Compositions comprising an rnase inhibitor

Abstract

The present invention relates to lyo-ready biocompatible solutions and lyophilisates comprising an activeRNaseInh(-SH)n, kits comprising solutions and / or lyophilisates of the present invention and methods of producing solutions and / or lyophilisates of the present invention. The present invention further relates to the use of the herein disclosed solutions, kits and / or lyophilisates for inhibiting an RNase.

Description

[0001] COMPOSITIONS COMPRISING AN RNASE INHIBITOR

[0002] Field of the invention

[0003] The present invention relates to lyo-ready biocompatible solutions and lyophilisates comprising an activeRNase!nh(-SH)n, kits comprising solutions and / or lyophilisates of the present invention and methods of producing solutions and / or lyophilisates of the present invention. The present invention further relates to the use of the herein disclosed solutions, kits and / or lyophilisates for inhibiting an RNase.

[0004] Background of the invention

[0005] RNases are universally present and when working with RNA, for example, reverse transcribing RNA to produce cDNA via RT-PCR, it is necessary to prevent degradation of RNA by these RNases.

[0006] RNase inhibitors are proteins that can be used when working with RNA to inhibit RNases. Most RNase inhibitors irreversibly inhibit RNases and thus can protect RNA from degradation by RNases. One possibility to increase storage stability and / or decrease storage costs and certain requirements for specialized compositions is freeze-drying, also known as lyophilisation. Storage in form of a lyophilisate, i.e. in the form of a powder, increases stability. However, freeze-drying itself stresses proteins and may lead to a loss of activity by degradation and / or precipitation. A common cryoprotectant in aqueous compositions is glycerol which is capable of preventing damage to proteins during freezing. However, glycerol interferes with any freeze-drying process due to a low glass transition temperature. In order to provide aqueous compositions with an RNase inhibitor which can be freeze-dried, lyo-ready compositions that stabilize the contained RNase inhibitor during the lyophilisation process are desired. Further, it is desired that the RNase inhibitor is active after resolubilisation of a lyophilisate obtainable from the desired compositions, such that the resolubilised RNase inhibitor is again capable of protecting RNA from degradation. RNase inhibitors typically contain reduced cysteine residues, and therefore during production and storage a reducing agent is required to keep the RNase inhibitors active and prevent oxidation of the cysteines. Maintaining the sulfhydryl (-SH) groups of cysteine residues in their reduced state increases the stability of the RNase inhibitors. Commonly, dithiothreitiol (DTT) is used as the standard reducing agent in biochemistry because it is cheap and it does not form mixed-disulfide species, in contrast to, for example, glutathione. In addition, DTT is deemed more convenient because it does not smell as strongly as other reducing agents, particularly betamercaptoethanol.

[0007] WO 2022 / 114981 Al discloses compositions with recombinant human RNase inhibitor which are reported to be suitable for lyophilisation and freeze-dried preparations thereof. Disclosed ingredients do not contain glycerol but buffer, salt and DTT as reducing agent.

[0008] In view of the desire to provide improved solutions that can be used to improve production of freeze-dried preparations with an RNase inhibitor, the so far known technical solutions only provide compositions which have disadvantages. Among these, insufficient stabilization of RNase inhibitor activity in a freeze-dried preparation is of particular importance.

[0009] Thus, there remains a need to provide compositions, such as solutions or lyophilisates, comprising an RNase inhibitor with an increased stability of its activity, in particular when stored for longer time and / or when exposed to stressors, such as increased temperature and / or lyophilisation.

[0010] Summary of the invention

[0011] In a first aspect, the present invention relates to a lyo-ready biocompatible solution comprising

[0012] (i) an active RNase inhibitor comprising at least one cysteine residue with a reduced sulfhydryl group (activeRNaselnh(-SH)n) and

[0013] (ii) tris(2-carboxyethyl)phosphine (TCEP). The inventors have surprisingly found that, generally, the stability of the activity of RNase inhibitors in lyo-ready biocompatible solutions can be increased by including tris(2-carboxyethyl)phosphine (TCEP) in the solution. In particular this applies to solutions intended to be used for freeze-drying. As shown in the examples, the stability of the activity of an RNase inhibitor exposed to different stressors is higher in compositions comprising TCEP than in compositions comprising DTT. The latter is known to the skilled person as the standard reducing agent in biochemistry.

[0014] In a second aspect, the present invention relates to a lyo-ready biocompatible solution consisting of an activeRNaseInh(-SH)n, a reducing agent, a buffering agent, a monovalent salt, trehalose, a nonionic surfactant and water, wherein the reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

[0015] In a third aspect, the present invention relates to a lyophilisate comprising an activeRNaseInh(-SH)n and TCEP.

[0016] In a fourth aspect, the present invention relates to a kit comprising in a container at least one lyo-ready biocompatible solution of the first or second aspect, or at least one lyophilisate of the third aspect.

[0017] In a fifth aspect, the present invention relates to a method of producing a lyophilisate comprising an activeRNaseInh(-SH)n, wherein the method comprises the step of lyophilising a lyo-ready biocompatible solution comprising an activeRNaseInh(-SH)n and TCEP, in particular a lyo-ready biocompatible solution of the first or second aspect.

[0018] In a sixth aspect, the present invention relates to a method of producing a solution comprising an activeRNaseInh(-SH)n and TCEP, wherein the method comprises the step of resolubilising a lyophilisate of the third aspect or obtainable, in particular obtained, by a method of the fifth aspect.

[0019] In a seventh aspect, the present invention relates to a solution obtainable, in particular obtained, by a method of the sixth aspect, wherein the RNase inhibition activity of the solution is at least 90 % after lyophilisation and a storage of the lyophilisate for at most 3 days compared to the same solution before lyophilisation.

[0020] In an eighth aspect, the present invention relates to a solution obtainable, in particular obtained, by a method of the sixth aspect, wherein the RNase inhibition activity of the solution is at least 75 % after 3 weeks of storage of the lyophilisate at 35 °C compared to the same solution before lyophilisation.

[0021] In a ninth aspect, the present invention relates to a solution obtainable, in particular obtained, by a method of the sixth aspect, wherein the RNase inhibition activity of the solution is at least 77 % after lyophilisation and a storage of the lyophilisate for at most 3 days and storage of the solution for 6 months compared to a second solution comprising the same ingredients at the same amounts after storage of the second solution at -20 °C for 7 months.

[0022] In a tenth aspect, the present invention relates to a solution obtainable, in particular obtained, by a method of the sixth aspect, wherein the RNase inhibition activity of the solution is at least 90 % after 3 weeks of storage of the lyophilisate at 35 °C and subsequent 5 months of storage of the lyophilisate at -20 °C compared to a second solution comprising the same ingredients at the same amounts after storage of the second solution at -20 °C for 7 months.

[0023] In an eleventh aspect, the present invention relates to the use of a lyo-ready biocompatible solution of the first, second, a solution of the seventh, eighth, ninth or tenth aspect, a lyophilisate of the third aspect or a kit of the fourth aspect for the inhibition of an RNase.

[0024] In a twelfth aspect, the present invention relates to the use of a lyo-ready biocompatible solution for producing a lyophilisate comprising an activeRNaseInh(-SH)n, in particular of the third aspect, wherein the lyo-ready biocompatible solution comprises an activeRNaseInh(-SH)n and tris(2- carboxyethyl)phosphine (TCEP). List of figures

[0025] FIG. 1 shows a photograph of two solutions comprising an RNAse Inhibitor, formulation SI (left) and formulation S2 (right), obtained by resolubilisation of previously stressed lyophilisates, wherein a clouding is visible in formulation S2.

[0026] Detailed Description of the invention

[0027] Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular embodiments and examples described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0028] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

[0029] In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise. The following definitions and embodiments apply to the present disclosure in its entirety, especially to all aspects and embodiments of the invention.

[0030] As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents, unless the content clearly dictates otherwise.

[0031] The word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0032] The use of the alternative (e.g. “or”) should be understood to mean either one, both or any combination thereof of the alternatives.

[0033] The term “and / or” should be understood to mean either one or both of the alternatives.

[0034] Percentages, concentrations, amounts and other numerical data may be expressed or presented herein in a “range” format. It is to be understood that such a range format is used merely for convenience and brevity, and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of "4 % to 20 %" should be interpreted to include not only the explicitly recited values of 4 % to 20 %, but to also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 4, 5, 6, 7, 8, 9, 10, ... 18, 19, 20 % and sub-ranges such as from 4-10 %, 5-15 %, 10-20 %, etc. This same principle applies to ranges reciting minimal or maximal values. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.

[0035] As used herein and unless stated otherwise, it is to be understood that the term “about” is used synonymously with the term “approximately”. Illustratively and unless stated otherwise, the use of the term “about” when used in conjunction with a stated numerical value or range denotes somewhat more or somewhat less than the stated value or range, to within a range of ±15% of that stated, ±10% of that stated, ±5% of that stated, or conveniently ± 2% of that stated. Such values are thus encompassed by the scope of the claims reciting the terms “about” or “approximately”.

[0036] The term “solution” as used herein refers to a composition comprising one or more solvent(s), wherein each solvent is in the liquid state of matter under ambient conditions, and one or more solutes, wherein each solute in the absence of a solvent is in the solid or gaseous state of matter under ambient conditions. Examples of solvents include water, ethanol, glycerol, DMSO (Dimethyl sulfoxide) or THF (T etrahydrofuran) .

[0037] A “biocompatible” solution is a solution that comprises water and one or more solutes in homogeneous mixture, wherein each solute is present at a particular concentration, wherein all compounds of the solution together are suited to provide conditions capable of supporting and / or preserving and / or maintaining structural integrity, activity and / or function of a given biomolecule, such as polypeptide or nucleic acid. Such conditions vary depending on the respective biomolecule under consideration. Specifically, active polypeptide compounds such as but not limited to enzymes and compounds involved in intramolecular interaction (e.g. receptors, ligands, activators, inhibitors, etc.) might require, depending on the enzyme, in the biocompatible solution a certain pH which is advantageously buffered, one or more salts and frequently other additive(s), too. Examples for additives are surfactants and compounds to maintain in the solution a certain redox potential.

[0038] A “lyo-ready” solution is an aqueous solution which can be lyophilized so that a solid cake can be obtained. In particular, it refers to a solution which is substantially free of or which contains at most 0.5 % (w / v), typically at most 0.1 % (w / v), of a lyo-inhibiting solvent, or is substantially free of various lyo-inhibiting solvents or each at a maximum of 0.5 % (w / v). A “lyo-inhibiting solvent” is understood to refer to a solvent that differs substantially from water with respect to either or both its boiling point and vapour pressure. In an embodiment under ambient conditions the boiling point of the lyo-inhibiting solvent is 0.01 % to 10 % higher than that of water, in a specific embodiment it is 0.1 % to 5 % higher than that of water, and in a more specific embodiment it is 0.1% to 1% higher than that of water. In another embodiment under ambient conditions the vapour pressure of the lyo-inhibiting solvent is 0.01 % to 10 % less than that of water, in a specific embodiment it is 0.1 % to 5 % less than that of water, and in a more specific embodiment it is 0.1% to 1% less than that of water. Examples of lyo-inhibiting solvents include glycerol and other polyols, or some ionic liquids. A lyo-ready solution is in particular a glycerol- free solution.

[0039] The term "glycerol-free" refers to a property of a composition, e.g. a solution or a lyophilisate, and refers that the composition does comprise at most 0.5 % (w / v), typically at most 0.1 % (w / v), glycerol. Glycerol as a cryoprotectant is commonly used to increase the stability of proteins, such as enzymes, during storage. However, it may also interfere with lyophilisation. It is therefore recommended to avoid glycerol in compositions that are to be lyophilised.

[0040] A “lyophilisate” is a powder with a low water content. Typically the water content of a lyophilisate 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).

[0041] As used in the present disclosure, "% (w / v)" refers to weight by volume percent, which is a unit of concentration measuring the amount of solute, for example, in grams (g), expressed as a percent of the total volume of solution, for example, in milliliters (ml).

[0042] As used in the present disclosure, "% (v / v)" refers to volume by volume percent, which is a unit of concentration measuring the amount of a specific solute, for example, in milliliters (ml) expressed as a percent of the total volume of solution, for example, in milliliters (ml).

[0043] As used in the present disclosure, "% (w / w)" refers to weight by weight percent, which is a unit of measuring the amount of a specific substance, for example, in grams (g) expressed as a percent of the total weight of a mixture, for example, in gram (g), which can be a powder or solution.

[0044] The term "lyophilising" or "lyophilisation" refers to the freeze-drying of a substance by freezing it and then reducing the surrounding pressure (e.g. below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or less) to allow the frozen medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilising" and "freeze-drying" are used herein interchangeably.

[0045] The terms "reconstitute" or “resolubilise” relate to adding a solvent, such as water, to a dried product, such as a lyophilisate, to return it to a liquid state such as its original liquid state.

[0046] The term "recombinant" in the context of the present disclosure means "made through genetic engineering". In some embodiments, a "recombinant protein" in the context of the present disclosure is not occurring naturally.

[0047] As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and refer to temperatures from at least about 15 °C, e.g. 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.

[0048] As used herein, the term “ambient conditions” refers to a condition with an ambient temperature and an atmospheric pressure of around 1013 ± 50 hPa.

[0049] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring. The term "found in nature" means "present in nature" and includes known objects as well as objects that have not yet been discovered and / or isolated from nature, but that may be discovered and / or isolated in the future from a natural source. The term “native” refers to a naturally occurring object.

[0050] According to the present invention, the term "peptide" refers to molecules which comprise about two or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150, consecutive amino acids linked to one another via peptide bonds. The term "polypeptide" refers to large peptides, in particular peptides having at least about 151 amino acids. "Peptides" and "polypeptides" are both protein molecules. Thus, the terms "peptide", "protein" and "polypeptide" are used herein usually as synonyms.

[0051] For the purposes of the present invention, "variants" of an amino acid sequence (peptide or polypeptide) may comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, in particular those which are naturally occurring. The term "variant" includes, in particular, fragments of an amino acid sequence. Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid addition variants comprise amino- and / or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein. Amino acid deletion variants that comprise the deletion at the N-terminal and / or C-terminal end of the protein are also called N-terminal and / or C- terminal truncation variants. Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous peptides or polypeptides and / or to replacing amino acids with other ones having similar properties. In some embodiments, amino acid changes in peptide and polypeptide variants are conservative amino acid changes, i.e. substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In some embodiments, conservative amino acid substitutions include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0052] "Sequence similarity" indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences. An alignment for determining sequence similarity, such as sequence identity can be done with art known tools, such as using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS ::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0053] The terms "% identical" and "% identity" or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or "window of comparison", in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of algorithms, e.g. the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, the local homology algorithm by Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website. In some embodiments, the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match / Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. In some embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment.

[0054] Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g. the number of positions in the reference sequence) and multiplying this result by 100.

[0055] The amino acid sequence variants described herein may readily be prepared by the skilled person, for example, by recombinant DNA manipulation. The manipulation of DNA sequences for preparing peptides or polypeptides having substitutions, additions, insertions or deletions, is described in detail in Molecular Cloning: A Laboratory Manual, 4th Edition, M.R. Green and J. Sambrook et al. (1989), eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012, for example. Furthermore, the peptides, polypeptides and amino acid variants described herein may be readily prepared with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar methods. In some embodiments, a fragment or variant of an amino acid sequence (peptide or polypeptide) is a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e. it is functionally equivalent. With respect to sequences of binding agents such as antibodies, one particular function is one or more binding activities displayed by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant", as used herein, in particular refers to a variant molecule or sequence that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that is still capable of fulfilling one or more of the functions of the parent molecule or sequence, e.g. binding to a target molecule. In some embodiments, the modifications in 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, e.g. 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 the parent molecule or sequence. However, in other embodiments, function of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0056] According to various embodiments of the present disclosure, a nucleic acid encoding a peptide or polypeptide is taken up by or introduced, i.e. transfected or transduced, into a cell which cell may be present in vitro or in a subject, resulting in expression of said peptide or polypeptide. The cell may, e.g. 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 the surface. According to the present disclosure, terms such as "nucleic acid expressing" and "nucleic acid encoding" or similar terms are used interchangeably herein and with respect to a particular peptide or polypeptide mean that the nucleic acid, if present in the appropriate environment, e.g. within a cell, can be expressed to produce said peptide or polypeptide.

[0057] The term "expression" as used herein includes the transcription and / or translation of a particular nucleotide sequence.

[0058] In the context of the present disclosure, the term "transcription" relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA (especially mRNA). Subsequently, the RNA may be translated into peptide or polypeptide. With respect to RNA, the term "expression" or "translation" relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or polypeptide.

[0059] The term "DTT- and / or glutathione-free" refers to a property of a composition, e.g. a solution or a lyophilisate, and refers that the composition does comprise at most 0.5 mM, typically at most 0.1 mM, typically at most 0.01 mM, DTT- and / or glutathione.

[0060] An “active” RNase Inhibitor according to the present invention is an RNase Inhibitor that is capable of inhibiting the degradation of RNA in solution. The amount of activity can be measured in the form of units. In particular, an RNase inhibitor comprising at least one cysteine in its polypeptide chain and 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 sulfhydryl form. The term “activeRNase!nh(-SH)n” refers to an active RNase inhibitor with n cysteines, wherein the cysteines are reduced and n is one or more cysteines.

[0061] The terms "unit" or “international unit” with respect to an RNase inhibitor refers to the amount of RNase inhibitor required to inhibit an RNase. One unit of an RNase inhibitor is that amount required to inhibit by 50 % the activity of 5 ng 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 units comprised in a solution, can be determined by measuring the inhibition of hydrolysis of 2',3'-cyclic cytidine monophosphate, for example as described by Peter Blackburn (see Blackburn P., Ribonuclease Inhibitor from Human Placenta: Rapid Purification and Assay, J Biol Chem, Vol. 254, No. 24, pp. 12484 - 12487, 1979).

[0062] The term “stability of the activity” refers to the property of a solution or a lyophilisate that can prevent at least partially the loss of active RNase inhibitor in the solution or lyophilisate for a certain time. The extent to which a solution or lyophilisate has to prevent the loss of active RNase inhibitor in order to stabilize the activity of an RNase inhibitor depends on the conditions to which the solution is exposed. For example, a solution with 100 units of an RNase inhibitor is thought to stabilize the activity of the RNase inhibitor when the activity of the RNase inhibitor is at, e.g, 80 units in a solution with the same volume obtained after resolubilisation of a lyophilisate obtained from freeze-drying of the solution with 100 units and storage of the lyophilisate for 3 weeks at ambient conditions.

[0063] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0064] In a first aspect, the present invention relates to a lyo-ready biocompatible solution comprising

[0065] (i) an active RNase inhibitor comprising at least one cysteine residue with a reduced sulfhydryl group (activeRNaselnh(-SH)n) and

[0066] (ii) tris(2-carboxyethyl)phosphine (TCEP).

[0067] In some embodiments of the present invention, the lyo-ready biocompatible solution is an aqueous solution. In some embodiments of the present invention, the lyo-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) other solvents.

[0068] In some embodiments of the present invention, the lyo-ready biocompatible solution is for use in the production of a lyophilisate. In some embodiments of the present invention, the lyo-ready biocompatible solution is for use in the production of a lyophilisate comprising an activeRNaseInh(-SH)n and TCEP.

[0069] In some embodiments of the present invention, the RNase inhibition activity of the lyo-ready biocompatible solution is at least 90 % after 3 weeks of storing the solution at 35 °C compared to the same solution before storage.

[0070] In some embodiments of the present invention, the RNase inhibition activity is measured by measuring the RNase inhibition activity by a method known to the skilled person, in particular a method according to Blackburn P., Ribonuclease Inhibitor from Human Placenta: Rapid Purification and Assay, J Biol Chem, Vol. 254, No. 24, pp. 12484 - 12487, 1979. In some embodiments of the present invention, the lyo-ready biocompatible solution has a pH of 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 lyo-ready biocompatible solution is dithiothreitol (DTT)- and / or glutathione-free.

[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 present invention, the lyo-ready biocompatible solution further comprises a buffering agent.

[0074] In some embodiments of the present invention, the buffering agent is selected from the group consisting of TES (2-{[l,3-Dihydroxy-2-(hydroxymethyl)propan-2- yl] amino} ethane- 1 -sulfonic acid), MOPS (3-(7V-morpholino)propanesulfonic acid), HEPES (4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid), Tris

[0075] (tris(hydroxymethyl)aminomethane), phosphate and combinations thereof. In some embodiments of the present invention, the buffering agent is HEPES. In some embodiments of the present invention, the concentration of the buffering agent is 5 to 100 mM, 10 to 50 mM or 20 mM.

[0076] In some embodiments of the present invention, the lyo-ready biocompatible solution further comprises a salt.

[0077] In some embodiments of the present invention, the lyo-ready biocompatible solution further comprises a monovalent salt.

[0078] In some embodiments of the present invention, the monovalent salt is NaCl and / or KC1. In some embodiments of the present invention, the monovalent salt is NaCl or KC1. In some embodiments of the present invention, the monovalent salt is NaCl. In some embodiments of the present invention, the monovalent salt is KC1. 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. In some embodiments of the present invention, the lyo-ready biocompatible solution further comprises trehalose. In some embodiments of the present invention, the concentration of trehalose is 10 to 40 % (w / v), 15 to 35 % (w / v) or 20 to 30 % (w / v). Trehalose, in comparison to other carbohydrates, in particular sucrose, may help increase the stability of the activity of an RNase inhibitor in the compositions of the present invention further.

[0079] In some embodiments of the present invention, the lyo-ready biocompatible solution further comprises a nonionic surfactant. In some embodiments of the present invention, the nonionic surfactant is selected from the group consisting of triton X- 100, polysorbate 20, polysorbate 60, polysorbate 80, polydocanol and combinations thereof. In some embodiments of the present invention, the nonionic surfactant is polysorbate 20. In some embodiments of the present 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 present invention, the concentration of the nonionic surfactant is 0.05 to 0.1 % (v / v).

[0080] In some embodiments of the present invention, the lyo-ready biocompatible solution comprises an activeRNaseInh(-SH)n, tris(2-carboxyethyl)phosphine (TCEP), a buffering agent, a salt, trehalose and water.

[0081] In some embodiments of the present invention, the lyo-ready biocompatible solution comprises an activeRNaseInh(-SH)n, tris(2-carboxyethyl)phosphine (TCEP), a buffering agent, a salt, trehalose, a nonionic surfactant and water.

[0082] In some embodiments of the present invention, the lyo-ready biocompatible solution comprises an activeRNaseInh(-SH)n, tris(2-carboxyethyl)phosphine (TCEP), a buffering agent, a monovalent salt, trehalose, a nonionic surfactant and water.

[0083] In some embodiments of the present invention, the lyo-ready biocompatible solution comprises TCEP, HEPES, KC1, trehalose and polysorbate 20.

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

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

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

[0087] In some embodiments of the present invention, the lyo-ready biocompatible solution comprises 5 mM TCEP, 20 mM HEPES, 100 mM KC1, 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 lyo-ready biocompatible solution comprises 5 mM TCEP, 20 mM HEPES, 100 mM KC1, 20 to 30 % (w / v) trehalose and 0.05 to 0.1 % (v / v) polysorbate 20.

[0089] In some embodiments of the present invention, the activeRNaseInh(-SH)n is an RNase inhibitor from Mus musculus, Capra hircus, Xenopus laevis, Homo sapiens or Rattus norvegicus. In some embodiments of the present invention, the activeRNaseInh(-SH)n is an RNase inhibitor from Hattus norvegicus.

[0090] In some embodiments of the present invention, the activeRNaseInh(-SH)n is a recombinant protein. In some embodiments of the present invention, the 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 proteintag, avi-tag and strep-tag. The tag might be connected to the activeRNaseInh(-SH)n with a linker, in particular a linker cleavable by a protease to remove the tag. The activeRNaseInh(-SH)n included in solutions or lyophilisates of the present invention may be obtained by methods known to the skilled person. In some embodiments of the present invention, the activeRNaseInh(-SH)n included in solutions or lyophilisates of the present invention may be obtained by expression in prokaryotic or eukaryotic expression system. The prokaryotic or eukaryotic expression systems may comprise a nucleic acid coding for an activeRNase!nh(-SH)n. The activeRNase!nh(-SH)n may be purified by methods known to the skilled person. For example, the activeRNase!nh(-SH)n may be purified using crystallization, affinity chromatography, ion exchange chromatography and / or size exclusion chromatography.

[0091] In some embodiments of the present invention, the activeRNaseInh(-SH)n comprises or 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, the activeRNaseInh(-SH)n comprises or consists of the amino acid sequence of SEQ ID NO: 1.

[0092] In some embodiments of the present invention, the 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, the activeRNaseInh(-SH)n consists of the amino acid sequence of SEQ ID NO: 1.

[0093] In some embodiments of the present invention, the solution comprises at least 30 units of the activeRNaseInh(-SH)n per pl, at least 40 units of the activeRNaseInh(-SH)n per pl, at least 100 units of the activeRNaseInh(-SH)n per pl, at least 200 units of the activeRNaseInh(-SH)n per pl, at least 300 units of the activeRNaseInh(-SH)n per pl or at least 400 units of the activeRNaseInh(-SH)n per pl. In some embodiments of the present invention, the solution comprises at least400 units of the activeRNaseInh(-SH)n per pl.

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

[0095] All embodiments mentioned for the first aspect of the invention above apply for the second aspect of the invention and vice versa. In a third aspect, the present invention relates to a lyophilisate comprising an activeRNase!nh(-SH)n and TCEP.

[0096] In some embodiments of the present invention, the lyophilisate further comprises a buffering agent, a monovalent salt, trehalose and / or a nonionic surfactant.

[0097] In some embodiments of the present invention, the lyophilisate is DTT- and / or glutathione-free.

[0098] All embodiments mentioned for the first and / or second aspect of the invention above apply for the third aspect of the invention and vice versa.

[0099] In a fourth aspect, the present invention relates to a kit comprising in a container at least one lyo-ready biocompatible solution of the first or second aspect or at least one lyophilisate of the third aspect.

[0100] All embodiments mentioned for the first, second and / or third aspect of the invention above apply for the fourth aspect of the invention and vice versa.

[0101] Suitable container for a solid or liquid composition are known to the skilled person and may be of glass, optionally with a plastic cap, or plastic. The kit may comprise the at least one lyo-ready biocompatible solution of the first or second aspect frozen. The kit may further comprise suitable means for keeping the solution cool or frozen, such as dry ice, frozen water or cold gel pack. The kit may be kept for storage at a suitable temperature, in particular for keeping the solution frozen, such as -20 °C.

[0102] In a fifth aspect, the present invention relates to a method of producing a lyophilisate comprising an activeRNaseInh(-SH)n, wherein the method comprises the step of lyophilising a lyo-ready biocompatible solution comprising an activeRNaseInh(- SH)n and TCEP, in particular a lyo-ready biocompatible solution of the first or second aspect.

[0103] All embodiments mentioned for the first, second, third and / or fourth aspect of the invention apply for the fifth aspect of the invention and vice versa. In a sixth aspect, the present invention relates to a method of producing a solution comprising an activeRNaseInh(-SH)n and TCEP, wherein the method comprises the step of: resolubilising a lyophilisate of the third aspect or obtainable, in particular obtained, by a method of the fifth aspect.

[0104] All embodiments mentioned for the first, second, third, fourth and / or fifth aspect of the invention apply for the sixth aspect of the invention and vice versa.

[0105] In some embodiments of the present invention, the lyophilisate is resolubilised with a solution comprising at least one salt, at least one buffering agent, at least one nonionic surfactant and / or at least one reducing agent.

[0106] In a seventh aspect, the present invention relates to a solution obtainable, in particular obtained, by a method of the sixth aspect, wherein the RNase inhibition activity of the solution is at least 90 % after lyophilisation and a storage of the lyophilisate for at most 3 days compared to the same solution before lyophilisation.

[0107] All embodiments mentioned for the first, second, third, fourth, fifth and / or sixth aspect of the invention apply for the seventh aspect of the invention and vice versa.

[0108] In some embodiments of the present invention, the RNase inhibition activity is at least 95 % after lyophilisation and a storage of the lyophilisate for at most 3 days compared to the same solution before lyophilisation.

[0109] In some embodiments of the present invention, the RNase inhibition activity is at least 90 % or at least 95 % after lyophilisation and a storage of the lyophilisate for at most 1 or 2 days, or at most 12 h, compared to the same solution before lyophilisation.

[0110] In some embodiments of the present invention, the RNase inhibition activity after lyophilisation is measured by resolubilising the lyophilisate and measuring the RNase inhibition activity by a method known to the skilled person, in particular a method according to Blackburn P., Ribonuclease Inhibitor from Human Placenta: Rapid Purification and Assay, J Biol Chem, Vol. 254, No. 24, pp. 12484 - 12487, 1979. In some embodiments of the present invention, the lyophilisate is resolubilized with a solution comprising at least one salt, at least one buffering agent, at least one non-ionic surfactant and / or at least one reducing agent. In some embodiments of the present invention, the lyophilisate is resolubilised in a solution comprising the same ingredients as the solution used for producing the lyophilisate, in particular the same concentrations and / or amounts of the ingredients. In some embodiments of the present invention, the lyophilisate is resolubilised to obtain a solution with the same amount of RNase inhibitor as has been used in the solution before lyophilisation, e.g. the solution used for measuring the RNase inhibition activity before lyophilisation has a concentration of 4000 units / ml in a volume of 1 ml, which is used to produce a lyophilisate with the same amount of RNase inhibitor, which is resolubilised to 1 ml and thus comprises the same amount of RNase inhibitor as the solution before lyophilisation, but might have a different activity and therefore less units per volume.

[0111] In an eighth aspect, the present invention relates to a solution obtainable, in particular obtained, by a method of the sixth aspect, wherein the RNase inhibition activity of the glycerol-free solution is at least 75 % after 3 weeks of storage of the lyophilisate at 35 °C compared to the same solution before lyophilisation.

[0112] All embodiments mentioned for the first, second, third, fourth, fifth, sixth and / or seventh aspect of the invention apply for the eighth aspect of the invention and vice versa.

[0113] In some embodiments of the present invention, the RNase inhibition activity is at least 77 % or at least 80 % after 3 weeks of storage of the lyophilisate at 35 °C compared to the same solution before lyophilisation.

[0114] In a ninth aspect, the present invention relates to a solution obtainable, in particular obtained, by a method of the sixth aspect, wherein the RNase inhibition activity of the solution is at least 77 % after lyophilisation and a storage of the lyophilisate for at most 3 days and storage of the solution for 6 months compared to a second solution comprising the same ingredients at the same amounts after storage of the second solution at -20 °C for 7 months. All embodiments mentioned for the first, second, third, fourth, fifth, sixth, seventh and / or eighth aspect of the invention apply for the ninth aspect of the invention and vice versa.

[0115] In some embodiments of the present invention, the second solution used for comparison originates from the same solution used for obtaining the lyophilisate employed in a method of the sixth aspect, which second solution may be stored at - 20 °C during storage of the lyophilisate and / or the solution obtainable, in particular obtained, from the lyophilisate.

[0116] In a tenth aspect, the present invention relates to a solution obtainable, in particular obtained, by a method of the sixth aspect, wherein the RNase inhibition activity of the solution is at least 90 % after 3 weeks of storage of the lyophilisate at 35 °C and subsequent 5 months of storage of the lyophilisate at -20 °C compared to a second solution comprising the same ingredients at the same amounts after storage of the second solution at -20 °C for 7 months.

[0117] All embodiments mentioned for the first, second, third, fourth, fifth, sixth, seventh eighth and / or ninth aspect of the invention apply for the tenth aspect of the invention and vice versa.

[0118] In a eleventh aspect, the present invention relates to the use of a lyo-ready biocompatible solution of the first, second, a solution of the seventh or eighth aspect, a lyophilisate of the third aspect or a kit of the fourth aspect for the inhibition of an RNase.

[0119] All embodiments mentioned for the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and / or tenth aspect of the invention apply for the eleventh aspect of the invention and vice versa.

[0120] In some embodiments of the present invention, the use includes adding a lyo-ready biocompatible solution or lyophilisate of the present invention to a composition, in particular the compositions is a solution. In some embodiments of the present invention, an RNase is present in the composition, in particular solution. In some embodiments of the present invention, the composition, in particular solution, does not contain an RNase, but is at risk of contamination with an RNase. In some embodiments of the present invention, the composition is for use in a reverse transcription reaction or in vitro transcription, e.g. production of mRNA in vitro.

[0121] In a twelfth aspect, the present invention relates to the use of a lyo-ready biocompatible solution for producing a lyophilisate comprising an activeRNaseInh(-SH)n, in particular of the third aspect, wherein the lyo-ready biocompatible solution comprises an activeRNaseInh(-SH)n and tris(2- carboxyethyl)phosphine (TCEP).

[0122] All embodiments mentioned for the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and / or eleventh aspect of the invention apply for the twelfth aspect of the invention and vice versa.

[0123] In further embodiments, the present invention relates to the following items:

[0124] 1. A lyo-ready biocompatible solution comprising

[0125] (i) an active RNase inhibitor comprising at least one cysteine residue with a reduced sulfhydryl group (activeRNaselnh(-SH)n) and

[0126] (ii) tris(2-carboxyethyl)phosphine (TCEP).

[0127] 2. A lyo-ready biocompatible solution consisting of an activeRNaseInh(-SH)n, a reducing agent, a buffering agent, a monovalent salt, trehalose, a nonionic surfactant and water, wherein the reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

[0128] 3. The lyo-ready biocompatible solution of item 1 or 2 for use in the production of a lyophilisate, in particular a lyophilisate comprising an activeRNaseInh(-SH)n and TCEP.

[0129] 4. The lyo-ready biocompatible solution of any one of items 1 to 3, wherein the RNase inhibition activity of the solution is at least 90 % after 3 weeks of storing the solution at 35 °C compared to the same solution before storage.

[0130] 5. The lyo-ready biocompatible solution of 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. 6. The lyo-ready biocompatible solution of any one of items 1 to 5, wherein the solution is dithiothreitol (DTT)- and / or glutathione-free.

[0131] 7. The lyo-ready biocompatible solution of 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.

[0132] 8. The lyo-ready biocompatible solution of any one of items 1 or 3-7, wherein the solution further comprises a buffering agent.

[0133] 9. The lyo-ready biocompatible solution of item 2 or 8, wherein the buffering agent is selected from the group consisting of TES, MOPS, HEPES, Tris, phosphate and combinations thereof, typically HEPES.

[0134] 10. The lyo-ready biocompatible solution of any one of items 2 or 8-9, wherein the concentration of the buffering agent is 5 to 100 mM, typically 10 to 50 mM, typically 20 mM.

[0135] 11. The lyo-ready biocompatible solution of any one of items 1 or 3-10, wherein the solution further comprises a monovalent salt.

[0136] 12. The lyo-ready biocompatible solution of item 2 or 11, wherein the monovalent salt is NaCl and / or KC1.

[0137] 13. The lyo-ready biocompatible solution of any one of items 2 or 11-12, 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.

[0138] 14. The lyo-ready biocompatible solution of any one of items 1 or 3-13, wherein the solution further comprises trehalose.

[0139] 15. The lyo-ready biocompatible solution of any one of items 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).

[0140] 16. The lyo-ready biocompatible solution of any one of items 1 or 3-15, wherein the solution further comprises a nonionic surfactant. 17. The lyo-ready biocompatible solution of item 2 or 16, wherein the nonionic surfactant is selected from the group consisting of triton X-100, polysorbate 20, polysorbate 60, polysorbate 80, polydocanol and combinations thereof.

[0141] 18. The lyo-ready biocompatible solution of any one of items 2 or 16-17, 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).

[0142] 19. The lyo-ready biocompatible solution of any one of items 1 or 3-18 comprising TCEP, HEPES, KC1, trehalose and polysorbate 20.

[0143] 20. The lyo-ready biocompatible solution of any one of items 1 or 3-19 comprising 1 to 20 mM TCEP, 5 to 100 mM of a buffering agent, 5 to 300 mM of a monovalent salt, 10 to 40 % (w / v) trehalose and 0.005 to 0.5 % (v / v) of a nonionic surfactant.

[0144] 21. The lyo-ready biocompatible solution of any one of items 1 or 3-20 comprising 5 mM TCEP, 20 mM of a buffering agent, 100 mM of a monovalent salt, 20 to 30 % (w / v) trehalose and 0.01 to 0.1 % (v / v) of a nonionic surfactant.

[0145] 22. The lyo-ready biocompatible solution of any one of items 1 or 3-21 comprising 5 mM TCEP, 20 mM HEPES, 100 mM KC1, 20 to 30 % (w / v) trehalose and 0.01 to 0.1 % (v / v) polysorbate 20.

[0146] 23. The lyo-ready biocompatible solution of any one of items 1 to 22, wherein the activeRNase!nh(-SH)n is an RNase inhibitor from Rattus norvegicus.

[0147] 24. The lyo-ready biocompatible solution of any one of items 1 to 23, wherein the activeRNase!nh(-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.

[0148] 25. The lyo-ready biocompatible solution of any one of items 1 to 23, wherein the activeRNase!nh(-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. 26. The lyo-ready biocompatible solution of any one of items 1 to 23, wherein the activeRNase!nh(-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.

[0149] 27. The lyo-ready biocompatible solution of any one of items 1 to 23, wherein the activeRNaseInh(-SH)n comprises, in particular consists of, the amino acid sequence of SEQ ID NO: 1.

[0150] 28. The lyo-ready biocompatible solution of any one of items 1 to 27, wherein the solution comprises at least 30 units of the activeRNase!nh(-SH)n per pl, typically at least 40 units of the activeRNase!nh(-SH)n per pl, typically at least 100 units of the activeRNase!nh(-SH)n per hl, typically at least 200 units of the activeRNase!nh(-SH)n per typically at least 300 units of the activeRNase!nh(-SH)n per typically at least 400 units of the activeRNase!nh(-SH)n per pl.

[0151] 29. A lyophilisate comprising an activeRNaseInh(-SH)n and TCEP.

[0152] 30. The lyophilisate of item 29 further comprising a buffering agent, a monovalent salt, trehalose and / or a nonionic surfactant.

[0153] 31. The lyophilisate of item 29 or 30, wherein the lyophilisate is DTT- and / or glutathione-free.

[0154] 32. The lyophilisate of item 30 or 31, wherein the buffering agent is selected from the group consisting of TES, MOPS, HEPES, Tris, phosphate and combinations thereof, typically HEPES.

[0155] 33. The lyophilisate of any one of items 30 to 32, wherein the monovalent salt is NaCl and / or KC1.

[0156] 34. The lyophilisate of 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, polydocanol and combinations thereof. 35. The lyophilisate of any one of items 29 to 34 comprising TCEP, HEPES, KC1, trehalose and polysorbate 20.

[0157] 36. The lyophilisate of any one of items 29 to 35, wherein the activeRNase!nh(-SH)n is an RNase inhibitor from Rattus norvegicus.

[0158] 37. The lyophilisate of any one of items 29 to 36, wherein the activeRNase!nh(-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.

[0159] 38. The lyophilisate of any one of items 29 to 36, wherein the activeRNase!nh(-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.

[0160] 39. The lyophilisate of any one of items 29 to 36, wherein the 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.

[0161] 40. The lyophilisate of any one of items 29 to 36, wherein the activeRNaseInh(-SH)n comprises or consists of the amino acid sequence of SEQ ID NO: 1.

[0162] 41. A kit comprising in a container at least one lyo-ready biocompatible solution of any one of items 1 to 28 or at least one lyophilisate of any one of items 29 to 40.

[0163] 42. A method of producing a lyophilisate comprising an activeRNaseInh(-SH)n, wherein the method comprises the step of: lyophilising a lyo-ready biocompatible solution comprising an activeRNaseInh(- SH)n and TCEP, in particular a lyo-ready biocompatible solution of any one of items 1 to 28.

[0164] 43. A method of producing a lyo-ready biocompatible solution comprising an activeRNaseInh(-SH)n and TCEP, wherein the method comprises the step of: resolubilising a lyophilisate of any one of items 29 to 40 or obtainable, in particular obtained, by a method of item 42. 44. A solution obtainable, in particular obtained, by a method of item 43, wherein the RNase inhibition activity of the solution is at least 90 % after lyophilisation and a storage of the lyophilisate for at most 3 days compared to the same solution before lyophilisation.

[0165] 45. The solution of item 44, wherein the RNase inhibition activity is at least 95 %.

[0166] 46. A solution obtainable, in particular obtained, by a method of item 43, wherein the RNase inhibition activity of the solution is at least 75 % after 3 weeks of storage of the lyophilisate at 35 °C compared to the same solution before lyophilisation.

[0167] 47. The solution of item 46, wherein the RNase inhibition activity is at least 77 %.

[0168] 48. The solution of item 46 or 47, wherein the RNase inhibition activity is at least 80 %.

[0169] 49. A solution obtainable, in particular obtained, by a method of item 43, wherein the RNase inhibition activity of the solution is at least 77 % after lyophilisation and a storage of the lyophilisate for at most 3 days and storage of the solution for 6 months compared to a second solution comprising the same ingredients at the same amounts after storage of the second solution at -20 °C for 7 months.

[0170] 50. A solution obtainable, in particular obtained, by a method of item 43 wherein the RNase inhibition activity of the solution is at least 90 % after 3 weeks of storage of the lyophilisate at 35 °C and subsequent 5 months of storage of the lyophilisate at - 20 °C compared to a second solution comprising the same ingredients at the same amounts after storage of the second solution at -20 °C for 7 months.

[0171] 51. Use of a lyo-ready biocompatible solution of any one of items 1-28, a solution of any one of items 44-50, a lyophilisate of any one of items 29 to 40 or a kit of item 41 for the inhibition of an RNase.

[0172] 52. The use of item 51, wherein the use includes adding a lyo-ready biocompatible solution or lyophilisate of the present invention to a composition, wherein the composition comprises an RNase or is at risk of contamination with an RNase. 53. Use of a lyo-ready biocompatible solution for producing a lyophilisate comprising an activeRNase!nh(-SH)n, in particular of any one of items 29 to 40, wherein the lyo-ready biocompatible solution comprises an activeRNase!nh(-SH)n and tris(2-carboxyethyl)phosphine (TCEP).

[0173] 54. The use of item 53, wherein the solution has 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.

[0174] 55. The use of item 53 or 54, wherein the solution is DTT- and / or glutathione-free.

[0175] 56. The use of 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.

[0176] 57. The use of any one of items 53 to 56, wherein the solution further comprises a buffering agent.

[0177] 58. The use of item 57, wherein the buffering agent is selected from the group consisting of TES, MOPS, HEPES, Tris, phosphate and combinations thereof, typically HEPES.

[0178] 59. The use of item 57 or 58, wherein the concentration of the buffering agent is 5 to 100 mM, typically 10 to 50 mM, typically 20 mM.

[0179] 60. The use of any one of items 53 to 59, wherein the solution further comprises a monovalent salt.

[0180] 61. The use of item 60, wherein the monovalent salt is NaCl and / or KC1.

[0181] 62. The use of item 60 or 61, 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.

[0182] 63. The use of any one of items 53 to 62, wherein the solution further comprises trehalose.

[0183] 64. The use of item 63, wherein the concentration of trehalose is 10 to 40 % (w / v), typically 15 to 35 % (w / v), typically 20 to 30 % (w / v). 65. The use of any one of items 53 to 64, wherein the solution further comprises a nonionic surfactant.

[0184] 66. The use of item 65, wherein the nonionic surfactant is selected from the group consisting of triton X-100, polysorbate 20, polysorbate 60, polysorbate 80 and combinations thereof.

[0185] 67. The use of 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).

[0186] 68. The use of any one of items 53 to 67, wherein the solution comprises TCEP, HEPES, KC1, trehalose and polysorbate 20.

[0187] 69. The use of any one of items 53 to 68, wherein the solution comprises 1 to 20 mM TCEP, 5 to 100 mM of a buffering agent, 5 to 300 mM of a monovalent salt, 10 to 40 % (w / v) trehalose and 0.005 to 0.5 % (v / v) of a nonionic surfactant.

[0188] 70. The use of any one of items 53 to 69, wherein the solution comprises 5 mM TCEP, 20 mM of a buffering agent, 100 mM of a monovalent salt, 20 to 30 % (w / v) trehalose and 0.01 to 0.1 % (v / v) of a nonionic surfactant.

[0189] 71. The use of any one of items 53 to 70, wherein the solution comprises 5 mM TCEP, 20 mM HEPES, 100 mM KC1, 20 to 30 % (w / v) trehalose and 0.01 to 0. 1 % (v / v) polysorbate 20.

[0190] 72. The use of any one of items 53 to 71, wherein the activeRNase!nh(-SH)n is an RNase inhibitor from Rattus norvegicus.

[0191] 73. The use of any one of items 53 to 72, wherein the activeRNase!nh(-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.

[0192] 74. The use of any one of items 53 to 72, wherein the activeRNase!nh(-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. 75. The use of any one of items 53 to 72, wherein the activeRNase!nh(-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.

[0193] 76. The use of any one of items 53 to 72, wherein the activeRNaseInh(-SH)n comprises or consists of the amino acid sequence of SEQ ID NO: 1.

[0194] 77. The use of any one of items 53 to 76, wherein the solution comprises at least 30 units of the activeRNaseInh(-SH)n per pl, typically at least 40 units of the activeRNaseInh(-SH)n per pl, typically at least 100 units of the activeRNaseInh(-SH)n per pl, typically at least 200 units of the activeRNaseInh(-SH)n per pl, typically at least 300 units of the activeRNaseInh(-SH)n per pl, typically at least 400 units of the activeRNaseInh(-SH)n per pl.

[0195] Sequence List

[0196] SEQ

[0197] ID Name Sequence

[0198] NO

[0199] MSLDIQCEQLSDARWTELLPLIQ

[0200] QYQVVRLDDCGLTEVRCKDIRSA

[0201] IQANPALTELSLRTNELGDAGVG

[0202] LVLQGLQNPTCKIQKLSLQNCSL

[0203] TEAGCGVLPDVLRSLSTLRELHL

[0204] NDNPLGDEGLKLLCEGLRDPQCR

[0205] LEKLQLEYCNLTATSCEPLASVL RVKPDFKEL VL SNNDFHE AGIHT

[0206] LCQGLKDSACQLESLKLENCGIT

[0207] RNase SANCKDLCDVVASKASLQELDLG

[0208] 1 inhibitor SNKLGNTGIAALCSGLLLPSCRLR

[0209] TLWLWDCDVTAEGCKDLCRVLR

[0210] AKQSLKELSLAGNELKDEGAQLL

[0211] CESLLEPGCQLESLWVKTCSLTA

[0212] ASCPHFCSVLTKNRSLFELQMSS

[0213] NPLGDSGVVELCKALGYPDTVLR

[0214] VLWLGDCDVTDSGCSSLATVLLA

[0215] NRSLRELDLSNNCMGDNGVLQLL

[0216] ESLKQPSCILQQLVLYDIYWTDE

[0217] VEDQLRALEEERPSLRIIS Examples

[0218] The following examples are provided to illustrate, but not to limit the presently claimed invention.

[0219] Example 1

[0220] Four different formulations with RNase inhibitor from rat (SEQ ID NO: 1) were freshly prepared and evaluated: 20% or 30% trehalose and 5 mM DTT or TCEP respectively.

[0221] Final buffer formulation:

[0222] 20 mM Hepes, 100 mM KC1, 20% or 30% trehalose, 5 mM DTT or TCEP, 0.01 % Tween 20, pH 7.60 / 4.0 °C

[0223] The inhibition of RNase A by RNase inhibitor is measured by its ability to inhibit the hydrolysis of cyclic 2’,3’-CMP as substrate (Blackburn P.: Ribonuclease Inhibitor from Human Placenta: Rapid Purification and Assay. J Biol Chem. 1979 Dec 25;254(24): 12484-7).

[0224] The measurement of RNase A inhibition by RNase inhibitor is conducted in a photometer (UVIKON 930 and higher, Kontron Instruments; and Cary 1, Varian) at OD286.

[0225] To a 3-ml cuvette (quartz / plastic), 0.3 ml of Tris / acetate buffer (1 M Tris / acetate, pH 6.5 with 0.2 mM EDTA), 0.3 ml of 10 mM cyclic 2’,3’-CMP in H20, and additional H20 to give a final volume of 2.940 ml were added.

[0226] The ingredients were mixed in the cuvette and incubated for 5 min in the photometer before the reaction was initiated by addition of 3 pg of RNase A and the solutions were mixed again.

[0227] The photometer temperature was kept constant at a temperature of 25 °C by connecting it to a 25 °C water bath. The temperature was checked before and after each assay. The temperature was 25 °C before RNase A was added. The rate of hydrolysis of the cyclic 2’, 3’- CMP was determined from the rate of increase in absorption produced at 286 nm. Therefore, AE at 286 nm for 10 min was measured and the result was used to determine AE per min.

[0228] The amount of inhibitor assayed was determined from the percentage inhibition of the RNase A contained in the assay.

[0229] The inhibition of RNase A, by different concentrations of inhibitor, is linear with respect to the concentration of the inhibitor in the assay with cyclic 2’,3’-CMP as substrate. Thus, a portion of inhibitor solution could serve, by linear interpolation, to estimate the units of activity in the sample (see above publication by Blackbum).

[0230] For evaluation, only the values which reduce the AE to 75.0 - 50.0 % were used.

[0231] By definition, 1 U is defined as the amount of protein required to inhibit 5 ng of RNase A by 50 %. Thus, 200 U = 1 pg RNase A and 600 U = 3 pg RNase A inhibited by 50 %.

[0232] Stability monitoring of RNase formulations

[0233] Volume activity was measured according to the method of Blackburn (see above) with or without treatment of stress testing at 35 °C and / or lyophilisation of the samples.

[0234] Volume Activity of RNase Inhibitor formulations with various amounts of trehalose and either DTT or TCEP as reductant was measured unstressed in their liquid form (see column A, table 1) prior to being stressed for 3 weeks at 35 °C in their liquid form (see column B, table 1), lyophilised over night and resolubilised the next day (see column C, table 1), or lyophilised and stressed 3 weeks at 35 °C (see column D, table 1). Lyophilisation was performed with a Christ Beta 2-8 LD Plus Freeze Dryer at standard conditions according to manufacturer’s instructions with 1.5 ml vials. Table 1 :

[0235] A B C D

[0236] 20 % Trehalose / DTT [U / ml] 929000 749000 787000 494000

[0237] 30 % Trehalose / DTT [U / ml] 876000 680000 728000 620000

[0238] 20 % Trehalose / TCEP [U / ml] 751000 728000 681000 600000

[0239] 30 % Trehalose / TCEP [U / ml] 686000 724000 689000 529000

[0240] 20 % Trehalose / DTT [%] 100 81 85 53

[0241] 30 % Trehalose / DTT [%] 100 78 83 71

[0242] 20 % Trehalose / TCEP [%] 100 97 91 80

[0243] 30 % Trehalose / TCEP [%] 100 106 100 77

[0244] RNase Inhibitor is stabilized by formulations containing TCEP (compared to identical formulation containing DTT) in all three conditions tested: liquid storage at 35 °C, after lyophilisation and also after storage of the lyophilised formulations at 35 °C.

[0245] Example 2

[0246] Commonly sucrose is used as a lyoprotectant. It was therefore also tested to use sucrose instead of trehalose. However, formulations comprising sucrose were not stable when stressed: Two formulations comprising the RNase Inhibitor as used in Example 1 and sucrose, SI (20 mM Hepes, 100 mM potassium chloride, 20 % Sucrose, 5 mM TCEP, 0.05 % Tween 20, pH 7,6 @ 4 °C) and S2 (20 mM Hepes, 100 mM potassium chloride, 20 % Sucrose, 5 mM DTT, 0.05 % Tween 20, pH 7,6 @ 4 °C), were subjected to lyophilisation and subsequent storage at 35 °C for 3 weeks. In none of these two formulations, SI and S2, the lyophilisate itself was stable, but clumping of the lyophilisate appeared with both formulations. The resolubilisation required more time for the lyophilisate of both formulations, too. In addition, a clouding was visible for formulation S2 (see FIG. 1).

[0247] Compared to sucrose with either DTT or TCEP, the combination of trehalose with TCEP was able to keep the formulation stable in that no clumping of the lyophilisate or clouding of the resolubilised solution appeared, and in that the lyophilisate can be resolubilised more easily.

[0248] Example 3

[0249] In order to compare the stability of RNase Inhibitor activity during stressed longterm storage in formulations comprising the RNase Inhibitor as used in Example 1 together with TCEP or DTT, two formulations, with TCEP (Formulation A: 20 mM Hepes, 100 mM potassium chloride, 20 % Trehalose, 5 mM TCEP, 0.01 % Tween 20, pH 7,6 @ 4 °C) and with DTT (Formulation B: 20 mM Hepes, 100 mM potassium chloride, 20 % Trehalose, 5 mM DTT, 0.01 % Tween 20, pH 7,6 @ 4 °C), were subjected to different stressing conditions. Condition I included lyophilisation and resolubilisation the next day with subsequent storage of the resolubilised solution at -20 °C for 6 months and condition II included lyophilisation, storage of the lyophilisate at 35 °C for 3 weeks, subsequent storage of the lyophilisate for 5 months at -20 °C and resolubilisation. The solutions exposed to conditions I and II originated from the same solution, the rest of which was stored without any stress for 7 months total at -20 °C (control condition) and used to compare it with the solutions exposed to stressed long-term conditions. The activity was measured as described in Example 1. Table 2:

[0250] Sample Volume Concentration Specific Residual Volume activity [mg / ml] activity activity [%] activity

[0251] [U / pl] [U / mg] normalised to concentration of 6 mg / ml [U / pl]

[0252] Control 835 7.7 108441.56 100.00 650.649 condition / Formulation A

[0253] Condition I / 666 7.7 86493.51 79.76 518.961

[0254] Formulation A

[0255] Condition II 773 7.7 100389.61 92.57 602.338

[0256] /

[0257] Formulation

[0258] A

[0259] Control 743 7.9 94050.63 100.00 564.304 condition / Formulation B

[0260] Condition I / 557 7.9 70506.33 74.97 423.038

[0261] Formulation B

[0262] Condition II 611 7.9 77341.77 82.23 464.051

[0263] /

[0264] Formulation

[0265] B As can be seen from the residual activity, formulations comprising DTT fared much worse when stressed than formulations comprising TCEP, which even after lyophilisation, storage at elevated temperature and subsequent freezing, still retains more than 90 % of activity (see Table 2). Also the specific activity was higher in formulations comprising TCEP compared to formulations comprising DTT. This shows that TCEP can better stabilise the activity of the RNase Inhibitor, not only for short term storage, but also for long term storage, especially when stressed.

Claims

Claims1. A lyo-ready biocompatible solution comprising(i) an active RNase inhibitor comprising at least one cysteine residue with a reduced sulfhydryl group (activeRNaselnh(-SH)n) and(ii) tris(2-carboxyethyl)phosphine (TCEP).

2. The lyo-ready biocompatible solution of claim 1 for use in the production of a lyophilisate.

3. The lyo-ready biocompatible solution of claim 1 or 2, wherein the solution is dithiothreitol (DTT)- and / or glutathione-free.

4. The lyo-ready biocompatible solution of any one of claims 1 to 3, wherein the solution further comprises a buffering agent, a monovalent salt, trehalose and / or a nonionic surfactant.

5. The lyo-ready biocompatible solution of any one of claims 1 to 4, wherein the activeRNase!nh(-SH)n is an RNase inhibitor from Rattus norvegicus.

6. A lyophilisate comprising an activeRNase!nh(-SH)n and TCEP.

7. The lyophilisate of claim 6 further comprising a buffering agent, a monovalent salt, trehalose and / or a nonionic surfactant.

8. A kit comprising in a container at least one lyo-ready biocompatible solution of any one of claims 1 to 5 or at least one lyophilisate of claim 6 or 7.

9. A method of producing a solution comprising an activeRNase!nh(-SH)n and TCEP, wherein the method comprises the step of: resolubilising a lyophilisate of claim 6 or 7.

10. A solution obtainable by a method of claim 9, wherein the RNase inhibition activity of the solution is at least 90 % after lyophilisation and a storage of thelyophilisate for at most 3 days compared to the same solution before lyophilisation.

11. A solution obtainable by a method of claim 9, wherein the RNase inhibition activity of the solution is at least 75 % after 3 weeks of storage of the lyophilisate at 35 °C compared to the same solution before lyophilisation.

12. Use of a lyo-ready biocompatible solution of any one of claims 1 to 5, a lyophilisate of claim 6 or 7 or a kit of claim 8 for the inhibition of an RNase.

13. The use of claim 12, wherein the use includes adding a lyo-ready biocompatible solution or lyophilisate of the present invention to a composition, wherein the composition comprises an RNase or is at risk of contamination with an RNase.

14. Use of a lyo-ready biocompatible solution for producing a lyophilisate comprising an activeRNase!nh(-SH)n, in particular of claim 6 or 7, wherein the lyo-ready biocompatible solution comprises an activeRNase!nh(-SH)n and tris(2-carboxyethyl)phosphine (TCEP).

15. The use of claim 14, wherein the solution further comprises a buffering agent, a monovalent salt, trehalose and / or a nonionic surfactant.

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