, the lyophilized-ready biocompatible solution comprises 5 mM TCEP, 20 mM buffer, 100 mM monovalent salt, 20% to 30% (w / v) trehalose, and 0.01% to 0.1% (v / v) nonionic surfactant.

[0086] In some embodiments of the present invention, the lyophilized-ready biocompatible solution comprises 5 mM TCEP, 20 mM buffer, 100 mM monovalent salt, 20% to 30% (w / v) trehalose, and 0.05% to 0.1% (v / v) nonionic surfactant.

[0087] In some embodiments of the present invention, the lyophilized ready biocompatible solution comprises 5 mM TCEP, 20 mM HEPES, 100 mM KCl, 20% to 30% (w / v) trehalose and 0.01% to 0.1% (v / v) polysorbate 20.

[0088] In some embodiments of the present invention, the freeze-dried ready biocompatible solution comprises 5 mM TCEP, 20 mM HEPES, 100 mM KCl, 20% to 30% (w / v) trehalose and 0.05% to 0.1% (v / v) polysorbate 20.

[0089] In some embodiments of the present invention, activeRNaseInh(-SH)n is an RNase inhibitor derived from mice, goats, African clawed frogs, Homo sapiens, or brown rats. In some embodiments of the present invention, activeRNaseInh(-SH)n is an RNase inhibitor derived from brown rats.

[0090] In some embodiments of the present invention, activeRNaseInh(-SH)n is a recombinant protein. In some embodiments of the present invention, activeRNaseInh(-SH)n is recombinantly fused with a protein tag. Examples of protein tags may include His-tag, HA-tag, Fc-tag, MBP (maltose-binding protein)-tag, avi-tag, and strep-tag. The tag may be attached to activeRNaseInh(-SH)n using a adapter, particularly an adapter that can be cleaved by a protease to remove the tag. The activeRNaseInh(-SH)n contained in the solutions or lyophilized products of the present invention can be obtained by methods known to those skilled in the art. In some embodiments of the present invention, the activeRNaseInh(-SH)n contained in the solutions or lyophilized products of the present invention can be obtained by expression in a prokaryotic or eukaryotic expression system. The prokaryotic or eukaryotic expression system may contain nucleic acids encoding activeRNaseInh(-SH)n. activeRNaseInh(-SH)n can be purified by methods known to those skilled in the art. For example, active RNase Inh(-SH)n can be purified using crystallization, affinity chromatography, ion exchange chromatography, and / or size exclusion chromatography.

[0091] In some embodiments of the present invention, activeRNaseInh(-SH)n comprises or is composed of the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least 85%, at least 90%, or at least 95% sequence identity. In some embodiments of the present invention, activeRNaseInh(-SH)n comprises or is composed of the amino acid sequence of SEQ ID NO: 1.

[0092] In some embodiments of the present invention, activeRNaseInh(-SH)n consists of the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least 85%, at least 90%, or at least 95% sequence identity. In some embodiments of the present invention, activeRNaseInh(-SH)n consists of the amino acid sequence of SEQ ID NO: 1.

[0093] In some embodiments of the present invention, the solution contains at least 30 units of activeRNaseInh(-SH)n per µl, at least 40 units of activeRNaseInh(-SH)n per µl, at least 100 units of activeRNaseInh(-SH)n per µl, at least 200 units of activeRNaseInh(-SH)n per µl, at least 300 units of activeRNaseInh(-SH)n per µl, or at least 400 units of activeRNaseInh(-SH)n per µl. In some embodiments of the present invention, the solution contains at least 400 units of activeRNaseInh(-SH)n per µl.

[0094] In a second aspect, the present invention relates to a freeze-dried ready biocompatible solution comprising activeRNaseInh(-SH)n, a reducing agent, a buffer, a monovalent salt, trehalose, a nonionic surfactant, and water, wherein the reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

[0095] All embodiments mentioned above in connection with the first aspect of the present invention are applicable to the second aspect of the present invention, and vice versa.

[0096] In a third aspect, the present invention relates to lyophilized products containing activeRNaseInh(-SH)n and TCEP.

[0097] In some embodiments of the present invention, the lyophilized product further comprises a buffer, a monovalent salt, trehalose, and / or a nonionic surfactant.

[0098] In some embodiments of the present invention, the lyophilized product does not contain DTT and / or glutathione.

[0099] All embodiments mentioned above with respect to the first and / or second aspects of the present invention are applicable to the third aspect of the present invention, and vice versa.

[0100] In a fourth aspect, the present invention relates to a kit comprising, in a container, at least one lyophilized biocompatible solution of the first or second aspect or at least one lyophilized product of the third aspect.

[0101] All embodiments mentioned above in connection with the first, second, and / or third aspects of the present invention are applicable to the fourth aspect of the present invention, and vice versa.

[0102] Suitable containers for solid or liquid compositions are known to those skilled in the art and may be glass, optionally with a plastic cap, or plastic. The kit may contain at least one lyophilized, biocompatible solution of the first or second aspect. The kit may further contain suitable means for keeping the solution cool or frozen, such as dry ice, chilled water, or cryogel packs. The kit may be intended for storage at suitable temperatures, particularly for keeping the solution frozen, for example, -20°C.

[0103] In a fifth aspect, the present invention relates to a method for producing a lyophilized product containing active RNaseInh(-SH)n, wherein the method comprises the following steps: Lyophilized lyophilized biocompatible solutions containing active RNase Inh(-SH)n and TCEP, particularly lyophilized biocompatible solutions of the first or second aspect.

[0104] All embodiments mentioned in the first, second, third and / or fourth aspects of the present invention are applicable to the fifth aspect of the present invention, and vice versa.

[0105] In a sixth aspect, the present invention relates to a method for producing a solution comprising active RNase Inh(-SH)n and TCEP, wherein the method comprises the following steps: To reconstitute the lyophilized product obtained by the third aspect or, in particular, by the fifth aspect.

[0106] All embodiments mentioned in the first, second, third, fourth and / or fifth aspects of the present invention are applicable to the sixth aspect of the present invention, and vice versa.

[0107] In some embodiments of the present invention, the lyophilized product is reconstituted with a solution containing at least one salt, at least one buffer, at least one nonionic surfactant and / or at least one reducing agent.

[0108] In a seventh aspect, the present invention relates to a solution that can be obtained by, in particular by, the method of the sixth aspect, wherein, compared with the same solution before lyophilization, the RNase inhibitory activity of the solution is at least 90% after lyophilization and storage of the lyophilized product for up to 3 days.

[0109] All embodiments mentioned in the first, second, third, fourth, fifth and / or sixth aspects of the present invention are applicable to the seventh aspect of the present invention, and vice versa.

[0110] In some embodiments of the invention, the RNase inhibitory activity is at least 95% after lyophilization and storage of the lyophilized product for up to 3 days, compared to the same solution before lyophilization.

[0111] In some embodiments of the invention, the RNase inhibitory activity is at least 90% or at least 95% after lyophilization and storage of the lyophilized product for at most 1 or 2 days, or at most 12 hours, compared to the same solution prior to lyophilization.

[0112] In some embodiments of the invention, the RNase inhibitory activity of the lyophilized product is measured by reconstituted lyophilized material and by methods known to those skilled in the art, particularly according to the method of Blackburn P., *Ribonucase Inhibitor from Human Placenta: Rapid Purification and Assay*, JBiol Chem, Vol. 254, No. 24, pp. 12484-12487, 1979. In some embodiments of the invention, the lyophilized product is reconstituted with a solution containing at least one salt, at least one buffer, at least one nonionic surfactant, and / or at least one reducing agent. In some embodiments of the invention, the lyophilized product is reconstituted in a solution containing the same components (particularly the same concentration and / or amount) as the solution used to produce the lyophilized product. In some embodiments of the invention, the lyophilized product is reconstituted to obtain a solution containing the same amount of RNase inhibitor as used in the solution before lyophilization. For example, the solution used to measure the RNase inhibitory activity before lyophilization has a concentration of 4000 units / ml in a 1 ml volume, which is used to produce a lyophilized product containing the same amount of RNase inhibitor. This lyophilized product is reconstituted to 1 ml and thus contains the same amount of RNase inhibitor as the solution before lyophilization, but may have different activities and therefore fewer units per volume.

[0113] In an eighth aspect, the present invention relates to a solution that can be obtained by, in particular by, the method of the sixth aspect, wherein the RNase inhibitory activity of the glycerol-free solution is at least 75% after the lyophilized product is stored at 35°C for 3 weeks, compared to the same solution before lyophilization.

[0114] All embodiments mentioned in the first, second, third, fourth, fifth, sixth and / or seventh aspects of the present invention are applicable to the eighth aspect of the present invention, and vice versa.

[0115] In some embodiments of the present invention, the RNase inhibitory activity is at least 77% or at least 80% after the lyophilized product is stored at 35°C for 3 weeks, compared to the same solution before lyophilization.

[0116] In a ninth aspect, the present invention relates to a solution that can be obtained by, in particular by, the method of the sixth aspect, wherein, compared with a second solution containing the same amount of the same components after being stored at -20°C for 7 months, the RNase inhibitory activity of the solution is at least 77% after being lyophilized and the lyophilized product is stored for at most 3 days and the solution is stored for 6 months.

[0117] All embodiments mentioned in the first, second, third, fourth, fifth, sixth, seventh and / or eighth aspects of the present invention are applicable to the ninth aspect of the present invention, and vice versa.

[0118] In some embodiments of the invention, the second solution used for comparison is derived from the same solution used to obtain the lyophilized material in the method of the sixth aspect, and the second solution may be stored at -20°C during the storage of the lyophilized material and / or during the storage of the solution that can be obtained from, in particular, the lyophilized material.

[0119] In a tenth aspect, the present invention relates to a solution that can be obtained by, in particular by, the method of the sixth aspect, wherein, compared with a second solution containing the same amount of the same components after being stored at -20°C for 7 months, the RNase inhibitory activity of the solution is at least 90% after being stored at 35°C for 3 weeks and subsequently at -20°C for 5 months.

[0120] All embodiments mentioned in the first, second, third, fourth, fifth, sixth, seventh, eighth and / or ninth aspects of the present invention are applicable to the tenth aspect of the present invention, and vice versa.

[0121] In the eleventh aspect, the present invention relates to the use of the lyophilized biocompatible solutions of the first and second aspects, the solutions of the seventh or eighth aspect, the lyophilized products of the third aspect, or the kits of the fourth aspect for inhibiting RNases.

[0122] All embodiments mentioned in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and / or tenth aspects of the present invention are applicable to the eleventh aspect of the present invention, and vice versa.

[0123] In some embodiments of the invention, the use includes adding the lyophilized, biocompatible solution or lyophilized product of the invention to a composition, particularly, the composition being a solution. In some embodiments of the invention, RNase is present in the composition, particularly in the solution. In some embodiments of the invention, the composition, particularly the solution, is RNase-free, but there is a risk of RNase contamination. In some embodiments of the invention, the composition is used for reverse transcription or in vitro transcription, for example, to produce mRNA in vitro.

[0124] In a twelfth aspect, the present invention relates to the use of lyophilized biocompatible solutions for producing lyophilized products (particularly the third aspect) containing activeRNaseInh(-SH)n, wherein the lyophilized biocompatible solution contains activeRNaseInh(-SH)n and tris(2-carboxyethyl)phosphine (TCEP).

[0125] All embodiments mentioned in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and / or eleventh aspects of the present invention are applicable to the twelfth aspect of the present invention, and vice versa.

[0126] In a further embodiment, the present invention relates to the following items: 1. A lyophilized, biocompatible solution comprising... (i) An active RNase inhibitor containing at least one cysteine ​​residue having a reduced thiol group, and (ii) Tris(2-carboxyethyl)phosphine (TCEP).

[0127] 2. A freeze-dried ready biocompatible solution comprising active RNase Inh(-SH)n, a reducing agent, a buffer, a monovalent salt, trehalose, a nonionic surfactant, and water, wherein the reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

[0128] 3. A lyophilization-ready biocompatible solution according to Project 1 or 2, used to produce lyophilized products, particularly lyophilized products containing active RNase Inh(-SH)n and TCEP.

[0129] 4. A freeze-dried, biocompatible solution according to any one of items 1 to 3, wherein the RNase inhibitory activity of the solution is at least 90% after being stored at 35°C for 3 weeks, compared with the same solution before storage.

[0130] 5. A lyophilized, biocompatible solution according to any one of items 1 to 4, having a pH of 5.0 to 9.0 at 4°C, typically 7.0 to 8.0 at 4°C, typically 7.6 at 4°C.

[0131] 6. A freeze-dried, biocompatible solution according to any one of items 1 to 5, wherein the solution is free of dithiothreitol (DTT) and / or glutathione.

[0132] 7. A lyophilized, biocompatible solution according to any one of items 1 to 6, wherein the concentration of TCEP is 1 to 20 mM, typically 5 to 10 mM, typically 5 mM.

[0133] 8. A lyophilized, biocompatible solution according to any one of items 1 or 3 to 7, wherein the solution further comprises a buffer.

[0134] 9. A lyophilized, biocompatible solution according to item 2 or 8, wherein the buffer is selected from the group consisting of: TES, MOPS, HEPES, Tris, phosphates and combinations thereof, typically HEPES.

[0135] 10. A lyophilized, biocompatible solution according to any one of items 2 or 8 to 9, wherein the concentration of the buffer is 5 to 100 mM, typically 10 to 50 mM, typically 20 mM.

[0136] 11. A freeze-dried, biocompatible solution according to any one of items 1 or 3 to 10, wherein the solution further comprises a monovalent salt.

[0137] 12. A freeze-dried, biocompatible solution according to item 2 or 11, wherein the monovalent salt is NaCl and / or KCl.

[0138] 13. A lyophilized, biocompatible solution according to any one of Item 2 or 11 to 12, wherein the concentration of the monovalent salt is 1 to 500 mM, typically 5 to 300 mM, typically 5 to 250 mM, typically 50 to 150 mM, typically 100 mM.

[0139] 14. A freeze-dried, biocompatible solution according to any one of items 1 or 3 to 13, wherein the solution further comprises trehalose.

[0140] 15. A freeze-dried, biocompatible solution according to any one of Item 2 or 14, wherein the concentration of trehalose is 10% to 40% (w / v), typically 15% to 35% (w / v), typically 20% to 30% (w / v).

[0141] 16. A freeze-dried, biocompatible solution according to any one of items 1 or 3 to 15, wherein the solution further comprises a nonionic surfactant.

[0142] 17. A freeze-dried, biocompatible solution according to item 2 or 16, wherein the nonionic surfactant is selected from the group consisting of: Triton X-100, polysorbate 20, polysorbate 60, polysorbate 80, polydextrose, and combinations thereof.

[0143] 18. A lyophilized, biocompatible solution according to any one of Item 2 or 16 to 17, wherein the concentration of the nonionic surfactant is 0.005% to 0.5% (v / v), typically 0.005% to 0.2% (v / v), typically 0.01% to 0.1% (v / v), typically 0.05% to 0.1% (v / v).

[0144] 19. A lyophilized, biocompatible solution according to any one of items 1 or 3 to 18, comprising TCEP, HEPES, KCl, trehalose, and polysorbate 20.

[0145] 20. A lyophilized, biocompatible solution according to any one of items 1 or 3 to 19, comprising 1 to 20 mM TCEP, 5 to 100 mM buffer, 5 to 300 mM monovalent salt, 10% to 40% (w / v) trehalose and 0.005% to 0.5% (v / v) nonionic surfactant.

[0146] 21. A lyophilized, biocompatible solution according to any one of items 1 or 3 to 20, comprising 5 mM TCEP, 20 mM buffer, 100 mM monovalent salt, 20% to 30% (w / v) trehalose and 0.01% to 0.1% (v / v) nonionic surfactant.

[0147] 22. A lyophilized, biocompatible solution according to any one of items 1 or 3 to 21, comprising 5 mM TCEP, 20 mM HEPES, 100 mM KCl, 20% to 30% (w / v) trehalose and 0.01% to 0.1% (v / v) polysorbate 20.

[0148] 23. A lyophilized, biocompatible solution according to any one of items 1 to 22, wherein activeRNaseInh(-SH)n is an RNase inhibitor derived from brown rats.

[0149] 24. A lyophilized, biocompatible solution according to any one of items 1 to 23, wherein activeRNaseInh(-SH)n comprises or consists of the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least 85% sequence identity.

[0150] 25. A lyophilized, biocompatible solution according to any one of items 1 to 23, wherein activeRNaseInh(-SH)n comprises or consists of the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least 90% sequence identity.

[0151] 26. A lyophilized, biocompatible solution according to any one of items 1 to 23, wherein activeRNaseInh(-SH)n comprises or consists of the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least 95% sequence identity.

[0152] 27. A lyophilized, biocompatible solution according to any one of items 1 to 23, wherein activeRNaseInh(-SH)n comprises, and in particular consists of, the amino acid sequence of SEQ ID NO: 1.

[0153] 28. A lyophilized, biocompatible solution according to any one of items 1 to 27, wherein the solution contains at least 30 units of activeRNaseInh(-SH)n per µl, typically at least 40 units of activeRNaseInh(-SH)n per µl, typically at least 100 units of activeRNaseInh(-SH)n per µl, typically at least 200 units of activeRNaseInh(-SH)n per µl, typically at least 300 units of activeRNaseInh(-SH)n per µl, typically at least 400 units of activeRNaseInh(-SH)n per µl.

[0154] 29. A lyophilized product comprising active RNase Inh(-SH)n and TCEP.

[0155] 30. The lyophilized product according to item 29 further comprises a buffer, a monovalent salt, trehalose, and / or a nonionic surfactant.

[0156] 31. The lyophilized product according to item 29 or 30, wherein the lyophilized product is free of DTT and / or glutathione.

[0157] 32. The lyophilized product according to item 30 or 31, wherein the buffer is selected from the group consisting of: TES, MOPS, HEPES, Tris, phosphates and combinations thereof, typically HEPES.

[0158] 33. The freeze-dried product according to any one of items 30 to 32, wherein the monovalent salt is NaCl and / or KCl.

[0159] 34. The lyophilized product according to any one of items 30 to 33, wherein the nonionic surfactant is selected from the group consisting of: Triton X-100, polysorbate 20, polysorbate 60, polysorbate 80, polydextrose, and combinations thereof.

[0160] 35. The lyophilized product according to any one of items 29 to 34, comprising TCEP, HEPES, KCl, trehalose and polysorbate 20.

[0161] 36. The lyophilized product according to any one of items 29 to 35, wherein activeRNaseInh(-SH)n is an RNase inhibitor derived from brown rats.

[0162] 37. The lyophilized product according to any one of items 29 to 36, wherein activeRNaseInh(-SH)n comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least 85% sequence identity.

[0163] 38. The lyophilized product according to any one of items 29 to 36, wherein activeRNaseInh(-SH)n comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least 90% sequence identity.

[0164] 39. The lyophilized product according to any one of items 29 to 36, wherein activeRNaseInh(-SH)n comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least 95% sequence identity.

[0165] 40. The lyophilized product according to any one of items 29 to 36, wherein activeRNaseInh(-SH)n comprises or is composed of the amino acid sequence of SEQ ID NO: 1.

[0166] 41. A kit comprising, in a container, at least one lyophilized ready biocompatible solution of any one of items 1 to 28 or at least one lyophilized product of any one of items 29 to 40.

[0167] 42. A method for producing a lyophilized product containing active RNase Inh(-SH)n, wherein the method comprises the following steps: Lyophilized lyophilized biocompatible solutions containing active RNase Inh(-SH)n and TCEP, particularly lyophilized biocompatible solutions according to any one of items 1 to 28.

[0168] 43. A method for producing a lyophilized, biocompatible solution containing active RNase Inh(-SH)n and TCEP, wherein the method comprises the following steps: To reconstitute the lyophilized product according to any one of items 29 to 40 or that can be obtained by, in particular by, the method according to item 42.

[0169] 44. A solution that can be obtained, in particular, by the method according to item 43, wherein the RNase inhibitory activity of the solution is at least 90% after lyophilization and storage of the lyophilized product for up to 3 days, compared to the same solution before lyophilization.

[0170] 45. The solution according to item 44, wherein the RNase inhibitory activity is at least 95%.

[0171] 46. ​​A solution that can be obtained, in particular, by the method according to item 43, wherein the RNase inhibitory activity of the solution is at least 75% after the lyophilized product is stored at 35°C for 3 weeks, compared to the same solution before lyophilization.

[0172] 47. The solution according to item 46, wherein the RNase inhibitory activity is at least 77%.

[0173] 48. The solution according to item 46 or 47, wherein the RNase inhibitory activity is at least 80%.

[0174] 49. A solution that can be obtained, in particular, by the method according to item 43, wherein, compared with a second solution containing the same amount of the same components after being stored at -20°C for 7 months, the RNase inhibitory activity of the solution is at least 77% after lyophilization and storage of the lyophilized product for up to 3 days and after storage of the solution for 6 months.

[0175] 50. A solution that can be obtained, in particular, by the method according to item 43, wherein, compared with a second solution containing the same amount of the same components after being stored at -20°C for 7 months, the RNase inhibitory activity of the solution is at least 90% after being stored at 35°C for 3 weeks and subsequently at -20°C for 5 months.

[0176] 51. Use of the lyophilized, biocompatible solution according to any one of items 1 to 28, the solution according to any one of items 44 to 50, the lyophilized product according to any one of items 29 to 40, or the kit according to item 41 for inhibiting RNase.

[0177] 52. The use according to item 51, wherein the use includes adding the lyophilized ready biocompatible solution or lyophilized product of the present invention to a composition, wherein the composition contains RNase or is at risk of RNase contamination.

[0178] 53. Use of a lyophilized-ready biocompatible solution for producing lyophilized products containing activeRNaseInh(-SH)n, particularly for producing lyophilized products according to any one of items 29 to 40, wherein the lyophilized-ready biocompatible solution contains activeRNaseInh(-SH)n and tris(2-carboxyethyl)phosphine (TCEP).

[0179] 54. As described in item 53, the solution has a pH of 5.0 to 9.0 at 4°C, typically 7.0 to 8.0 at 4°C, and typically 7.6 at 4°C.

[0180] 55. As described in item 53 or 54, the solution is free of DTT and / or glutathione.

[0181] 56. The use according to any one of items 53 to 55, wherein the concentration of TCEP is 1 to 20 mM, typically 5 to 10 mM, typically 5 mM.

[0182] 57. The use according to any one of items 53 to 56, wherein the solution further comprises a buffer.

[0183] 58. As described in item 57, the buffer is selected from the group consisting of: TES, MOPS, HEPES, Tris, phosphates and combinations thereof, typically HEPES.

[0184] 59. As described in item 57 or 58, wherein the concentration of the buffer is 5 to 100 mM, typically 10 to 50 mM, typically 20 mM.

[0185] 60. The use according to any one of items 53 to 59, wherein the solution further comprises a monovalent salt.

[0186] 61. According to the uses described in item 60, the monovalent salt is NaCl and / or KCl.

[0187] 62. As described in item 60 or 61, the concentration of the monovalent salt is 1 to 500 mM, typically 5 to 300 mM, typically 5 to 250 mM, typically 50 to 150 mM, typically 100 mM.

[0188] 63. The use according to any one of items 53 to 62, wherein the solution further comprises trehalose.

[0189] 64. As described in item 63, the concentration of trehalose is 10% to 40% (w / v), typically 15% to 35% (w / v), typically 20% to 30% (w / v).

[0190] 65. The use according to any one of items 53 to 64, wherein the solution further comprises a nonionic surfactant.

[0191] 66. As described in item 65, the nonionic surfactant is selected from the group consisting of: Triton X-100, polysorbate 20, polysorbate 60, polysorbate 80, and combinations thereof.

[0192] 67. As described in item 65 or 66, wherein the concentration of the nonionic surfactant is 0.005% to 0.5% (v / v), typically 0.005% to 0.2% (v / v), typically 0.01% to 0.1% (v / v), typically 0.05% to 0.1% (v / v).

[0193] 68. The use according to any one of items 53 to 67, wherein the solution further comprises TCEP, HEPES, KCl, trehalose and polysorbate 20.

[0194] 69. The use according to any one of items 53 to 68, wherein the solution comprises 1 to 20 mM TCEP, 5 to 100 mM buffer, 5 to 300 mM monovalent salt, 10% to 40% (w / v) trehalose and 0.005% to 0.5% (v / v) nonionic surfactant.

[0195] 70. The use according to any one of items 53 to 69, wherein the solution comprises 5 mM TCEP, 20 mM buffer, 100 mM monovalent salt, 20% to 30% (w / v) trehalose and 0.01% to 0.1% (v / v) nonionic surfactant.

[0196] 71. The use according to any one of items 53 to 70, wherein the solution comprises 5 mM TCEP, 20 mM HEPES, 100 mM KCl, 20% to 30% (w / v) trehalose and 0.01% to 0.1% (v / v) polysorbate 20.

[0197] 72. The use according to any one of items 53 to 71, wherein activeRNaseInh(-SH)n is an RNase inhibitor derived from brown rats.

[0198] 73. The use according to any one of items 53 to 72, wherein activeRNaseInh(-SH)n comprises or consists of the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least 85% sequence identity.

[0199] 74. The use according to any one of items 53 to 72, wherein activeRNaseInh(-SH)n comprises or consists of the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least 90% sequence identity.

[0200] 75. The use according to any one of items 53 to 72, wherein activeRNaseInh(-SH)n comprises or consists of the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least 95% sequence identity.

[0201] 76. The use according to any one of items 53 to 72, wherein activeRNaseInh(-SH)n comprises or is composed of the amino acid sequence of SEQ ID NO: 1.

[0202] 77. The use according to any one of items 53 to 76, wherein the solution contains at least 30 units of activeRNaseInh(-SH)n per µl, typically at least 40 units of activeRNaseInh(-SH)n per µl, typically at least 100 units of activeRNaseInh(-SH)n per µl, typically at least 200 units of activeRNaseInh(-SH)n per µl, typically at least 300 units of activeRNaseInh(-SH)n per µl, typically at least 400 units of activeRNaseInh(-SH)n per µl.

[0203] sequence list

[0204] Example

[0205] The following examples are provided to illustrate, rather than limit, the inventions claimed herein.

[0206] Example 1

[0207] Four different formulations containing a rat-derived RNase inhibitor (SEQ ID NO: 1) were freshly prepared and evaluated: 20% or 30% trehalose and 5 mM DTT or TCEP, respectively.

[0208] Final buffer formulation: 20 mM Hepes, 100 mM KCl, 20% or 30% Trehalose, 5 mM DTT or TCEP, 0.01% Tween 20, pH 7.60 / 4.0℃ The inhibition of RNase A by RNase inhibitors is measured by their ability to inhibit the hydrolysis of cyclic 2',3'-CMP, which is the substrate (Blackburn P.: Ribonuclease Inhibitor from Human Placenta: Rapid Purification and Assay. J Biol Chem. 1979 Dec 25;254(24):12484-7).

[0209] The inhibition of RNase A by RNase inhibitors was measured in OD using photometers (UVIKON 930 and higher, Kontron Instruments; and Cary 1, Varian). 286 The following will proceed.

[0210] Add 0.3 ml of Tris / acetate buffer (1 M Tris / acetate, pH 6.5, 0.2 mM EDTA), 0.3 ml of 10 mM cyclic 2',3'-CMP H2O solution, and bring the volume up to a final volume of 2.940 ml to a 3 ml cuvette (quartz / plastic).

[0211] The components were mixed in a cuvette and incubated in a photometer for 5 min. The reaction was then initiated by adding 3 µg of RNase A and the solution was mixed again.

[0212] The photometer was connected to a 25°C water bath to maintain a constant temperature of 25°C. The temperature was checked before and after each measurement. The temperature was 25°C before adding RNase A.

[0213] The hydrolysis rate of cyclic 2',3'-CMP was determined by the rate of increase in absorption at 286 nm. Therefore, ΔE at 286 nm was measured over 10 min, and the results were used to determine ΔE per min.

[0214] The amount of inhibitor measured was determined by the percentage of inhibition of RNase A contained in the assay.

[0215] In assays using cyclic 2',3'-CMP as a substrate, the inhibition of RNase A by different concentrations of inhibitor was linear with respect to the inhibitor concentration. Therefore, the active units in the sample can be estimated using a portion of the inhibitor solution via linear interpolation (see Blackburn's publication above).

[0216] For the evaluation, only values ​​that reduce ΔE to 75.0% to 50.0% are used.

[0217] By definition, 1 U is defined as the amount of protein required to inhibit 50% of RNase A. Therefore, 200 U = 1 μg RNase A, and 600 U = 3 µg RNase A is inhibited by 50%.

[0218] Stability monitoring of RNA enzyme preparations

[0219] Volumetric activity was measured according to Blackburn’s method (see above), with or without stress testing at 35°C and / or lyophilization of the samples.

[0220] The volumetric activity of RNase inhibitor formulations containing different amounts of trehalose and DTT or TCEP as reducing agents was measured in liquid form under unstressed conditions (see Table 1, column A), in liquid form before stress treatment at 35°C for 3 weeks (see Table 1, column B), after overnight lyophilization and reconstitution the next day (see Table 1, column C), or after lyophilization and stress treatment at 35°C for 3 weeks (see Table 1, column D). Lyophilization was performed using a Christ Beta 2-8 LD Plus freeze dryer under standard conditions using 1.5 ml vials according to the manufacturer's instructions.

[0221] Table 1:

[0222] The RNase inhibitors were stable under all three conditions tested, with the formulation containing TCEP (compared to the same formulation containing DTT): liquid storage at 35°C, after lyophilization, and lyophilized formulation stored at 35°C.

[0223] Example 2

[0224] Sucrose is commonly used as a freeze-drying protectant. Therefore, the use of sucrose instead of trehalose was also tested. However, formulations containing sucrose are unstable under stress. Two formulations containing the RNase inhibitor and sucrose used in Example 1, S1 (20 mM Hepes, 100 mM potassium chloride, 20% sucrose, 5 mM TCEP, 0.05% Tween 20, pH 7.6 @ 4℃) and S2 (20 mM Hepes, 100 mM potassium chloride, 20% sucrose, 5 mM DTT, 0.05% Tween 20, pH 7.6 @ 4℃), were lyophilized and subsequently stored at 35℃ for 3 weeks. In both formulations S1 and S2, the lyophilized products were unstable, but clumping occurred in both formulations. Reconstitution of the lyophilized products from both formulations also required more time. Furthermore, turbidity was observed in formulation S2 (see [link to example]). Figure 1 ).

[0225] Compared to sucrose with DTT or TCEP, the combination of trehalose and TCEP maintains formulation stability because there is no clumping of the lyophilized material or turbidity in the reconstituted solution, and because the lyophilized material can be reconstituted more easily.

[0226] Example 3

[0227] To compare the stability of RNase inhibitor activity during long-term stress storage of formulations containing the RNase inhibitor used in Example 1 and formulations containing TCEP or DTT, two formulations—one containing TCEP (Formulation A: 20 mM Hepes, 100 mM potassium chloride, 20% trehalose, 5 mM TCEP, 0.01% Tween 20, pH 7.6 @ 4℃) and the other containing DTT (Formulation B: 20 ​​mM Hepes, 100 mM potassium chloride, 20% trehalose, 5 mM DTT, 0.01% Tween 20, pH 7.6 @ 4℃)—were subjected to different stress conditions. Condition I included lyophilization and reconstitution the following day, followed by storage of the reconstituted solution at -20℃ for 6 months; Condition II included lyophilization, storage of the lyophilized product at 35℃ for 3 weeks, followed by storage of the lyophilized product at -20℃ for 5 months and reconstitution. Solutions subjected to conditions I and II were derived from the same solution, while the remaining solutions were stored at -20°C for a total of 7 months without any stress (control condition) and used for comparison with solutions subjected to long-term stress conditions. Activity was measured as described in Example 1.

[0228] Table 2:

[0229] The residual activity results show that formulations containing DTT performed significantly worse under stress than those containing TCEP, which retained over 90% of its activity even after lyophilization, high-temperature storage, and subsequent freezing (see Table 2). The specific activity of formulations containing TCEP was also higher than that of formulations containing DTT. This indicates that TCEP can better stabilize the activity of RNase inhibitors, making it suitable not only for short-term storage but also for long-term storage, especially under stress.

Claims

1. A lyophilized, biocompatible solution comprising... (i) An active RNase inhibitor containing at least one cysteine ​​residue having a reduced thiol group, and (ii) Tris(2-carboxyethyl)phosphine (TCEP).

2. The lyophilization-ready biocompatible solution according to claim 1, used to produce lyophilized products.

3. The freeze-dried, biocompatible solution according to claim 1 or 2, wherein the solution is free of dithiothreitol (DTT) and / or glutathione.

4. The lyophilized, biocompatible solution according to any one of claims 1 to 3, wherein the solution further comprises a buffer, a monovalent salt, trehalose, and / or a nonionic surfactant.

5. The lyophilization-ready biocompatible solution of any one of claims 1 to 4, wherein the active RNase Inh(-SH)n is a RNase inhibitor from Brown rat Angelman Syndrome.

6. A lyophilized product comprising active RNase Inh(-SH)n and TCEP.

7. The lyophilized product according to claim 6, further comprising a buffer, a monovalent salt, trehalose, and / or a nonionic surfactant.

8. A kit comprising, in a container, at least one lyophilized ready biocompatible solution according to any one of claims 1 to 5 or at least one lyophilized product according to claim 6 or 7.

9. A method for producing a solution containing active RNase Inh(-SH)n and TCEP, wherein the method comprises the following steps: Reconstitute the lyophilized product according to claim 6 or 7.

10. A solution obtainable by the method according to claim 9, wherein the RNase inhibitory activity of the solution is at least 90% after lyophilization and storage of the lyophilized product for up to 3 days, compared to the same solution before lyophilization.

11. A solution obtainable by the method according to claim 9, wherein the RNase inhibitory activity of the solution is at least 75% after the lyophilized product is stored at 35°C for 3 weeks, compared to the same solution before lyophilization.

12. Use of the lyophilized ready biocompatible solution according to any one of claims 1 to 5, the lyophilized product according to claim 6 or 7, or the kit according to claim 8 for inhibiting RNase.

13. The use according to claim 12, wherein the use includes adding the lyophilized ready biocompatible solution or lyophilized product of the present invention to the composition, wherein the composition contains RNase or is at risk of RNase contamination.

14. A lyophilized-ready biocompatible solution for producing lyophilized products containing activeRNaseInh(-SH)n, particularly for producing lyophilized products according to claim 6 or 7, wherein the lyophilized-ready biocompatible solution contains activeRNaseInh(-SH)n and tris(2-carboxyethyl)phosphine (TCEP).

15. The use according to claim 14, wherein the solution further comprises a buffer, a monovalent salt, trehalose, and / or a nonionic surfactant.

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