Method and kit for measuring and reducing protein impurities

Abstract

The present invention relates to a method of measuring protein impurities in an RNA composition. In particular, the present invention relates to methods of incubating the RNA composition with a protease of the protease family S8A, such that the protein impurities in the RNA composition are digested to form peptide products. The present invention also relates to methods for reducing protein impurities in an RNA composition and for producing RNA compositions that are suitable for in vivo administration.

Description

[0001] MEASURING AND REDUCING PROTEIN IMPURITIES FIELD OF THE INVENTION The present invention relates to a method of measuring protein impurities in an RNAcomposition. In particular, the present invention relates to methods of incubating an RNAcomposition with a protease of the protease family S8A, such that the protein impurities in the RNA composition are digested to form peptide products. The present invention also relates tomethods for reducing protein impurities in an RNA composition and for producing RNAcompositions that are suitable for in vivo administration.BACKGROUND TO THE INVENTIONTherapeutic RNA (e.g. mRNA) is produced by in vitro transcription (IVT). Typically, enzymesand other proteins (such as T7 RNA Polymerase, inorganic Pyrophosphatase, DNase 1 andan RNAse Inhibitor) may be employed in this reaction to generate and stabilize the RNA, todegrade the DNA template and to degrade the by-product pyrophosphate. After completion of the reaction, these proteins as well as lower molecular weight substances need to be removed from the RNA composition that has been produced. Both ultrafiltration and solid-phase chromatography methods are employed in the art for drug substance purification. The efficiency of the former can be increased substantially by prior digestion of the proteins intoshort peptides using the fungal protease Proteinase K. Contamination of RNA compositions(e.g. RNA drug substance) by traces of the enzymes and other proteins (or of their peptidefragments thereof) may cause toxic or immunogenic effects in vivo.Protein impurities of biopharmaceuticals are typically identified and quantified by means ofmass spectrometry. Since specific proteolytic fragments (i.e. peptide products) can bedetected with substantially higher sensitivity than their parent proteins, these peptide productscan be measured as protein surrogates (Kang et al.2020, Biomedical chromatography : BMC34 (1), e4633; Pilely et al.2021, Analytical and bioanalytical chemistry. DOI: 10.1007 / s00216-021-03648-2). This approach only provides an accurate and precise quantification if therespective surrogate peptide product is generated in a reproducible, stochiometric proportionto its parent protein.Quality-relevant product attributes need to be monitored and minimized through processimprovement (Challener 2022, Pharmaceutical Technology 46 (2), pp.16–21). Importantly,this also includes the levels of Proteinase K itself. There is a need for improved methods for quality control of RNA compositions and products. SUMMARY OF THE INVENTION The present inventors have surprisingly found that proteases of the protease family S8A, otherthan Proteinase K, can be used to degrade proteins (and their peptide fragments thereof) usedduring the production of an RNA composition to a stable endpoint, in a consistent andreproducible manner,Accordingly, in a first aspect, the present invention provides a method of measuring protein impurities in an RNA composition comprising the steps of: (i) incubating the RNA composition with a protease of the protease family S8A, suchthat the protein impurities in the RNA composition are digested to form peptide products; followed by (ii) incubating the RNA composition with an agent that precipitates the RNA in the RNAcomposition; (iii) purifying the peptide products in the RNA composition and / or concentrating thepeptide products in the RNA composition; and (iv) measuring the peptide products by resolving and quantifying the peptide products.In some embodiments, the protease of the protease family S8A of step (i) of the methods ofthe first aspect according to the present invention may not be Proteinase K.In some embodiments, the protease of the protease family S8A of step (i) of the methods ofthe first aspect according to the present invention may be a Subtilisin protease.In some embodiments, step (ii) may be a step of acidification.In some embodiments, step (ii) may be a step of precipitation with high salt concentration. Insome embodiments, the salt may be lithium chloride.In some embodiments, the step of purifying the peptide products in step (iii) may be by solidphase extraction.In some embodiments, the step of concentrating the peptide in step (iii) may be by vacuum-centrifugation. In some embodiments, the step of concentrating the peptide in step (iii) may be by lyophilisation.In some embodiments, resolving the peptide products in step (iv) may be by using liquidchromatography (LC), isoelectric focusing, capillary electrophoresis, direct-infusion massspectrometry or Ion Mobility Separation, or combinations thereof. In some embodiments, theLC may be ion exchange chromatography, hydrophilic interaction chromatography, orreversed-phase liquid chromatography.In some embodiments, quantifying the peptide products in step (iv) may be by massspectrometry (MS). In some embodiments, the mass spectrometry (MS) may be selected fromthe list consisting of: multiple reaction monitoring mass spectrometry (MRM-MS), singlereaction monitoring (SRM-MS), parallel reaction monitoring mass spectrometry (PRM-MS),data-dependent-acquisition high resolution mass spectrometry (DDA-HRMS), or data-independent acquisition high resolution mass spectrometry (DIA-HRMS).In some embodiments, the peptide products may be normalized to a standard and absolutequantities may be derived from a calibration with identical, synthetic peptides or their parentalproteins. In a second aspect, the present invention provides a method of reducing protein impurities in an RNA composition comprising the step of (i) incubating the RNA composition with a protease such that the protein impurities inthe RNA composition are digested to form peptide products; wherein the protease is aprotease of the protease family S8A.In some embodiments, the protease of the protease family S8A of step (i) of the methods ofthe second aspect according to the present invention may not be Proteinase K. In some embodiments, the protease of the protease family S8A of step (i) of the methods ofthe second aspect according to the present invention may be a Subtilisin protease.In a third aspect, the present invention provides a method of producing an RNA productsuitable for in vivo administration, comprising the steps of:(i) incubating an RNA composition with a protease such that the protein impurities inthe RNA composition are digested to form peptide products; wherein the protease is aprotease of the protease family S8A; and (ii) obtaining an RNA product that is suitable for in vivo administration.In some embodiments, the protease of the protease family S8A of step (i) of the methods ofthe third aspect according to the present invention may not be Proteinase K.In some embodiments, the protease of the protease family S8A of step (i) of the methods ofthe third aspect according to the present invention may be a Subtilisin protease.In a fourth aspect, the present invention provides a use of a protease to digest proteinimpurities in an RNA composition, wherein the protease is a protease of the protease familyS8A.In some embodiments, the protease of the protease family S8A of the use of the fourth aspectaccording to the present invention may not be Proteinase K. In some embodiments, the protease of the protease family S8A of the use of the fourth aspectaccording to the present invention may be a Subtilisin protease.In some embodiments of the first, second, third and / or fourth aspect according to the invention,the protease may be selected from the list consisting of: Subtilisin A, Subtilisin DY, SubtilisinBNP’, Subtilisin E, and Subtilisin NAT, Subtilisin BL, Subtilisin Savinase, Tk-subtilisin,Subtilisin J, or any variant thereof.In some embodiments, the protease may be Subtilisin A derived from Bacillus licheniformis.In some embodiments, the Subtilisin A may comprise or consist of an amino acid sequencehaving at least 80% identity to the amino acid sequence of SEQ ID NO: 1.In some embodiments, the Subtilisin A may comprise or consist of an amino acid sequencehaving at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments of the first, second, and / or third aspect according to the invention, step(i) may be preceded by a prior step of in vitro transcription to produce the RNA composition,wherein the step may comprise transcribing RNA from a DNA template in vitro.In some embodiments, the prior step of transcribing RNA from a DNA template in vitro maybe carried out in the presence of an RNA polymerase, a pyrophosphatase, and / or an RNAseinhibitor to produce an intermediate RNA / DNA mixture.In some embodiments, the prior step of transcribing RNA from a DNA template in vitro maybe followed by an additional step of incubation with a DNAse and / or Proteinase K and a furtherstep of ultrafiltration, prior to step (i) of the method according to the first, second, and / or thirdaspect according to the invention. In some embodiments of the first, second, and / or third aspect according to the invention, theprotein impurities may comprise intact or digested enzyme reagents and / or proteins used inthe production of the RNA composition. In some embodiments of the first, second, and / or third aspect according to the invention, theprotein impurities may be derived from one or more proteins selected from the list consistingof: an RNA polymerase, a pyrophosphatase, an RNAse inhibitor, a DNAse and Proteinase K.In some embodiments of the first, second, and / or third aspect according to the invention, step(i) may be performed for:(i) less than 1 minute to about 60 minutes; or (ii) about 1 hour to about 5 hours; or (iii) more than 5 hours.In some embodiments of the first, second, and / or third aspect according to the invention, theincubation in step (i) may be performed for less than 10 minutes to more than 5 hours. In someembodiments, the incubation in step (i) may be performed for about 1 hour to about 5 hours.In some embodiments, the incubation in step (i) may be performed for about 1 hour to about4 hours. In some embodiments, the incubation in step (i) may be performed for about 1 hourto about 3 hours. In some embodiments, the incubation in step (i) may be performed for about1 hour to about 2 hours.In some embodiments of the first, second, and / or third aspect according to the invention, theincubation in step (i) may be performed for about 1.5 hours to about 5 hours. In someembodiments, the incubation in step (i) may be performed for about 1.5 hours to about 4.5hours. In some embodiments, the incubation in step (i) may be performed for about 1.5 hoursto about 4 hours. In some embodiments, the incubation in step (i) may be performed for about1.5 hours to about 3.5 hours. In some embodiments, the incubation in step (i) may beperformed for about 1.5 hours to about 3 hours. In some embodiments, the incubation in step(i) may be performed for about 1.5 hours to about 2.5 hours. In some embodiments, theincubation in step (i) may be performed for about 1.5 hours to about 2 hours.In some embodiments of the first, second, and / or third aspect according to the invention, step(i) may be performed for less than 10 minutes; about 10 to about 15 minutes, about 15 toabout 20 minutes, about 20 to about 25 minutes, about 25 to about 30 minutes, about 30 to about 35 minutes, about 35 to about 40 minutes, about 40 to about 45 minutes, about 45 toabout 50 minutes, about 50 to about 55 minutes, about 55 to about 60 minutes.In some embodiments of the first, second, and / or third aspect according to the invention, step(i) may be performed for about 1 hour to about 1.5 hours, about 1.5 to about 2 hours, about 2to about 2.5 hours, about 2.5 to about 3 hours, about 3 to about 3.5 hours, about 3.5 to about 4 hours, about 4 to about 4.5 hours, or about 4.5 to about 5 hours. In some embodiments of the first, second, and / or third aspect according to the invention, step(i) may be performed at less than 18°C, at about 18°C to about 30°C, or at more than 30°C.In some embodiments, step (i) may be performed at 18°C, 19°C, 20°C, 21°C, 22°C, 23°C,24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C.In some embodiments of any aspect according to the invention, the RNA may be suitable fortherapeutic administration.In some embodiments of any aspect according to the invention, the RNA may be any form ofRNA.In some embodiments of any aspect according to the invention, the RNA may be mRNA. In a fourth aspect, the present invention also provides an RNA product suitable for in vivoadministration, wherein the RNA product is obtainable by the method according the second,and / or third aspect according to the invention.In a fifth aspect, the present invention also provides a kit comprising:(i) a protease of the protease family S8A; (ii) an RNA polymerase, a pyrophosphatase, an RNAse inhibitor, and / or a DNAse;In some embodiments of the fifth aspect according to the invention, the kit further comprises(iii) Proteinase K and / or (iv) instructions for using the kit to reduce and / or quantify proteinimpurities in an RNA product or RNA composition.In some embodiments of the fifth aspect according to the invention, the protease of theprotease family S8A in (i) of the kit according to the invention may not be Proteinase K.In some embodiments of the fifth aspect according to the invention, the protease of theprotease family S8A in (i) of the kit according to the invention may be a Subtilisin protease.In some embodiments of the fifth aspect according to the invention, the protease may beselected from the list consisting of: Subtilisin A, Subtilisin DY, Subtilisin BNP’, Subtilisin E, andSubtilisin NAT, Subtilisin BL, Subtilisin Savinase, Tk-subtilisin, Subtilisin J, or any variantthereof.In some embodiments of the fifth aspect according to the invention, the protease may beSubtilisin A derived from Bacillus licheniformis. In some embodiments of the fifth aspect according to the invention, the Subtilisin A may comprise or consist of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments of the fifth aspect according to the invention, the Subtilisin A may comprise or consist of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 1. BRIEF DESCRIPTION OF THE FIGURESFigure 1: Assay principle: An S8A-family protease, like Subtilisin A, is used in during analyticalsample preparation to drive the digestion of the target proteins to a reproducible endpoint resulting in a quantitative production of the surrogate peptide that is quantified by LC-MS. Figure 2: Assay Workflow: Overview of the major steps of sample preparation, measurement and data analysis. DETAILED DESCRIPTION OF THE INVENTION Protein impuritiesProteins (e.g. enzymes) used in in vitro RNA production processes are commonly removedfrom the generated RNA composition following completion of the in vitro transcription process.However, contamination of RNA compositions and RNA drug products by traces of theproteins (or their peptide fragments thereof) used in RNA production processes may causetoxic or immunogenic effects in vivo. The presence of these proteins in an RNA compositionor RNA drug product can be termed “protein impurities”. As such, it is an important step ofquality control to measure levels of proteins impurities in order to ensure that RNAcompositions and RNA drug products meet the necessary safety requirements for use in vivo.As used herein, proteins used in RNA production processes can be termed “protein reagents”, which include inter alia enzymes.Without wishing to be bound by theory, the term “protein impurities” or “protein impurity” mayrefer to: (i) intact protein reagent(s) used in the production of an RNA composition; and (ii) atleast partially digested protein reagent(s) that are peptides derived from protein reagent(s)used in the production of an RNA composition. Examples of protein reagents used in theproduction of an RNA composition include enzymes that serve to catalyse reactions requiredfor the production of RNA from a DNA template.In the context the present disclosure, the terms “digest”, “digestion”, “digested”, “degrade”,“degraded”, “cleave”, and “cleavage” etc. refer to proteins that have undergone proteolysis,e.g. by enzymatic cleavage. As such, these terms can be considered as interchangeable. Standard methods for protein quantification include the surrogate peptide approach wherebyproteins are degraded to specific peptide products which can be detected with substantiallyhigher sensitivity (e.g. using mass spectrometry) compared to detection of the proteins priorto degradation. It will be understood that a protein, prior to degradation to specific peptideproducts, may be termed a “parent protein”. As such, levels of these peptide products can actas a surrogate marker for levels of the parent proteins. Proteinase K treatment can be employed during production of an RNA composition to degrade enzymes / other proteins used in the RNA production process. However, cleavage byProteinase K may be inconsistent and can result in a random collection of peptide fragmentsnot compatible with the standard methods for protein quantification. The primary reasons for this may be because: (1) the cleavage of the proteins by Proteinase K may be incomplete due to the limited concentration of the Proteinase K and sub-optimal reaction conditions; and / or (2) the cleavage sites may be used (at least in part) stochastically. Since the distancebetween potential cleavage sites is typically smaller than the minimal substrate length, the temporal order of the cleavage events determines the peptide products that are produced, which may be inconsistent in size. The present invention is based upon the surprising finding that proteases of the proteasefamily S8A, other than Proteinase K, can be used to degrade proteins (and their peptidefragments thereof) used during the production of an RNA composition to a stable endpoint,whereby peptide products derived from the degraded proteins are reproducibly generated ina consistent manner, which is independent of the efficiency of proteolysis initiated byProteinase K that may be used initially during the production of an RNA composition. This isdue to predictable cleavage patterns by proteases of the protease family S8A, other than Proteinase K, which result in consistent peptide products. These peptide products are of aconsistent size (i.e. amino acid length) and can be detected in a quality control assay, servingas surrogate markers of the parent protein(s).The accuracy and precision of this approach is based on the fact that the quantity and nature(e.g. size) of the peptide products, produced by digestion of the protein impurities using themethods and use according to the invention, are consistent and reproducible.As used herein, it will be understood that an “RNA composition” may encompass an RNAsample. As used herein, it will also be understood that an “RNA composition” may be used to make and / or be further processed into an “RNA product”, which may also be termed an “RNAdrug substance” or an “RNA drug product”. In other words, incubating an RNA compositionwith a protease of the protease family S8A as defined herein may result in an RNA product,e.g. an RNA product that is suitable for in vivo administration.It will be understood that the specific protein impurities generated during the production of anRNA composition will depend on the proteins (e.g. enzymes and other proteins) that are usedin the RNA production process.In some embodiments, a protein impurity may be a full-length protein or an intact protein. Itwill be understood that a full-length protein or intact protein may be hundreds of amino acidsin length or more.In some embodiments, a protein impurity may be a fragment of a protein. Such a fragmentmay have any length, provided it is shorter than the full-length protein. It should be noted that a full-length protein may give rise to a number of protein impurities of differing or identical lengths.In some embodiments, the protein impurities may already be at least partially digested andthus may be in the form of peptides (i.e. peptide impurities) derived from the protein impurities.As used herein, the terms “protein impurity” and “peptide impurity” may be used interchangeably, and preferred usage of each term will typically depend on the size of the protein / peptide impurity in question.In some embodiments, a protein impurity may be a peptide (i.e. a peptide impurity). In someembodiments, the peptide impurity may be less than 100 amino acids in length, such as lessthan 50 amino acids in length, such as less than 40 amino acids in length, such as less than 30 amino acids in length, such as less than 25 amino acids in length, such as less than 20 amino acids in length, such as less than 15 amino acids in length, such as less than 10 amino acids in length, such as less than 5 amino acids in length.In some embodiments, the protein impurities may comprise (i) intact protein reagents (e.g.enzymes) used in the production of an RNA composition; and / or (ii) peptides derived from atleast partially-digested proteins reagents (e.g. enzymes) used in the production of an RNAcomposition.In some embodiments, the protein impurities may comprise one or more proteins selectedfrom the list consisting of: RNA polymerase, pyrophosphatase, RNAse inhibitor, DNAse andProteinase K, or combinations thereof. Suitable RNA polymerases, pyrophosphatases, RNAse inhibitors, and DNAses will be known in the art.In some embodiments, the protein impurities may comprise all of the proteins selected fromthe list consisting of: RNA polymerase, pyrophosphatase, RNAse inhibitor, DNAse andProteinase K. As used herein, the term “peptide product” refers to the digested fragments that are producedby digestion of the protein impurities and / or peptide impurities using a protease of the proteasefamily S8A according to the methods and use according to the invention. It will be understoodthat a peptide product will be smaller in size (e.g. the number of amino acids in length)compared to the size of the peptide impurity (i.e. the parent peptide) or protein impurity (i.e.the parent protein). In some embodiments, the peptide product may be less than 100 amino acids in length, such as less than 50 amino acids in length, such as less than 40 amino acids in length, such as less than 30 amino acids in length, such as less than 25 amino acids in length, such as less than 20 amino acids in length, such as less than 15 amino acids in length, such as less than10 amino acids in length, such as less than 7 amino acids in length, such as less than 6 aminoacids in length, such as less than 5 amino acids in length, or such as less than 4 amino acidsin length. In some embodiments, the peptide product may be 1-100 amino acids in length. In some embodiments, the peptide product may be 4-100 amino acids in length. In some embodiments, the peptide product may be 50-100 amino acids in length. In someembodiments, the peptide product may be 1-50 amino acids in length. In some embodiments,the peptide product may be 4-50 amino acids in length. In some embodiments, the peptide product may be 1-40 amino acids in length. In some embodiments, the peptide product may be 4-40 amino acids in length. In some embodiments, the peptide product may be 1-30 amino acids in length. In some embodiments, the peptide product may be 4-30 amino acids in length. In some embodiments, the peptide product may be 1-25 amino acids in length. In some embodiments, the peptide product may be 4-25 amino acids in length.In some embodiments, the peptide product may be 1-20 amino acids in length. In someembodiments, the peptide product may be 4-20 amino acids in length. In some embodiments, the peptide product may be 1-15 amino acids in length. In some embodiments, the peptide product may be 4-15 amino acids in length.In some embodiments, the peptide product may be 1-10 amino acids in length. In someembodiments, the peptide product may be 4-10 amino acids in length.In some embodiments, the peptide product may be 1-5 amino acids in length. In someembodiments, the peptide product may be 4-5 amino acids in length. In some embodiments, the peptide product may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more than 50 amino acids in length.In some embodiments, the peptide product may be 4-7 amino acids in length.In some embodiments, the peptide product may be 4 amino acids in length. In someembodiments, the peptide product may be 5 amino acids in length. In some embodiments, thepeptide product may be 6 amino acids in length. In some embodiments, the peptide productmay be 7 amino acids in length.In some embodiments, the peptide product may be 10-50 amino acids in length. In some embodiments, the peptide product may be 10-40 amino acids in length. In some embodiments, the peptide product may be 10-30 amino acids in length. In some embodiments, the peptide product may be 10-20 amino acids in length. In some embodiments, the peptide product may be 20-40 amino acids in length. In some embodiments, the peptide product may be 10-40 amino acids in length. In some embodiments, the peptide product may be 20-30 amino acids in length.It will be understood that a peptide product may be a single amino acid in length.Measuring the protein impuritiesThe protease of the protease family S8A as described herein may be used for the purposesof quality control, in order to measure the levels of protein impurities in an RNA composition. Accordingly, in a first aspect, the present invention provides a method of measuring proteinimpurities in an RNA composition, comprising the steps of:(i) incubating the RNA composition with a protease of the protease family S8A, such that the protein impurities in the RNA composition are digested to form peptide products; followed by (ii) incubating the RNA composition with an agent that precipitates the RNA in the RNA composition; (iii) purifying the peptide products in the RNA composition and / or concentrating the peptide products in the RNA composition; and (iv) measuring the peptide products by resolving and quantifying the peptide products. It will be understood that the steps (i) to (iv) are to be performed in a sequential manner.It will be understood that in the first aspect of the present invention, the protease of theprotease family S8A is incubated with an RNA composition in step (i) that has already beenproduced. Such RNA compositions are described herein. In some embodiments, the RNA composition that has already been produced may have been produced using Proteinase K.As such, in some embodiments, the protease of the protease family S8A used in step (i) ofthe method according to the first aspect of the invention may be a protease that is notProteinase K. In other words, Proteinase K may be used initially during the production of theRNA composition to begin proteolysis of protein reagents used in the production thereof, buta different protease of the protease family S8A (e.g. a Subtilisin protease) may be used tocomplete the digestion of the protein impurities in step (i) of the method according to the firstaspect of the invention. Accordingly, in some embodiments, Proteinase K may be used duringthe production of the RNA composition prior to step (i) of the method according to the firstaspect of the invention, but may not be used in step (i) of the method according to the first aspect of the invention. Step (i)Step (i) of the method according to the first aspect of the invention is a step of incubating theRNA composition with a protease of the protease family S8A, such that the protein impurities in the RNA composition are digested to form peptide products.In some embodiments, the incubation in step (i) may be performed for less than 1 minute toabout 60 minutes. In some embodiments, the incubation in step (i) may be performed for about 1 hour to about 5 hours. In some embodiments, the incubation in step (i) may be performedfor more than 5 hours.In some embodiments, the incubation in step (i) may be performed for less than 10 minutes tomore than 5 hours. In some embodiments, the incubation in step (i) may be performed for about 1 hour to about 5 hours. In some embodiments, the incubation in step (i) may beperformed for about 1 hour to about 4 hours. In some embodiments, the incubation in step (i)may be performed for about 1 hour to about 3 hours. In some embodiments, the incubation instep (i) may be performed for about 1 hour to about 2 hours.In some embodiments, the incubation in step (i) may be performed for about 1.5 hoursto about 5 hours. In some embodiments, the incubation in step (i) may be performed for about 1.5 hours to about 4.5 hours. In some embodiments, the incubation in step(i) may be performed for about 1.5 hours to about 4 hours. In some embodiments, theincubation in step (i) may be performed for about 1.5 hours to about 3.5 hours. In someembodiments, the incubation in step (i) may be performed for about 1.5 hours to about3 hours. In some embodiments, the incubation in step (i) may be performed for about1.5 hours to about 2.5 hours. In some embodiments, the incubation in step (i) may beperformed for about 1.5 hours to about 2 hours.In some embodiments, the incubation in step (i) may be performed for less than 10 minutes;about 10 to about 15 minutes, about 15 to about 20 minutes, about 20 to about 25 minutes, about 25 to about 30 minutes, about 30 to about 35 minutes, about 35 to about 40 minutes, about 40 to about 45 minutes, about 45 to about 50 minutes, about 50 to about 55 minutes, about 55 to about 60 minutes.In some embodiments, the incubation in step (i) may be performed for about 10 minutes, about15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about40 minutes, about 45 minutes, about 50 minutes, or about 55 minutes.In some embodiments, the incubation in step (i) may be performed for about 1 hour to about1.5 hours, about 1.5 to about 2 hours, about 2 to about 2.5 hours, about 2.5 to about 3 hours,about 3 to about 3.5 hours, about 3.5 to about 4 hours, about 4 to about 4.5 hours, or about 4.5 to about 5 hours. In some embodiments, the incubation in step (i) may be performed for about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about4.5 hours, or about 5 hours.In some embodiments, the incubation in step (i) may be performed for about 2 hours.In some embodiments, the incubation in step (i) may be performed for about 2.5 hours.In some embodiments, the incubation in step (i) may be performed at less than 18°C. In someembodiments, the incubation in step (i) may be performed at about 18°C to about 30°C. Insome embodiments, the incubation in step (i) may be performed or at more than 30°C.In some embodiments, the incubation in step (i) may be performed at about 18°C, about 19°C,about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about27°C, about 28°C, about 29°C, or about 30°C.In some embodiments, the incubation in step (i) may be performed at room temperature. It willbe understood that room temperature may be considered as a temperature in the range of 18-25°C, such as about 20°C, about 21°C or about 22°C.In some embodiments, the incubation in step (i) may be performed at 18-25°C. In someembodiments, the incubation in step (i) may be performed at 18-20°C. In someembodiments, the incubation in step (i) may be performed at 21-25°C.In some embodiments, the incubation in step (i) may be performed at 25°C.In some embodiments, the incubation in step (i) may be performed for 2 hours at atemperature of 18, 19, 20, 21, 22, 23, 24, or 25°C.In some embodiments, the incubation in step (i) may be performed for 2.5 hours at atemperature of 18, 19, 20, 21, 22, 23, 24, or 25°C. Step (ii)Step (ii) of the method according to the first aspect of the invention is a step of incubating theRNA composition with an agent that precipitates the RNA in the RNA composition. The agent used to precipitate the RNA in step (ii) may be any agent that is suitable for precipitating RNA. In some embodiments, step (ii) may be a step of acidification. In other words, the agent that causes precipitation of the RNA may be an agent that precipitates the RNA by acidification.In some embodiments, step (ii) may be a step of precipitation with high salt concentration. Inother words, the agent that causes precipitation of the RNA may be an agent that precipitatesthe RNA using a high salt concentration. It will be understood that “high salt concentration”encompasses any salt concentration that is suitable to precipitate the RNA, which may dependon the agent used. In some embodiments, the “high salt concentration” may be a concentrationof about 2 Molar (M), about 1.9 M, about 1.8 M, about 1.7 M, about 1.6 M, about 1.5 M, about1.4 M, about 1.3 M, about 1.2 M, about 1.1 M, about 1.0 M, about 0.9 M, about 0.8 M, about0.7 M, about 0.6 M, about 0.5 M, about 0.4 M, about 0.3 M, about 0.2 M or about 0.1 M.In some embodiments, the agent in step (ii) may be ethanol. In some embodiments, the ethanol may be 70% ethanol. In some embodiments, the agent in step (ii) may be trifluoroacetic acid. In some embodiments, the trifluoroacetic acid may have a final concentration of 1% v / v. In some embodiments, the agent in step (ii) may be formic acid (i.e. methanoic acid). In some embodiments, the agent in step (ii) may be ammonium acetate. In someembodiments, the concentration of the ammonium acetate may be 0.5 M.In some embodiments, the agent in step (ii) may be sodium acetate. In some embodiments,the concentration of the sodium acetate may be 0.3 M. In some embodiments, the agent in step (ii) may be lithium chloride. In some embodiments, the concentration of the lithium chloride may be 0.8 M. It will be understood that the incubation of step (ii) can be carried out at any temperature andfor any duration that suitably allows for precipitation of the RNA in the RNA composition, whichwill depend on the agent used in step (ii). In some embodiments, the incubation in step (ii) may be performed for less than 10 minutes to more than 2 hours. In some embodiments, the incubation in step (ii) may be performed forless than 10 minutes to more than 1.5 hours. In some embodiments, the incubation in step (ii)may be performed for less than 10 minutes to about 60 minutes.In some embodiments, the incubation in step (ii) may be performed for less than 10 minutes; about 10 to about 15 minutes, about 15 to about 20 minutes, about 20 to about 25 minutes,about 25 to about 30 minutes, about 30 to about 35 minutes, about 35 to about 40 minutes,about 40 to about 45 minutes, about 45 to about 50 minutes, about 50 to about 55 minutes, about 55 to about 60 minutes.In some embodiments, the incubation in step (ii) may be performed for about 1 minute, about2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45minutes, about 50 minutes, about 55 minutes, or about 60 minutes.In some embodiments, the incubation in step (ii) may be performed at less than 18°C. In someembodiments, the incubation in step (ii) may be performed at about 18°C to about 30°C. Insome embodiments, the incubation in step (ii) may be performed or at more than 30°C.In some embodiments, the incubation in step (ii) may be performed at a temperature of about5°C, about 4°C, about 3°C, about 2°C, or about 1°C.In some embodiments, the incubation in step (ii) may be performed at about 18°C, about 19°C,about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about27°C, about 28°C, about 29°C, or about 30°C.In some embodiments, the incubation in step (ii) may be performed at room temperature, asdefined above.In some embodiments, the incubation in step (ii) may be performed at 25°C.In some embodiments, the incubation in step (ii) may be performed for 5 minutes or less at atemperature of 18, 19, 20, 21, 22, 23, 24, or 25°C.In some embodiments, the incubation in step (ii) may be performed for 4 minutes at atemperature of 18, 19, 20, 21, 22, 23, 24, or 25°C. Step (iii)Step (iii) of the method according to the first aspect of the invention is a step of purifying thepeptide products in the RNA composition and / or concentrating the peptide products in the RNA composition. In some embodiments, step (iii) may comprise purifying the peptide products in the RNA composition. In some embodiments, step (iii) may comprise concentrating the peptide products in the RNA composition. In some embodiments, step (iii) may comprise purifying thepeptide products in the RNA composition and concentrating the peptide products in the RNAcomposition. In some embodiments, the step of purifying the peptide products in step (iii) may be by solidphase extraction. Suitable methods for solid phase extraction will be known in the art.In some embodiments, the step of concentrating the peptide in step (iii) may be by vacuum-centrifugation. In some embodiments, the step of concentrating the peptide in step (iii) maybe by lyophilisation. Suitable methods for vacuum-centrifugation and lyophilisation will beknown in the art. Step (iv) Step (iv) of the method according to the first aspect of the invention is a step of measuring the peptide products by resolving and quantifying the peptide products.In some embodiments, the step of resolving the peptide products in step (iv) may be by usingliquid chromatography (LC), isoelectric focusing, capillary electrophoresis, direct-infusionmass spectrometry or ion mobility separation, or combinations thereof.In some embodiments, the LC may be selected from the list consisting of: ion exchangechromatography, hydrophilic interaction chromatography, and reversed-phase liquidchromatography.Suitable methods for resolving the peptide products in step (iv) will be known in the art.In some embodiments, the step of resolving the peptide products in step (iv) may be by usingreversed-phase liquid chromatography.In some embodiments, the step of quantifying the peptide products in step (iv) may be byusing mass spectrometry (MS).In some embodiments, the mass spectrometry (MS) may be selected from the list consistingof: multiple reaction monitoring mass spectrometry (MRM-MS), single reaction monitoring(SRM-MS), parallel reaction monitoring mass spectrometry (PRM-MS), data-dependent-acquisition high resolution mass spectrometry (DDA-HRMS), or data-independent acquisitionhigh resolution mass spectrometry (DIA-HRMS).Suitable methods for quantifying the peptide products (e.g. by mass spectrometry) in step (iv)will be known in the art.In some embodiments, the step of quantifying the peptide products in step (iv) may be byusing multiple reaction monitoring mass spectrometry (MRM-MS).Normalisation of the peptide products may be performed for the purposes of quantification.In some embodiments, the peptide products may be normalised to a standard and absolutequantities may be derived from a calibration with identical, synthetic peptides or their parentalproteins. Methods of normalising the peptide products will be known in the art. For example,normalising the peptide products may be based on generation of a calibration curve with thepurified proteins that are used in the production of the RNA composition.Method of reducing protein impuritiesAs well as using the protease of the protease family S8A as described herein for the purposesof a quality control assessment of an RNA composition, it is also apparent that the proteaseof the protease family S8A as described herein may also be used to directly reduce proteinimpurities in an RNA composition.Accordingly, in a second aspect, the present invention provides a method of reducing proteinimpurities in an RNA composition comprising the step of: (i) incubating the RNA composition with a protease such that the protein impurities inthe RNA composition are digested to form peptide products; wherein the protease is a protease of the protease family S8A.It will be understood that in the second aspect of the present invention, the protease of theprotease family S8A may be incubated with an RNA composition in step (i) that has alreadybeen produced. Such RNA compositions are described herein. In some embodiments, the RNA composition that has already been produced may have been produced using Proteinase K.As such, in some embodiments, the protease of the protease family S8A used in step (i) ofthe method according to the second aspect of the invention may be a protease that is not Proteinase K. In other words, Proteinase K may be used initially during the production of theRNA composition to begin proteolysis of protein reagents used in the production thereof, buta different protease of the protease family S8A (e.g. a Subtilisin protease) may be used tocomplete the digestion of the protein impurities in step (i) of the method according to the second aspect of the invention. Accordingly, in some embodiments, Proteinase K may beused during the production of the RNA composition prior to step (i) of the method accordingto the second aspect of the invention, but may not be used in step (i) of the method according to the second aspect of the invention. Alternatively, in some embodiments of the second aspect of the present invention, ProteinaseK may not be used in the production of the RNA composition. In other words, there may be noinitial digestion prior step using Proteinase K. In some embodiments, step (i) of the method according to the second aspect of the inventionmay be performed as defined above according to step (i) of the method according to the firstaspect of the invention.Performing the method according to the second aspect of the invention may result in theprotein impurities in the RNA composition becoming reduced to a level that will not cause toxicor immunogenic effects in vivo or to a level where toxic or immunogenic effects in vivo arereduced, such as substantially reduced.It will be understood that further processes may be utilised to reduce the impurity level byremoving the peptide products. Such processes will be known in the art, and may include steps of filtration, bead purification, and / or chromatography. In some embodiments of the method according to the second aspect of the invention, a furtherstep of filtration, bead purification, and / or chromatography may be utilised.In one embodiment purification is performed by hydrophobic interaction chromatography (HIC). In one embodiment purification is performed by cellulose chromatography. In one embodiment purification is performed by tangential flow filtration (TFF). In one embodiment RNA is purified using magnetic bead purification. In one embodiment tangential flow filtration (TFF) is used for buffer exchange. In some embodiments, TFF buffer exchange is performed after another purification method, optionally HIC or cellulose chromatography.In some embodiments two or more of the RNA purification processes are used.In some embodiments one or more of the purification steps / processes is / are repeated. Insome embodiments one or more of the purification steps / processes is / are repeated at least twice. In one embodiment, the method of the invention comprises one or more purification steps, wherein said purification step is selected from any one or more of: (a) hydrophobic interaction chromatography (HIC); (b) tangential flow filtration (TFF);(c) cellulose chromatography; and / or(d) magnetic bead purification;Optionally, wherein any one or more of said purification steps may be performed one or more times. Hydrophobic interaction chromatography (HIC) is a versatile method for the purification andseparation of biomolecules by using the function of hydrophobicity. HIC utilizes a reversibleinteraction between the proteins and the hydrophobic ligand of a HIC resin. Tangential flow filtration (TFF) is a rapid and efficient method for the separation and purification of biomolecules by utilizing ultrafiltration membranes. The feed stream passes parallel to the membrane face as one portion passes through the membrane (permeate) andthe remainder (retentate) is recirculated back to the feed reservoir. In particular, TFF may alsobe used for buffer exchange after cellulose or HIC purification.RNA may be purified using magnetic bead purification. During magnetic bead purification, amagnetic force is applied to the sample mixture, and the molecule of interest (drug substance), which is attracted to the magnetic beads, is separated from the mixture.RNA may be purified by cellulose chromatography, which is a fast and cost-effective methodfor dsRNA removal. dsRNA binds selectively to cellulose in an ethanol-containing buffer, while ssRNA remains in the flow trough. In some embodiments of the second aspect of the present invention, the level of proteinimpurities in the RNA composition may be 1000 parts per million (ppm) or fewer, 900 ppm orfewer, 800 ppm or fewer, 700 ppm or fewer, 600 ppm or fewer, 500 ppm or fewer, 400 ppm orfewer, 300 ppm or fewer, 200 ppm or fewer, 100 ppm or fewer, 90 ppm or fewer, 80 ppm orfewer, 70 ppm or fewer, 60 ppm or fewer, 50 ppm or fewer, 40 ppm or fewer, 30 ppm or fewer,20 ppm or fewer, 10 ppm or fewer, 5 ppm or fewer, 4 ppm or fewer, 3 ppm or fewer, 2 ppm orfewer, 1 ppm or fewer, or 0.5 ppm or fewer.In some embodiments of the second aspect of the present invention, the level of proteinimpurities in the RNA composition may be 100 ppm or fewer.In some embodiments of the second aspect of the present invention, following incubation withthe protease of the protease family S8A as defined herein, the level of protein impurities withinthe RNA composition may be 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.9%or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less,0.2% or less, 0.1% or less, 0.09% or less, 0.08% or less, 0.07% or less, 0.06% or less, 0.05%or less, 0.04% or less, 0.03% or less, 0.02% or less, 0.01% or less, 0.009% or less, 0.008%or less, 0.007% or less, 0.006% or less, 0.005% or less, 0.004% or less, 0.003% or less,0.002% or less, 0.001% or less, 0.0009% or less, 0.0008% or less, 0.0007% or less, 0.0006%or less, 0.0005% or less, 0.0004% or less, 0.0003% or less, 0.0002% or less, or 0.0001% orless of the RNA composition.Method of producing an RNA product suitable for in vivo administrationAs the protease of the protease family S8A may be used to directly reduce protein impuritiesin an RNA composition, it is also apparent that the protease of the protease family S8A asdescribed herein may also be used in the production of an RNA product suitable for in vivoadministration, such as an RNA product that comprises low levels or no protein impurities.Due to the lack of (or reduced levels of) protein impurities, such an RNA product may bedeemed suitable for in vivo administration due to the reduced likelihood of toxic orimmunogenic effects in vivo.Accordingly, in a third aspect, the present invention provides a method of producing an RNAproduct suitable for in vivo administration, comprising the steps of:(i) incubating an RNA composition with a protease such that the protein impurities inthe RNA composition are digested to form peptide products; wherein the protease is aprotease of the protease family S8A; and (ii) obtaining an RNA product that is suitable for in vivo administration.It will be understood that in the third aspect of the present invention, the protease of theprotease family S8A may be incubated with an RNA composition in step (i) that has alreadybeen produced. Such RNA compositions are described herein. In some embodiments, the RNA composition that has already been produced may have been produced using Proteinase K. In some embodiments of the third aspect of the present invention, the RNA composition may be produced as described herein.In some embodiments of the third aspect of the present invention, the protease of the proteasefamily S8A used in step (i) of the method according to the third aspect of the invention may bea protease that is not Proteinase K. In other words, Proteinase K may be used initially duringthe production of the RNA composition to begin proteolysis of protein reagents used in theproduction thereof, but a different protease of the protease family S8A (e.g. a Subtilisinprotease) may be used to complete the digestion of the protein impurities in step (i) of themethod according to the third aspect of the invention. Accordingly, in some embodiments,Proteinase K may be used during the production of the RNA composition prior to step (i) ofthe method according to the third aspect of the invention, but may not be used in step (i) of the method according to the third aspect of the invention. Alternatively, in some embodiments of the third aspect of the present invention, Proteinase Kmay not be used in the production of the RNA composition. In other words, there may be noinitial digestion prior step using Proteinase K. In some embodiments, step (i) of the method according to the third aspect of the invention may be performed as defined above according to step (i) of the method according to the first aspect of the invention.Performing the method according to the third aspect of the invention may result in the proteinimpurities in the RNA product becoming reduced to a level that will not cause toxic orimmunogenic effects in vivo or to a level where toxic or immunogenic effects in vivo arereduced, such as substantially reduced.It will be understood that further processes may be utilised to reduce the impurity level byremoving the peptide products. Such processes will be known in the art, and may include steps of filtration, bead purification, and / or chromatography.In some embodiments of the method according to the third aspect of the invention, a furtherstep of filtration, bead purification, and / or chromatography may be utilised.Any one or more of the purification steps described in the previous section may also be used in accordance with the third aspect of the invention. In one embodiment, the method of the invention comprises one or more purification steps, wherein said purification step is selected from any one or more of: (a) hydrophobic interaction chromatography (HIC); (b) tangential flow filtration (TFF);(c) cellulose chromatography; and / or(d) magnetic bead purification; Optionally, wherein any one or more of said purification steps may be performed one or more times. In some embodiments of the third aspect of the present invention, the level of protein impuritiesin the RNA product may be 1000 parts per million (ppm) or fewer, 900 ppm or fewer, 800 ppmor fewer, 700 ppm or fewer, 600 ppm or fewer, 500 ppm or fewer, 400 ppm or fewer, 300 ppmor fewer, 200 ppm or fewer, 100 ppm or fewer, 90 ppm or fewer, 80 ppm or fewer, 70 ppm orfewer, 60 ppm or fewer, 50 ppm or fewer, 40 ppm or fewer, 30 ppm or fewer, 20 ppm or fewer,10 ppm or fewer, 5 ppm or fewer, 4 ppm or fewer, 3 ppm or fewer, 2 ppm or fewer, 1 ppm orfewer, or 0.5 ppm or fewer.In some embodiments of the third aspect of the present invention, the level of protein impuritiesin the RNA product may be 100 ppm or fewer.In some embodiments of the third aspect of the present invention, following incubation withthe protease of the protease family S8A as defined herein, the level of protein impurities withinthe RNA product may be 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.9% orless, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2%or less, 0.1% or less, 0.09% or less, 0.08% or less, 0.07% or less, 0.06% or less, 0.05% orless, 0.04% or less, 0.03% or less, 0.02% or less, 0.01% or less, 0.009% or less, 0.008% orless, 0.007% or less, 0.006% or less, 0.005% or less, 0.004% or less, 0.003% or less, 0.002%or less, 0.001% or less, 0.0009% or less, 0.0008% or less, 0.0007% or less, 0.0006% or less,0.0005% or less, 0.0004% or less, 0.0003% or less, 0.0002% or less, or 0.0001% or less ofthe RNA product. UseIt will be understood that the protease of the protease family S8A as described herein may beused to digest protein impurities in an RNA composition.Accordingly, in a fourth aspect, the present invention provides a use of a protease of theprotease family S8A to digest protein impurities in an RNA composition.In some embodiments, the protease of the protease family S8A of the use of the fourth aspectaccording to the present invention may not be Proteinase K. In some embodiments, the protease of the protease family S8A of the use of the fourth aspectaccording to the present invention may be a Subtilisin protease.It will be understood that incubation of an RNA composition with a protease of the proteasefamily S8A as described herein may result in digestion of protein impurities in the RNAcomposition, whereby the protein impurities are digested by the protease of the proteasefamily S8A as described herein to form peptide products.In some embodiments, the RNA composition may be produced as described herein.RNA product suitable for in vivo administrationIt will be understood that the method according to the second aspect of the invention and / or the method according to the third aspect of the invention can be used to obtain an RNA product that comprises low or no traces of protein impurities.As such, an RNA product that is obtained from the method according to the second aspect ofthe invention and / or the method according to the third aspect of the invention may be suitablefor in vivo administration due to reduced levels of protein impurities that are not sufficient tocause toxic or immunogenic effects in vivo.Accordingly, the present invention provides an RNA product suitable for in vivo administration,wherein the RNA product is obtainable by the method according to the second aspect of theinvention.Accordingly, the present invention provides an RNA product suitable for in vivo administration,wherein the RNA product is obtainable by the method according to the third aspect of theinvention.In some embodiments, the RNA product may be administered to a subject.In some embodiments, the subject may be a mammal. In some embodiments, the subject maybe a human. In some embodiments, the subject may a dog, a cat, a horse, a cow, or a pig.In some embodiments, the subject may be a patient. In some embodiments, the subject maybe a human patient. As used herein, it will be understood that the terms “patient” and “subject” are considered to be interchangeable.In some embodiments, the RNA of the RNA product may be any form of RNA.In some embodiments, the RNA of the RNA product may be mRNA.In some embodiments, the RNA of the RNA product may be rRNA.In some embodiments, the RNA of the RNA product may be tRNA.In some embodiments, the level of protein impurities in the RNA product may be 1000 partsper million (ppm) or fewer, 900 ppm or fewer, 800 ppm or fewer, 700 ppm or fewer, 600 ppmor fewer, 500 ppm or fewer, 400 ppm or fewer, 300 ppm or fewer, 200 ppm or fewer, 100 ppmor fewer, 90 ppm or fewer, 80 ppm or fewer, 70 ppm or fewer, 60 ppm or fewer, 50 ppm orfewer, 40 ppm or fewer, 30 ppm or fewer, 20 ppm or fewer, 10 ppm or fewer, 5 ppm or fewer,4 ppm or fewer, 3 ppm or fewer, 2 ppm or fewer, 1 ppm or fewer, or 0.5 ppm or fewer.In some embodiments, the level of protein impurities in the RNA product may be 100 ppm orfewer.In some embodiments, following incubation with the protease of the protease family S8A asdefined herein, the level of protein impurities within the RNA product may be 5% or less, 4%or less, 3% or less, 2% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% orless, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less of the RNA product.Kit In a fifth aspect, the present invention provides a kit comprising: (i) a protease of the protease family S8A; and(ii) an RNA polymerase, a pyrophosphatase, an RNAse inhibitor, and / or a DNAse.In some embodiments, the kit may further comprise Proteinase K. In some embodiments, theprotease of the protease family S8A in (i) of the kit according to the invention may not beProteinase K. In some embodiments of the fifth aspect according to the invention, the proteaseof the protease family S8A in (i) of the kit according to the invention may be a Subtilisinprotease. In some embodiments, the kit may comprise: (i) a protease of the protease family S8A; and(ii) an RNA polymerase, a pyrophosphatase, an RNAse inhibitor, and a DNAse.In some embodiments, the kit may further comprise Proteinase K. In some embodiments, theprotease of the protease family S8A in (i) of the kit according to the invention may not beProteinase K. In some embodiments of the fifth aspect according to the invention, the proteaseof the protease family S8A in (i) of the kit according to the invention may be a Subtilisinprotease.Suitable RNA polymerases, pyrophosphatases, RNAse inhibitors, and DNAses will be knownin the art. In some embodiments, the RNA polymerase may be a T7 polymerase. In some embodiments, the DNAse may be DNAse 1. It will be understood that the reagents (e.g. the enzymes and proteins) of the kit according to the invention may be contained in a suitable vial or container. In some embodiments, the kit may further comprise instructions for using the kit, for example,to reduce and / or quantify protein impurities in an RNA product or RNA composition.In some embodiments, the kit may comprise additional reagents. Protease family S8AThe protease family S8A (also known as the S8A subfamily of serine proteases) is a family ofserine proteases which encompasses Subtilisin proteases and also includes Subtilisin-likeproteases such as Proteinase K.The protease of the protease family S8A of any of the methods, the use, and / or the kitaccording to the invention may be any protease of the protease family S8A as describedherein.In some embodiments of any of the methods according to the present invention, the proteaseof the protease family S8A of step (i) of the any of the methods according to the presentinvention may not be Proteinase K.In some embodiments of the use according to the present invention, the protease of theprotease family S8A of the use according to the present invention may not be Proteinase K.In some embodiments of the kit according to the present invention, the protease of theprotease family S8A of (i) of the kit according to the present invention may not be ProteinaseK. In some embodiments of any of the methods and the use according to the present invention,Proteinase K may be used in combination with another protease of the protease family S8A(e.g. a Subtilisin protease).In some embodiments of any of the methods and the use according to the present invention,Proteinase K may be used in an initial step to begin proteolysis of protein reagents used in theproduction of the RNA composition, but another protease of the protease family S8A asdescribed herein (e.g. a Subtilisin protease) may be used according to the methods and useaccording to the present invention to complete the proteolysis process.In some embodiments, the protease of the protease family S8A may be a naturally occurring protease or a non-naturally occurring protease. In some embodiments, the protease may be a naturally occurring protease. In some embodiments, the protease may be a non-naturally occurring protease. It will be understood that a “naturally occurring protease” may include a protease that is derived or derivable from an organism, e.g. a mammal, a bacterium, a fungus, a plant. It will be understood that a “non-naturally occurring protease” may include a protease that has been generated synthetically using standard molecular biology and protein expression techniques. In some embodiments, the protease of the protease family S8A may be prepared by use ofrecombinant DNA techniques. It will be understood that recombinant DNA techniques forproducing enzymes are known in the art. As such, any recombinant DNA techniques that aresuitable for producing a protease of the protease family S8A according to the invention may be used.In some embodiments, the protease of the protease family S8A may be a Subtilisin protease.In some embodiments, the protease of the protease family S8A may be selected from the list of Subtilisin proteases consisting of: Subtilisin A, Subtilisin DY, Subtilisin BNP’, Subtilisin E,Subtilisin NAT, Subtilisin BL, Subtilisin Savinase, Tk-subtilisin, Subtilisin J, or any variantthereof. It will be understood that the Subtilisin proteases described above have similar cleavage patterns. Subtilisin A, frequently referred to as Subtilisin Carlsberg, is the type-example of the protease family S8A. Subtilisin A possess exceptional temperature stability and resistance to reducing conditions facilitating a rapid and complete digestion of substrates. In addition, it is largelyinsensitive to the sample matrix likely allowing the processing of a broad range of differentsample types. In some embodiments, the protease of the protease family S8A may be Subtilisin A. In some embodiments, Subtilisin A may be Subtilisin A derived from Bacillus licheniformis.In some embodiments, Subtilisin A may comprise or consist of an amino acid sequence havingat least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, Subtilisin A may comprise or consist of an amino acid sequence havingat least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% or 100% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, Subtilisin A may comprise an amino acid sequence having the amino acid sequence of SEQ ID NO: 1. In some embodiments, Subtilisin A may consist of an amino acid sequence having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the protease of the protease family S8A may comprise or consist ofan amino acid sequence having at least 80% identity to any one of the amino acid sequencesselected from the list consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. In some embodiments, the protease of the protease family S8A may comprise or consist of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the protease of the protease family S8A may comprise or consist of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the protease of the protease family S8A may comprise or consist of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the protease of the protease family S8A may comprise or consist of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the protease of the protease family S8A may comprise or consist of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO:6. In some embodiments, the protease of the protease family S8A may comprise or consist of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the protease of the protease family S8A may comprise or consist of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, the protease of the protease family S8A may comprise or consist ofan amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the protease of the protease family S8A may comprise or consist of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the protease of the protease family S8A may comprise an amino acid sequence having the amino acid sequence of any one of the sequences selected from the list consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. In some embodiments, the protease of the protease family S8A may consist of an amino acid sequence having the amino acid sequence of any one of the sequences selected from the list consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.SEQ ID NO: 1 (Subtilisin Carlsberg / Subtilisin A; UniProt: P00780):MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQ FRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVL DTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSG SGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYAKGVVVVAAAGNSGSSGNTNTIGYP AKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILS KHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQSEQ ID NO: 2 (Subtilisin BPN’; UniProt: P00782): MRGKKVWISLLFALALIFTMAFGSTSSAQAAGKSNGEKKYIVGFKQTMSTMSAAKKKDVISEKGGKVQ KQFKYVDAASATLNEKAVKELKKDPSVAYVEEDHVAHAYAQSVPYGVSQIKAPALHSQGYTGSNVKVA VIDSGIDSSHPDLKVAGGASMVPSETNPFQDNNSHGTHVAGTVAALNNSIGVLGVAPSASLYAVKVLG ADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGTSGSSSTV GYPGKYPSVIAVGAVDSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAAL ILSKHPNWTNTQVRSSLENTTTKLGDSFYYGKGLINVQAAAQSEQ ID NO: 3 (Subtilisin E; UniProt: P04189):MRSKKLWISLLFALTLIFTMAFSNMSAQAAGKSSTEKKYIVGFKQTMSAMSSAKKKDVISEKGGKVQK QFKYVNAAAATLDEKAVKELKKDPSVAYVEEDHIAHEYAQSVPYGISQIKAPALHSQGYTGSNVKVAV IDSGIDSSHPDLNVRGGASFVPSETNPYQDGSSHGTHVAGTIAALNNSIGVLGVAPSASLYAVKVLDS TGSGQYSWIINGIEWAISNNMDVINMSLGGPTGSTALKTVVDKAVSSGIVVAAAAGNEGSSGSTSTVG YPAKYPSTIAVGAVNSSNQRASFSSAGSELDVMAPGVSIQSTLPGGTYGAYNGTSMATPHVAGAAALI LSKHPTWTNAQVRDRLESTATYLGNSFYYGKGLINVQAAAQSEQ ID NO: 4 (Subtilisin NAT; UniProt: P35835):MRSKKLWISLLFALTLIFTMAFSNMSAQAAGKSSTEKKYIVGFKQTMSAMSSAKKKDVISEKGGKVQK QFKYVNAAAATLDEKAVKELKKDPSVAYVEEDHIAHEYAQSVPYGISQIKAPALHSQGYTGSNVKVAV IDSGIDSSHPDLNVRGGASFVPSETNPYQDGSSHGTHVAGTIAALNNSIGVLGVAPSASLYAVKVLDS TGSGQYSWIINGIEWAISNNMDVINMSLGGPTGSTALKTVVDKAVSSGIVVAAAAGNEGSSGSTSTVG YPAKYPSTIAVGAVNSSNQRASFSSVGSELDVMAPGVSIQSTLPGGTYGAYNGTSMATPHVAGAAALI LSKHPTWTNAQVRDRLESTATYLGNSFYYGKGLINVQAAAQSEQ ID NO: 5 (Subtilisin DY; UniProt: P00781):AQTVPYGIPLIKADKVQAQGYKGANVKVGIIDTGIAASHTDLKVVGGASFVSGESYNTDGNGHGTHVA GTVAALDNTTGVLGVAPNVSLYAIKVLNSSGSGTYSAIVSGIEWATQNGLDVINMSLGGPSGSTALKQ AVDKAYASGIVVVAAAGNSGSSGSQNTIGYPAKYDSVIAVGAVDSNKNRASFSSVGAELEVMAPGVSV YSTYPSNTYTSLNGTSMASPHVAGAAALILSKYPTLSASQVRNRLSSTATNLGDSFYYGKGLINVEAA AQSEQ ID NO: 6 (Subtilisin - Bacillus pumilus; UniProt: P07518):AQSVPYGISQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVRGGASFVPSETNPYQDGSSHGTHV AGTIAALNNSIGVLGVAPSSALYAVKVLDSTGSGQYSWIINGIEWAISNNMDVINMSLGGPTGSTALK TVVDKAVSSGIVVAAAAGNEGSSGSTSTVGYPAKYPSTIAVGAVNSANQRASFSSAGSELDVMAPGVS IQSTLPGGTYGAYNGTSMATPHVAGAAALILSKHPTWTNAQVRDRLESTATYLGSSFYYGKGLINVQA AAQSEQ ID NO: 7 (Subtilisin BL; UniProt: P29599):AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAG TIAALNNSIGVLGVAPSAELYAVKVLGADGRGAISSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQA VNSATSRGVLVVAASGNSGASSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYP GSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATRSEQ ID NO: 8 (Subtilisin Savinase; UniProt: P29600):AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAG TIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQA VNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYP GSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATRSEQ ID NO: 9 (Tk-subtilisin; UniProt: P58502):MKKSIALVLSIVLLAALFAVPASAGEQNTIRVIVSVDKAKFNPHEVLGIGGHIVYQFKLIPAVVVDVP ANAVGKLKKMPGVEKVEFDHQAVLLGKPSWLGGGSTQPAQTIPWGIERVKAPSVWSITDGSVSVIQVA VLDTGVDYDHPDLAANIAWCVSTLRGKVSTKLRDCADQNGHGTHVIGTIAALNNDIGVVGVAPGVQIY SVRVLDARGSGSYSDIAIGIEQAILGPDGVADKDGDGIIAGDPDDDAAEVISMSLGGPADDSYLYDMI IQAYNAGIVIVAASGNEGAPSPSYPAAYPEVIAVGAIDSNDNIASFSNRQPEVSAPGVDILSTYPDDS YETLMGTSMATPHVSGVVALIQAAYYQKYGKILPVGTFDDISKNTVRGILHITADDLGPTGWDADYGY GVVRAALAVQAALGSEQ ID NO: 10 (Subtilisin J; UniProt: P29142):MRSKKLWISLLFALTLIFTMAFSNMSVQAAGKSSTEKKYIVGFKQTMSAMSSAKKKDVISEKGGKVQK QFKYVNAAAATLDEKAVKELKKDPSVAYVEEDHIAHEYAQSVPYGISQIKAPALHSQGYTGSNVKVAV IDSGIDSSHPDLNVRGGASFVPSETNPYQDGSSHGTHVAGTIAALNNSIGVLGVSPSASLYAVKVLDS TGSGQYSWIINGIEWAISNNMDVINMSLGGPSGSTALKTVVDKAVSSGIVVAAAAGNEGSSGSSSTVG YPAKYPSTIAVGAVNSSNQRASFSSAGSELDVMAPGVSIQSTLPGGTYGAYNGTSMATPHVAGAAALI LSKHPTWTNAQVRDRLESTATYLGNSFYYGKGLINVQAAAQ The RNA composition It will be understood that production of an RNA composition may comprise the step of transcribing RNA from a DNA template in vitro.As used herein, it will be understood that an “RNA composition” may be an RNA sample. Asused herein, it will also be understood that an “RNA composition” may be used to make, and / orbe further processed into, an “RNA product”, which may also be termed an “RNA drugsubstance” or an “RNA drug product”. In other words, incubating an RNA composition with aprotease of the protease family S8A as defined herein may result in an RNA product, e.g. anRNA product that is suitable for in vivo administration.Accordingly, in some embodiments, step (i) according to the first aspect of the invention maybe preceded by a prior step of in vitro transcription to produce the RNA composition, whereinthe prior step comprises transcribing RNA from a DNA template in vitro. In some embodiments, step (i) according to the second aspect of the invention may bepreceded by a prior step of in vitro transcription to produce the RNA composition, wherein theprior step comprises transcribing RNA from a DNA template in vitro.In some embodiments, step (i) according to the third aspect of the invention may be precededby a prior step of in vitro transcription to produce the RNA composition, wherein the prior stepcomprises transcribing RNA from a DNA template in vitro.It will be understood that the process of transcribing RNA from a DNA template in vitro toproduce an RNA composition utilises a variety of protein reagents (e.g. enzymes), such asRNA polymerases, pyrophosphatases, and / or RNAse inhibitors. It will also be understood thattranscribing RNA from a DNA template in vitro in this manner may result in an intermediateRNA / DNA mixture. For example, T7 polymerase may be used to mediate RNA synthesis; a pyrophosphatase(such as an inorganic pyrophosphatase) may be used to degrade pyrophosphate; and anRNAse inhibitor may be used to inhibit the action of RNAses present during in vitro transcription.In some embodiments, the prior step of transcribing RNA from a DNA template in vitro maybe carried out in the presence of an RNA polymerase and / or a pyrophosphatase, and / or anRNAse inhibitor.In some embodiments, the prior step of transcribing RNA from a DNA template in vitro maybe carried out in the presence of an RNA polymerase and a pyrophosphatase, and an RNAseinhibitor. Additional enzymes and / or protein reagents may also be utilised during production of RNA from a DNA template. Enzymes that degrade the DNA template and / or enzymes that degradeproteins may be utilised in this process.For example, DNAse 1 may be used to degrade the DNA template; and Proteinase K may be used to (at least partially) degrade the enzymes and other proteins used in the in vitro transcription process.In some embodiments, the prior step of transcribing RNA from a DNA template in vitro mayfurther comprise a step of incubation with a DNAse.In some embodiments, the prior step of transcribing RNA from a DNA template in vitro mayfurther comprise a step of incubation with Proteinase K.In some embodiments, the prior step of transcribing RNA from a DNA template in vitro mayfurther comprise a step of incubation with a DNAse and a step of incubation with ProteinaseK. Without wishing to be bound by theory, Proteinase K may be used initially to begin proteolysisof protein reagents utilised in production of an RNA composition, which is then followed byincubating the RNA composition with a protease of the protease family S8A (i.e. a proteaseother than Proteinase K) according to step (i) of the methods of the first, second and / or thirdaspect(s) according to the invention and according to the use according to the invention.In some embodiments, the prior step of transcribing RNA from a DNA template in vitro mayfurther comprise a step of ultrafiltration.In some embodiments, the prior step of transcribing RNA from a DNA template in vitro mayfurther comprise a step of incubation with a DNAse and / or a step of incubation with ProteinaseK and / or a step of ultrafiltration.In some embodiments, the prior step of transcribing RNA from a DNA template in vitro mayfurther comprise a step of incubation with a DNAse and a step of incubation with ProteinaseK and a step of ultrafiltration.In some embodiments, the prior step of transcribing RNA from a DNA template in vitro mayfurther comprise a step of incubation with a DNAse and a step of ultrafiltration.In some embodiments, the prior step of transcribing RNA from a DNA template in vitro maybe carried out in the presence of an RNA polymerase, and / or a pyrophosphatase, and / or anRNAse inhibitor, and / or a DNAse, and / or Proteinase K and / or may comprise a step ofultrafiltration.In some embodiments, the prior step of transcribing RNA from a DNA template in vitro maybe carried out in the presence of an RNA polymerase, and a pyrophosphatase, and an RNAseinhibitor, and a DNAse, and Proteinase K, and may comprise a step of ultrafiltration.In some embodiments, the prior step of transcribing RNA from a DNA template in vitro maybe carried out in the presence of an RNA polymerase and a pyrophosphatase, and an RNAseinhibitor, and a DNAse, and may comprise a step of ultrafiltration.In some embodiments, the RNA composition may be suitable for therapeutic administrationand thus may be termed an “RNA product”, an “RNA drug substance” or an “RNA drugproduct”.In some embodiments, the RNA of the RNA composition may be any form of RNA.In some embodiments, the RNA of the RNA composition may be mRNA.In some embodiments, the RNA of the RNA composition may be rRNA.In some embodiments, the RNA of the RNA composition may be tRNA.Identity The terms “identity” and “% sequence identity” as used herein, may refer to the proportion of amino acids (expressed in percent) of an amino acid sequence in a peptide or protein, which across the amino acid sequence, are identical to a reference sequence. The percentage of identity is thus calculated by counting the number of aligned amino acids that are identical (a Match) between two sequences (in the amino acids sequence of the peptide or protein of the invention and in the reference sequence), dividing that number by the total number of aminoacids in the aligned region and multiplying by 100.Therefore, Percentage of Identity = (Matches divided by Length of the aligned region) multiplied by 100. Insertions and deletions are not allowed in the calculation the percentage of identity of an amino acid sequence. Any method of calculating percentage identity is permitted within the scope of the invention. General terms and definitions This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. The term “polypeptide” is used in the conventional sense to mean a series of amino acids, typically L-amino acids, connected one to the other, typically by peptide bonds between the α- amino and carboxyl groups of adjacent amino acids. The term “polypeptide” is used interchangeably with the terms “amino acid sequence”, “peptide” and / or “protein”. The term “residues” is used to refer to amino acids in an amino acid sequence. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure. It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The terms "comprising", "comprises" and "comprised of' as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms "comprising", "comprises" and "comprised of' also include the term "consisting of'.The term "variant" may refer to a polypeptide that has an equivalent function to the amino acidsequences described herein, but which includes one or more amino acid substitutions, insertions or deletions. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto. SUMMARY OF SEQUENCES SEQ ID NO Description1 Subtilisin Carlsberg / Subtilisin A2 Subtilisin BPN'3 Subtilisin E4 Subtilisin NAT5 Subtilisin DY6 Subtilisin - Bacillus pumilus7 Subtilisin BL8 Subtilisin Savinase9 Tk-subtilisin10 Subtilisin J

[0002] NUMBERED PARAGRAPHS The present invention may be described by way of the following numbered paragraphs:1. A method of measuring protein impurities in an RNA composition comprising the stepsof: (i) incubating the RNA composition with a protease of the protease family S8A, such that the protein impurities in the RNA composition are digested to form peptide products; followed by (ii) incubating the RNA composition with an agent that precipitates the RNA in the RNA composition; (iii) purifying the peptide products in the RNA composition and / or concentrating the peptide products in the RNA composition; and (iv) measuring the peptide products by resolving and quantifying the peptide products.2. The method according to paragraph 1, wherein step (ii) is a step of acidification.3. The method according to paragraph 1, wherein step (ii) is a step of precipitation withhigh salt concentration, optionally wherein the salt is lithium chloride.4. The method according to any one of paragraphs 1-3, wherein the step of purifying thepeptide products in step (iii) is by solid phase extraction.5. The method according to any one of paragraphs 1-3, wherein the step of concentratingthe peptide in step (iii) is by vacuum-centrifugation or lyophilisation.6. The method according to any one of paragraphs 1-5, wherein resolving the peptideproducts in step (iv) is by using liquid chromatography (LC), isoelectric focusing, capillary electrophoresis, direct-infusion mass spectrometry or Ion Mobility Separation, or combinations thereof; optionally wherein the LC is ion exchange chromatography, hydrophilic interaction chromatography, or reversed-phase liquid chromatography.7. The method according to paragraph 6, wherein the liquid chromatography (LC) isreversed-phase liquid chromatography.8. The method according to any one of paragraphs 1-7, wherein quantifying the peptideproducts in step (iv) is by mass spectrometry (MS).9. The method according to paragraph 8, wherein the mass spectrometry (MS) isselected from the list consisting of: multiple reaction monitoring mass spectrometry (MRM- MS), single reaction monitoring (SRM-MS), parallel reaction monitoring mass spectrometry (PRM-MS), data-dependent-acquisition high resolution mass spectrometry (DDA-HRMS), or data-independent acquisition high resolution mass spectrometry (DIA-HRMS).10. The method according to paragraph 8 or paragraph 9, wherein the mass spectrometry(MS) is multiple reaction monitoring mass spectrometry (MRM-MS).11. The method according to any one of paragraphs 1-10, wherein the peptide productsare normalized to a standard and absolute quantities are derived from a calibration with identical, synthetic peptides or their parental proteins.12. A method of reducing protein impurities in an RNA composition comprising the step of(i) incubating the RNA composition with a protease such that the protein impurities in the RNA composition are digested to form peptide products; wherein the protease is a protease of the protease family S8A.13. A method of producing an RNA product suitable for in vivo administration, comprisingthe steps of: (i) incubating an RNA composition with a protease such that the protein impurities in the RNA composition are digested to form peptide products; wherein the protease is a protease of the protease family S8A; and (ii) obtaining an RNA product that is suitable for in vivo administration.14. The method according to any one of paragraphs 1-13, wherein the protease is anaturally occurring protease or a non-naturally occurring protease.15. The method according to any one of paragraphs 1-14, wherein the protease isprepared by use of recombinant DNA techniques.16. The method according to any one of paragraphs 1-15, wherein the protease is selectedfrom the list consisting of: Subtilisin A, Subtilisin DY, Subtilisin BNP’, Subtilisin E, and Subtilisin NAT, Subtilisin BL, Subtilisin Savinase, Tk-subtilisin, Subtilisin J, or any variant thereof.17. The method according to any one of paragraphs 1-16, wherein the protease isSubtilisin A.18. The method according to any one of paragraphs 1-17, wherein the protease isSubtilisin A derived from Bacillus licheniformis.19. The method according to any one of paragraphs 16-18, wherein the Subtilisin Acomprises or consists of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 1.20. The method according to any one of paragraphs 16-19, wherein the Subtilisin Acomprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 1.21. The method according to any one of paragraphs 1-20, wherein step (i) is preceded bya prior step of in vitro transcription to produce the RNA composition, wherein the stepcomprises transcribing RNA from a DNA template in vitro.22. The method according to paragraph 21, wherein the prior step of transcribing RNAfrom a DNA template in vitro is carried out in the presence of an RNA polymerase, apyrophosphatase, and / or an RNAse inhibitor to produce an intermediate RNA / DNA mixture.23. The method according to paragraph 21 or paragraph 22, wherein the prior step oftranscribing RNA from a DNA template in vitro is followed by an additional step of incubationwith a DNAse and / or Proteinase K and a further step of ultrafiltration, prior to step (i) according to any one of paragraphs 1-20.24. The method according to any one of paragraphs 1-23, wherein the protein impuritiescomprise intact or digested enzyme reagents and / or proteins used in the production of the RNA composition.25. The method according to any one of paragraphs 1-24, wherein the protein impuritiesare derived from one or more proteins selected from the list consisting of: an RNA polymerase,a pyrophosphatase, an RNAse inhibitor, a DNAse and Proteinase K.26. The method according to any one of paragraphs 1-25, wherein step (i) is performedfor: (i) less than 1 minute to about 60 minutes; or (ii) about 1 hour to about 5 hours; or (iii) more than 5 hours.27. The method according to paragraph 26, wherein step (i) is performed for:(i) less than 10 minutes; about 10 to about 15 minutes, about 15 to about 20 minutes, about 20 to about 25 minutes, about 25 to about 30 minutes, about 30 to about 35 minutes, about 35 to about 40 minutes, about 40 to about 45 minutes, about 45 to about 50 minutes, about 50 to about 55 minutes, about 55 to about 60 minutes; or (ii) about 1 hour to about 1.5, about 1.5 to about 2 hours, about 2 to about 2.5 hours, about 2.5 to about 3 hours, about 3 to about 3.5 hours, about 3.5 to about 4 hours, about 4 to about 4.5 hours, or about 4.5 to about 5 hours.28. The method according to paragraph 27, wherein step (i) is performed for about 2 hours.29. The method according to any one of paragraphs 1-27, wherein step (i) is performed atless than 18°C, at about 18°C to about 30°C, or at more than 30°C.30. The method according to paragraph 29, wherein step (i) is performed at 18°C, 19°C,20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C.31. The method according to paragraph 29 or paragraph 30, wherein step (i) is performedat 25°C.32. The method according to any one of paragraphs 1-31, wherein the RNA is suitable fortherapeutic administration.33. The method according to any one of paragraphs 1-32, wherein the RNA is mRNA.34. An RNA product suitable for in vivo administration obtainable by the method accordingto any one of paragraphs 12-33.35. Use of a protease to digest protein impurities in an RNA composition, wherein theprotease is a protease of the protease family S8A.36. The use according to paragraph 35, wherein the protease is a naturally occurringprotease or a non-naturally occurring protease.37. The use according to paragraph 35 or paragraph 36, wherein the proteases is preparedby use of recombinant DNA techniques.38. The use according to any one of paragraphs 35-37, wherein the protease is selectedfrom the list consisting of: Subtilisin A, Subtilisin DY, Subtilisin BNP’, Subtilisin E, and Subtilisin NAT, Subtilisin BL, Subtilisin Savinase, Tk-subtilisin, Subtilisin J, or any variant thereof.39. The use according to any one of paragraphs 35-38, wherein the protease is SubtilisinA.40. The use according to any one of paragraphs 35-39, wherein the protease is SubtilisinA derived from Bacillus licheniformis.41. The use according to any one of paragraphs 38-40, wherein the Subtilisin A comprisesan amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 1.42. The use according to any one of paragraphs 38-41, wherein the Subtilisin A comprisesan amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 1.43. A kit comprising: (i) a protease of the protease family S8A;(ii) an RNA polymerase, a pyrophosphatase, an RNAse inhibitor, and / or a DNAse; andoptionally (iii) Proteinase K and / or (iv) instructions for using the kit to reduce and / or quantify proteinimpurities in an RNA product or RNA composition.44. The kit according to paragraph 43, wherein the protease is selected from the listconsisting of: Subtilisin A, Subtilisin DY, Subtilisin BNP’, Subtilisin E, and Subtilisin NAT, Subtilisin BL, Subtilisin Savinase, Tk-subtilisin, Subtilisin J, or any variant thereof. The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

[0003] EXAMPLES Example 1 Materials and Methods Brief summary of the assayFollowing adjustment of the reaction conditions and addition of isotope-labeled internalstandards for the IVT proteins, the RNA sample is in-solution digested with Subtilisin A (Figure1). Then, the mRNA is precipitated through acidification of the sample and the peptide products are purified from the supernatant by solid phase extraction (SPE) (Figure 2). The purified peptides are then quantified as for a standard surrogate peptide approach. Briefly, peptides are resolved by reversed-phase (RP) liquid chromatography and quantified by multiple reaction monitoring mass spectrometry. The measured intensity of the respective surrogate peptide is normalized to that of its internal standard and absolute quantities are derived from an external calibration with the purified target proteins. ReagentsUnless specified otherwise, reagents were purchased from Sigma Aldrich. Water was purifiedwith a Arium Pro VF system (Sartorius). Recombinant T7 RNA Polymerase, DNAse 1,inorganic pyrophosphatase, RNase Inhibitor Ribolock and Proteinase K were obtained fromThermo Fisher Scientific. 15N isotope-labeled protein internal standards were purchased fromrPeptide Inc. (T7 RNA Polymerase and inorganic pyrophosphatase) or Promise ProteomicsS.a.s (DNAse 1 and Proteinase K), respectively. The 13C15N isotope-labeled internal standardpeptide LLPL was purchased from JPT Peptide Technologies GmbH.In-solution subtilisin A digestion of protein impuritiesA 0.2 ml RNA DS (RNA drug substance) sample was spiked with isotope-labelled internalstandards, treated with TCEP (Tris(2-carboxyethyl)phosphine; 2 mM final concentration) andincubated in a thermomixer at 95°C for 10 min. After cooling, Subtilisin A (0.6 U / ml finalconcentration) was added and the sample was incubated for 2 hours in a thermomixer at25 °C. TFA (Trifluoroacetic acid) was added (1% v / v final concentration) to stop the digestionand precipitate the mRNA. For generation of an external calibration curve, an equimolarmixture of the recombinant proteins was treated identical to the RNA DS samples. Peptide concentrationAcidified digests were centrifuged at 3200 x g for 5 min at room temperature. The supernatantwas captured on a AttractSPE HLB microelution plate (Affinisep S.a.s), washed twice with0.2% TFA in water and then eluted with 25 % acetonitrile in water. The peptide eluate wasdiluted 1:1 with water. LC-MS / MS measurementThe diluted peptide eluate was separated on a Nexera XS UPLC system (Shimadzu) equippedwith a ACQUITY Premier Peptide HSS T3 column (2.1 x 50 mm, Waters Corporation) using a4 minute linear gradient of 10-23% (v / v) acetonitrile in water at a flow rate of 0.5 ml / min and40°C.0.1% (v / v) formic acid was used as a modifier in both mobile phases. Eluting target- andinternal standard peptides were detected on a LCMS-8060NX triple quadrupole massspectrometer (Shimadzu) operating in positive, scheduled multiple reaction monitoring (MRM)mode. Data analysis The MS / MS peak area of the respective analyte peptide was normalized to that of the corresponding internal standard and the absolute quantity of each analyte was calculated using the external calibration curve. Accuracy of the method was determined as the recovery of a defined concentration of recombinant analyte protein spiked into a representative RNA DS sample. ResultsA comprehensive characterization of the assay was conducted to determine key performanceparameters including specificity, lower limit of quantification (LLoQ), upper limit of quantification (ULoQ), calibration curve coefficient of determination (R2), autosampler stabilityof the analytes, carry-over as well as precision and accuracy of the assay at different analytelevels. In this study RNA DS after TFF+Cellulose purification was used.The results aresummarized in Table 1.Table 1: Assay performance parametersParameter Specificity Demonstrated Demonstrated Demonstrated 52% of LLoQ DemonstratedLLoQ (ng / mL) 0.43 1.48 0.44 0.74 0.48ULoQ (ng / mL) 104.04 355.88 104.63 176.48 115.86Coefficient of determination R20.9987 0.9999 0.9966 0.9955 0.9995Autosampler Stability 12°C, 48 hours (mean4 2 -2 -3change in %)Carry-over (%) 10.19 5.87 11.54 12.11 8.62Precision (%) System Precision (%) Method3.15 1.96 2.93 4.27 3.93Precision (%) Inter-operator6.73 0.74 31.79 1.84 2.90LLoQ Level Precision (%) Inter-operator10.91 5.34 3.47 18.34 13.60Low Level Precision (%) Inter-operator11.21 4.36 0.77 2.96 12.77Medium Level Precision (%) Inter-operator6.15 6.20 14.63 9.41 4.87High Level Precision (%) Inter-day25.73 3.66 9.73 12.10 15.13LLoQ Level Precision (%) Inter-day7.61 2.87 9.40 6.02 6.85Low Level Precision (%) Inter-day19.67 1.78 12.45 17.10 11.88Medium Level Precision (%) Inter-day6.68 2.49 6.29 2.91 4.98High Level Accuracy LLoQ Level80.68 90.64 80.45 80.36 81.47Accuracy Low Level91.45 96.76 106.06 93.84 103.30Accuracy Medium Level85.71 96.04 97.14 90.17 89.77Accuracy High Level 100.98 97.95 107.69* Surrogate peptide sequence used for quantificationng = nanogram; mL = milliliter; LLoQ = lower limit of quantification; ULoQ = upper limit of quantification.Furthermore, the suitability of the assay for different matrices was characterized throughfortification of RNA drug substance representative of seven typical production processescomprising IVT, purification and storage buffers (Table 2).Table 2: Purification processes and buffersProcess Purification type Buffer 1HIC / TFF 18 mM HEPES / 18 mM EDTA, pH 6.22 HIC / TFF 18 mM HEPES / 18 mM EDTA, pH 7.03 TFFonly 18 mM HEPES / 18 mM EDTA, pH 7.04 MagBeads 18 mM HEPES / 18 mM EDTA / 100 mM NaCl, pH 7.05 MagBeads / Cellulose / TFF 10 mM HEPES / 0.1 EDTA, pH 7.06 TFFonly 10 mM HEPES / 0.1 EDTA, pH 7.07 TFFonly 10 mM HEPES / 0.1 EDTA, pH 7.0HIC= hydrophobic interaction chromatography; TFF= tangential flow filtration: HEPES=4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); EDTA=Ethylenediaminetetraacetic acid; NaCl=Sodium chloride; MagBeads=Magnetic beadsThe accuracy, calculated based on the recovery of spiked analytes, is shown in Table 3.Table 3: Accuracy for matrices from different purification processes in percent (%)FortificatioProteinase KT7DNAse 1 Ribolock n level (fmol / µL) 10.4 91.8 97.1 75.5a) 94.8 93.71 0.8 84.3 92.0 73.9a) 91.6 73.7a)1 1.6 94.4 92.2 76.6a) 92.3 102.31 2.8 91.8 91.7 68.7a) 92.8 99.72 0.4 105.2 99.0 101.9 94.6 103.82 0.8 111.6 97.6 115.5 102.0 114.22 1.6 106.2 99.9 110.8 100.2 108.22 2.8 98.6 99.9 103.0 99.3 99.03 0.4 98.1 101.4 91.5 96.7 90.63 0.8 98.5 98.0 92.5 97.4 88.03 1.6 92.6 98.0 93.8 99.2 98.83 2.8 99.4 99.8 90.8 100.5 104.04 0.4 79.4a) 101.8 68.9a) 89.2 72.3a)4 0.8 100.4 100.9 101.4 103.4 104.44 1.6 109.5 99.8 111.8 102.9 122.9a)4 2.8 117.1 100.0 99.9 100.6 126.9a)5 0.4 97.8 87.6 75.7a) 75.3a) 72.5a)5 0.8 105.4 96.7 93.5 100.8 97.25 1.6 114.4 98.4 105.9 98.1 112.05 2.8 97.9 101.7 92.2 98.4 98.36 0.4 77.1a) b) 96.1 63.3a) 83.9 61.6a)6 0.8 94.6 97.9 91.0 99.0 87.16 1.6 97.1 101.6 93.8 101.0 95.16 2.8 100.3 99.5 90.5 99.7 101.47 0.4 90.6 100.0 83.4 90.6 98.97 0.8 116.0 103.0 96.5 92.3 114.07 1.6 109.6 102.4 100.3 96.7 112.97 2.8 105.8 104.0 94.3 98.0 104.9* Surrogate peptide sequence used for quantification a) Recovery outside the range of 80-120%; b) accuracy calculation impaired by high analyte levels in unspiked sample Discussion Despite the challenge that Proteinase K-mediated RNA purification poses to protein impurity quantification, the assay performance parameters are within the typical acceptance criteria forLC-MS / MS based protein quantification (Jenkins et al. 2015, The AAPS journal 17 (1), pp. 1– 16.). The quantitative assay range of approximately 1-100 ng of each individual IVT proteinper ml of RNA DS typically corresponds to an impurity content of 0.5 - 400 parts-per-million,which covers the range relevant for impurities in biotherapeutics very well. In contrast to threshold-based or total protein methods, respectively, the assay described heredelivers quantitative information for each individual protein used in the in vitro transcriptionprocess, which is not only key for assessing the safety profile of the RNA composition but also enables the optimization of the RNA purification processes. The assay generally provides residual protein quantification with acceptable accuracy independent of the RNA DS matrix. At the lowest fortification level, the accuracy for DNAse1, Proteinase K and inorganic Pyrophosphatase is reduced in some RNA DS matrices. However, these deviations correspond to absolute protein mass concentrations of only < 5 ng / ml, which corresponds to approximately 2-10 ppm of the RNA content. All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

CLAIMS1. A method of measuring protein impurities in an RNA composition comprising the stepsof: (i) incubating the RNA composition with a protease of the protease family S8A, suchthat the protein impurities in the RNA composition are digested to form peptide products; followed by (ii) incubating the RNA composition with an agent that precipitates the RNA in the RNAcomposition; (iii) purifying the peptide products in the RNA composition and / or concentrating thepeptide products in the RNA composition; and (iv) measuring the peptide products by resolving and quantifying the peptide products.

2. The method according to claim 1, wherein step (ii) is a step of acidification or is a stepof precipitation with high salt concentration, optionally wherein the salt is lithium chloride.

3. The method according to claim 1 or claim 2, wherein (a) the step of purifying thepeptide products in step (iii) is by solid phase extraction; or (b) the step of concentrating the peptide in step (iii) is by vacuum-centrifugation or lyophilisation.

4. The method according to any one of claims 1-3, wherein resolving the peptide productsin step (iv) is by using liquid chromatography (LC), isoelectric focusing, capillaryelectrophoresis, direct-infusion mass spectrometry or Ion Mobility Separation, or combinationsthereof; optionally wherein the LC is ion exchange chromatography, hydrophilic interactionchromatography, or reversed-phase liquid chromatography.

5. The method according to any one of claims 1-4, wherein quantifying the peptideproducts in step (iv) is by mass spectrometry (MS), optionally wherein the mass spectrometry(MS) is selected from the list consisting of: multiple reaction monitoring mass spectrometry(MRM-MS), single reaction monitoring (SRM-MS), parallel reaction monitoring massspectrometry (PRM-MS), data-dependent-acquisition high resolution mass spectrometry (DDA-HRMS), or data-independent acquisition high resolution mass spectrometry (DIA- HRMS).

6. The method according to any one of claims 1-5, wherein the peptide products arenormalized to a standard and absolute quantities are derived from a calibration with identical,synthetic peptides or their parental proteins.

7. A method of reducing protein impurities in an RNA composition comprising the step of(i) incubating the RNA composition with a protease such that the protein impurities inthe RNA composition are digested to form peptide products; wherein the protease is a protease of the protease family S8A.

8. A method of producing an RNA product suitable for in vivo administration, comprisingthe steps of:(i) incubating an RNA composition with a protease such that the protein impurities inthe RNA composition are digested to form peptide products; wherein the protease is aprotease of the protease family S8A; and (ii) obtaining an RNA product that is suitable for in vivo administration.

9. Use of a protease to digest protein impurities in an RNA composition, wherein theprotease is a protease of the protease family S8A.

10. The method according to any one of claims 1-8 or the use according to claim 9, whereinthe protease is selected from the list consisting of: Subtilisin A, Subtilisin DY, Subtilisin BNP’,Subtilisin E, and Subtilisin NAT, Subtilisin BL, Subtilisin Savinase, Tk-subtilisin, Subtilisin J, orany variant thereof.

11. The method or use according to claim 10, wherein the protease is Subtilisin A derivedfrom Bacillus licheniformis.

12. The method or use according to claim 10 or claim 11, wherein the Subtilisin Acomprises or consists of an amino acid sequence having at least 80% identity to the aminoacid sequence of SEQ ID NO: 1; optionally wherein the Subtilisin A comprises or consists ofan amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 1.

13. The method according to any one of claims 1-8 or 10-12, wherein step (i) is precededby a prior step of in vitro transcription to produce the RNA composition, wherein the stepcomprises transcribing RNA from a DNA template in vitro.

14. The method according to claim 13, wherein the prior step of transcribing RNA from aDNA template in vitro is carried out in the presence of an RNA polymerase, apyrophosphatase, and / or an RNAse inhibitor to produce an intermediate RNA / DNA mixture.

15. The method according to claim 13 or claim 14, wherein the prior step of transcribingRNA from a DNA template in vitro is followed by an additional step of incubation with a DNAseand / or Proteinase K and a further step of ultrafiltration, prior to step (i) of the method accordingto any one of claims 1-8 or 10-14.

16. The method according to any one of claims 1-8 or 10-15, wherein the protein impuritiescomprise intact or digested enzyme reagents and / or proteins used in the production of the RNA composition.

17. The method according to any one of claims 1-8 or 10-16, wherein the protein impuritiesare derived from one or more proteins selected from the list consisting of: an RNA polymerase,a pyrophosphatase, an RNAse inhibitor, a DNAse and Proteinase K.

18. The method according to any one of claims 1-8 or 10-17, wherein step (i) is performedfor: (i) less than 1 minute to about 60 minutes; or (ii) about 1 hour to about 5 hours; or(iii) more than 5 hours; optionally wherein step (i) is performed for:(iv) less than 10 minutes; about 10 to about 15 minutes, about 15 to about 20 minutes, about 20 to about 25 minutes, about 25 to about 30 minutes, about 30 to about 35 minutes, about 35 to about 40 minutes, about 40 to about 45 minutes, about 45 to about 50 minutes, about 50 to about 55 minutes, about 55 to about 60 minutes; or(v) about 1 hour to about 1.5, about 1.5 to about 2 hours, about 2 to about 2.5 hours, about 2.5 to about 3 hours, about 3 to about 3.5 hours, about 3.5 to about 4 hours, about 4 to about 4.5 hours, or about 4.5 to about 5 hours.

19. The method according to any one of claims 1-8 or 10-18, wherein step (i) is performedat less than 18°C, at about 18°C to about 30°C, or at more than 30°C; optionally wherein step (i) is performed at 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C.

20. The method according to any one of claims 1-8 or 10-19, wherein the RNA is suitablefor therapeutic administration.

21. The method according to any one of claims 1-8 or 10-20, wherein the RNA is mRNA.

22. An RNA product suitable for in vivo administration obtainable by the method accordingto any one of claims 7, 8 or 10-21.

23. A kit comprising:(i) a protease of the protease family S8A;(ii) an RNA polymerase, a pyrophosphatase, an RNAse inhibitor, and / or a DNAse;and optionally(iii) Proteinase K and / or (iv) instructions for using the kit to reduce and / or quantify proteinimpurities in an RNA product or RNA composition.

24. The kit according to claim 23, wherein the protease is selected from the list consistingof: Subtilisin A, Subtilisin DY, Subtilisin BNP’, Subtilisin E, and Subtilisin NAT, Subtilisin BL,Subtilisin Savinase, Tk-subtilisin, Subtilisin J, or any variant thereof.

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