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WO2025186218A1 - Purification et analyse d'arn - Google Patents

Purification et analyse d'arn

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Publication number
WO2025186218A1
WO2025186218A1 PCT/EP2025/055779 EP2025055779W WO2025186218A1 WO 2025186218 A1 WO2025186218 A1 WO 2025186218A1 EP 2025055779 W EP2025055779 W EP 2025055779W WO 2025186218 A1 WO2025186218 A1 WO 2025186218A1
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WO
WIPO (PCT)
Prior art keywords
rna
sample
mrna
column
volume
Prior art date
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Pending
Application number
PCT/EP2025/055779
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English (en)
Other versions
WO2025186218A8 (fr
Inventor
Hans Blom
Peter LUNDBÄCK
Wangshu JIANG
Birgit BLANK
Nils Norrman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cytiva Bioprocess R&D AB
Original Assignee
Cytiva Bioprocess R&D AB
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Filing date
Publication date
Application filed by Cytiva Bioprocess R&D AB filed Critical Cytiva Bioprocess R&D AB
Publication of WO2025186218A1 publication Critical patent/WO2025186218A1/fr
Publication of WO2025186218A8 publication Critical patent/WO2025186218A8/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase

Definitions

  • the present invention relates to methods for purifying RNA from a sample and, in particular from a sample obtainable by in vitro transcription of RNA.
  • the invention also relates to method of determining the concentration of RNA in a sample.
  • Reliable systems for purifying and separating biomolecules in a solution is a critical component of pharmaceutical and biotechnical industries. These industries put high demands on high quality products that are produced in a safe way.
  • mRNA technology enables rapid tailoring of the mRNA sequence in response to seasonal variations, which makes mRNA-based vaccines an attractive therapeutic strategy for future pandemics, as well as endemics and other infectious diseases such as rabies, Zika, and cytomegalovirus infection.
  • diseases such as HIV, various types of cancers, cystic fibrosis and diabetes.
  • mRNA vaccines are both safer, as the probability of random genome integration is virtually zero, and do not require nuclear entry for expression.
  • the mRNA only needs to reach the cytosol, where it is translated into the antigen using the cellular translation machinery. Furthermore, as the expression of the coded antigens is transient, the mRNA is generally quickly degraded within a few days at most by native cellular processes involving RNases. In addition, since the production of mRNA is based on a cell free system using IVT, safety concerns regarding the presence of cell-derived impurities and viral contaminants are virtually eliminated compared to the production of biologicals found in most other platforms as the mRNA cannot create any protein other than the protein for which it is coded.
  • mRNA technology has also shown to be a good candidate for use within personalized medicine.
  • Personalized medicine is an approach to healthcare that considers an individual's genetic makeup, lifestyle and environmental factors to develop personalized treatment plans.
  • the use of mRNA vaccines in personalized medicine involves the development of vaccines tailored to an individual's unique genetic profile.
  • cancer vaccines can be designed to target specific mutations or neoantigens found in a patient's tumor, providing a personalized treatment option.
  • vaccines for autoimmune diseases can be tailored to an individual's specific immune profile, improving their efficacy and reducing the risk of adverse effect.
  • RNA polymerase generally T7, SP6 or T3 RNA polymerases
  • NTPs nucleotide triphosphates
  • the IVT reaction requires other components such as the polymerase cofactor Mg 2+ and optimized buffer conditions at a suitable pH. Additional components and reagents may also be used, depending on the process design.
  • the template must be produced in advance, usually by enzymatic linearization of purified plasmid DNA, but it can also be produced by amplification of the region of interest using PCR.
  • capping of the 5'end increases stability of the mRNA by providing protection against exonuclease degradation, as well as improves protein translation. This can be performed for example during the IVT step by use of specific nucleotide such as the cap dinucleotide CleanCap® (TriLink BioTechnologies) or by using a separate post-IVT two-step enzymatic procedure based on the vaccinia capping enzyme (VCC).
  • VCC vaccinia capping enzyme
  • the IVT reaction can be performed within a few hours and generally produce milligram quantities of mRNA per millilitre, depending on scale and design. Compared to the timeconsuming processes of conventional vaccines, this provides an additional benefit as this significantly lowers the probability for contamination to occur. From a manufacturing perspective the flexible nature of the mRNA platform is highly valuable since the encoded antigen can be changed without affecting the physical-chemical characteristics of the mRNA backbone and as the performance of the IVT reaction is not dependent on the antigen encoded in the template this allows for standardized procedures. Thus, the combination of speed and flexibility associated with their development and production, the low costs and relatively simple manufacturing, as well as the ease of scalability makes the mRNA technology a very efficient platform to attack pandemics.
  • composition of the IVT reaction components may vary significantly depending on application, construct, enzymes used as well as other factors.
  • conditions for template digestion can vary significantly and is not restricted to the conditions shown in the examples hereinbelow, since the IVT material can have been processed/exposed to other additional steps e.g. capping or other chromatographic steps.
  • mRNA production process can generally be divided into upstream processing, which consists of the enzymatic generation of mRNA by IVT, and downstream processing which includes the unit operations required for purifying the mRNA product.
  • TFF also known as crossflow filtration
  • Microfiltration membranes typically have a pore size between 0.1 pm and 10 pm
  • ultrafiltration membranes have a pore size between 0.001 pm and 0.1 pm and are therefore often used for concentrating and desalting dissolved proteins, peptides, nucleic acids, carbohydrates and other molecules.
  • WO 2014/140211 teaches the use of core bead flow- through chromatography for RNA purification alone or in combination with TFF and/or hydroxyapatite chromatography.
  • the initial sample has to be diluted prior to loading, and the chromatography method requires a frequent harsh cleaning step (cleaning-in-place, CIP), e.g.
  • mRNA may be purified from IVT reaction mixtures using core bead flow-through chromatography, resulting in limited purity, such as about 90 % purity or less, and therefore core bead flow-through chromatography typically is required to be supplemented with additional methods for achieving pharmaceutical grade purity of mRNA.
  • Core bead chromatography often suffers from low capacity (or processability) when used in combination with complex start materials, i.e. when there is a lot of or a diversity in type of contaminants that need to be removed.
  • RNA purification methods wherein said method is cost-, time- and resource-efficient, but also allow for purification of very small sample volumes without compromising the pharmaceutical-grade purity and yield of the purified RNA.
  • Said method should be suitable for use when RNA is purified from an IVT reaction sample, and thus the sample is comprising several different biological and non- biological components.
  • One object of the invention is to provide methods and uses having unique and advantageous characteristics for purifying RNA from a sample.
  • Another object of the invention is to provide methods and uses for purifying RNA from a sample with a high recovery.
  • Another object of the invention is to provide methods and uses for purifying a sample containing RNA, such as an IVT reaction mixture, to yield RNA of high purity.
  • Another object of the invention is to provide methods and uses for purifying RNA from a sample, wherein said method has a short process time.
  • Another object of the invention is to provide methods and uses for purifying RNA from a sample, wherein said methods and uses are suitable for small-scale as well as for large-scale RNA purification.
  • Another object of the invention is to provide methods and uses for purifying mRNA from an IVT reaction sample.
  • Another object of the invention is to provide methods for purifying RNA from a sample, wherein said methods and uses comprise determining the concentration of the purified RNA.
  • Another object of the invention is to provide methods for purifying RNA from a sample, wherein said methods and uses comprise conditioning of said RNA.
  • Another object of the invention is to provide methods and uses for purifying RNA from a sample, wherein said methods and uses comprise conditioning of said RNA and determining the concentration of the purified RNA.
  • the present disclosure provides a method for purifying RNA from a sample comprising the RNA, DNA, protein and optionally further components, wherein said method comprises a) enzymatically digesting DNA in said sample to obtain a DNase-treated sample, and b) separating RNA of said DNase-treated sample from proteins and residual DNA fragments present in said DNase-treated sample by size exclusion chromatography (SEC) using a chromatography medium.
  • SEC size exclusion chromatography
  • step b) also separates the RNA of said DNase-treated sample from optional further components, including NTPs, buffer components, salts, detergents and additional reagents.
  • SEC also known as gel filtration
  • SEC is the mildest of all the chromatography techniques known in the art, which separates molecules by differences in size as they pass through a resin packed in a column.
  • SEC is widely used to partition proteins based on size. The present inventors have found that SEC is surprisingly beneficial for use in a method for purifying
  • the use of SEC in a method for purifying RNA is contemplated to offer advantages in terms of avoiding the drawbacks associated with existing methods known for RNA purification.
  • the method as disclosed herein provides a significantly reduced process time for RNA purification in comparison to that of known techniques.
  • many diafiltration volumes e.g. at least 5-7
  • the process time when using the method as disclosed herein may be at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90% shorter in comparison to the process time when using TFF for RNA purification.
  • the method according to the present invention can achieve the same purification in less than 2 minutes.
  • the present method offers a higher recovery yield of the RNA in comparison to that obtainable by existing methods, such as using TFF.
  • the method as disclosed herein is beneficial for purifying RNA to achieve RNA with high purity.
  • the present method is superior to known techniques as it is suitable for purifying RNA within a short period of time, while maintaining a high recovery yield and high purity of the RNA.
  • the method is easily scalable to different sample volumes, such as to sample volumes of the sub-mL range and above. Therefore, the method is suitable for both small- and large-scale RNA purification.
  • the method is thus provided for small- and large-scale RNA purification, wherein the volume of the sample is in the sub-mL range or higher.
  • the method is provided for large-scale RNA purification, wherein the volume of the sample is about 500 mL or higher. In fact, the method could be used for sample volumes of 10 L or higher.
  • the method is provided for small-scale RNA purification, wherein the volume of the sample is about 500 mL or less, such as about 100 mL or less, such as about 50 mL or less, such as about 10 mL or less, such as about 5 mL or less, such as about 4 mL or less, such as about 3 mL or less, such as about 2 mL or less, such as about 1 mL or less.
  • the method is particularly advantageous for small-scale RNA purification since the currently used preferred method for such purpose, wherein RNA is precipitated with lithium-chloride (LiCI) or TRIzol to remove components other than RNA present in a sample.
  • LiCI lithium-chloride
  • Precipitation with LiCI furthermore requires a prolonged process time as typically occurs overnight.
  • concentrations above 1 mg/ml are desirable due to the low recovery when having too low concentrations.
  • precipitation of RNA with other methods such as ammonium acetate and phenol/chloroform should preferably also be avoided.
  • Most, if not all, precipitation methods, not limited to the methods mentioned above are time-consuming and involve potentially toxic components. Furthermore, they require limited working volumes and are consequently difficult to scale-up for a large-scale production process.
  • the present method is advantageous in that any precipitation can be excluded from the process.
  • Another non-limiting reason for this particular advantage is that the present method is suitable for purifying RNA from sample volumes applicable for example in precision (also known as personalized) medicine.
  • the process may furthermore be GMP (Good Manufacturing Practice) compliant.
  • the method is suitable for use in existing chromatography systems, such as AKTA PureTM chromatography system (Cytiva®), thereby enabling its effortless introduction with wide applicability in established laboratories and industrial settings for RNA purification.
  • the method is also suitable for, and fully scalable to, bioprocess scale systems that are GMP compliant.
  • RNA sample is provided in a desired controlled environment e.g. with regard to buffer system, salt content, pH, etc.
  • a desired controlled environment e.g. with regard to buffer system, salt content, pH, etc.
  • I EX ion exchange chromatography
  • AC affinity chromatography
  • molecules do not bind to the chromatography resin, which means that buffer composition does not directly affect resolution (the degree of separation between peaks in a resulting chromatogram). Consequently, a significant advantage of SEC for use in RNA purification is that conditions can be varied to suit the type of sample or the requirements for further purification, analysis, or storage without altering the separation.
  • the present inventors have found that the method as disclosed herein is suitable for determining concentration of the RNA separated in step b) of the method as disclosed herein. It is also to be understood that in the method as disclosed herein, separation of RNA of a DNase-treated sample from proteins and residual DNA fragments present in said DNase-treated sample using SEC may occur simultaneously with conditioning of said RNA and/or determining the concentration of the RNA separated in step b) of the method as disclosed herein.
  • the present method for purifying RNA which may further comprise conditioning of said RNA and/or determination of the concentration of the RNA separated in step b) of the method as disclosed herein, may be achieved by subjecting a partially purified and/or a non-purified RNA sample to SEC.
  • partially purified RNA sample refers to a sample comprising RNA that has been subjected to at least one RNA purification step but still comprises at least one nondesired component, in particular protein and/or residual nucleic acid contaminants, which may be present in a sample comprising RNA prior to any RNA purification step.
  • non-purified RNA sample refers to a sample comprising RNA, which has not been subjected to any RNA purification step and comprises at least one non-desired component, in particular protein and/or residual nucleic acid contaminants, which may be present in a sample comprising RNA prior to any purification step.
  • the term may also be referred to as a "IVT reaction mixture".
  • at least one non-desired component refers to components which are not desired in the purified RNA sample obtained by the method as disclosed herein.
  • non-desired components may include buffer components, such as salts.
  • Non-desired components may be dependent on one or more subsequent processing steps of the purified RNA sample obtained by the method as disclosed herein.
  • the term "purified RNA sample obtained by the method as disclosed herein" refers to a sample comprising RNA that is essentially free of non-desired components, in particular any protein and residual nucleic acid contaminants, which may be present in a sample comprising RNA prior to any purification step.
  • RNA of the sample is beneficial for separating RNA of the sample from components other than RNA present in said sample.
  • these components include, beside proteins and residual DNA fragments, other components required for an IVT reaction, such as cofactors, salts and NTPs.
  • said step b) of the method as disclosed herein comprises separating RNA of said DNase-treated sample from proteins, residual DNA fragments and any additional components other than said RNA, said proteins and said residual DNA fragments present in said DNase-treated sample, such as salts and other buffer components and/or NTPs.
  • DNase-treated sample refers to a sample comprising RNA which has been subjected to DNase treatment.
  • the method as disclosed herein involves DNase-treatment of the sample according to step a) of the method. It is to be understood that said treatment results in the fragmentation of the DNA to an extent by which the size of the residual DNA fragments is substantially smaller than that of the RNA present in said sample, thus RNA and residual DNA fragments are separated from each other during SEC according to step b) of the method.
  • chromatography medium refers to the core of chromatography technology and is a critical raw material which serves as a stationary phase, through which the components of a mixture (such as a sample) is carried through.
  • Typical chromatography media are beaded resins, which are well known to those skilled in the art and are media used for example to capture and/or polish monoclonal antibodies, antibody fragments, vaccines and other biomolecules.
  • the chromatography medium may comprise beads, such as a matrix in the form of a plurality of beads, e.g. formed of polymeric material as detailed elsewhere herein. The beads are typically porous.
  • said chromatography medium comprises a homogeneous porous matrix.
  • the term "homogeneous matrix” as used herein refers to a matrix which is uniform.
  • the beads of the matrix may be homogeneous in that they are formed of a uniform material, a single layer of material, and/or have a uniform porosity.
  • homogeneous porous matrix refers to a matrix which can be characterized with one type of property, structure and material.
  • beads which comprise more than one layer, such as a core and a shell also referred to as "core beads”
  • core beads are not to be understood as being homogeneous beads or as forming a homogeneous porous matrix.
  • SEC resins are typically chromatography media formed of a homogeneous porous matrix.
  • said chromatography medium is a SEC resin comprising a homogeneous porous matrix.
  • a chromatography medium comprising porous beads
  • all liquids will enter the pores, and the largest molecules that are excluded from the pores will elute in the void volume (i.e., the elution volume of molecules that do not enter the pores and elute first), while the smallest molecules, such as salts, are eluted last.
  • the molecules in the void volume are larger than the largest pores. It is thus appreciated that the resolution of separation of RNA from proteins and residual DNA fragments present in a DNase-treated sample according to the method as disclosed herein may be affected by the pore size of the chromatography medium used in the method.
  • the resolution comprises three parts, i.e. selectivity, retention capacity and efficiency. Each of these terms is affected by the specific components of the method.
  • a column's particle size in particular, affects the efficiency term of the resolution equation.
  • Efficiency is ultimately derived from the theoretical plate model of chromatography.
  • a plate refers to one complete equilibrated transfer (or partition) of a solute between the mobile and stationary phases. Efficiency is a qualitative term used to measure the number of theoretical plates in a given column, or the degree to which an analyte partitions between the mobile and stationary phases.
  • efficiency is inversely proportional. As particle size decreases, efficiency increases, and more resolution is achieved. In contrast, efficiency is directly proportional to the column length. Shortening the analysis time with maintained resolution can be achieved by and decreasing the length of the column by the same factor as the particle size. It is also beneficial that efficiency is inversely proportional to the square of the peak width— higher efficiencies produce narrower peak widths. Narrow peak widths enhance resolution by lengthening the baseline between two adjacent peaks.
  • the present inventors demonstrate several types of chromatography media comprising a homogeneous porous matrix suitable for the method as disclosed herein and illustrate in the appended examples that depending on the size of the RNA, the most optimal chromatography medium may be selected.
  • the matrix typically comprises porous beads which may comprise a polymeric material, such as a polysaccharide and derivates thereof, e.g. agarose such as cross-linked agarose, or dextran, such as cross-linked dextran and combinations thereof.
  • a polymeric material such as a polysaccharide and derivates thereof, e.g. agarose such as cross-linked agarose, or dextran, such as cross-linked dextran and combinations thereof.
  • said matrix may comprise a polymeric material selected from the group consisting of crosslinked agarose, crosslinked dextran and crosslinked copolymer of allyl dextran and N,N'-methylene bisacrylamide.
  • said matrix is or comprises crosslinked agarose or crosslinked dextran.
  • said matrix is crosslinked agarose or crosslinked copolymer of allyl dextran and N,N'-methylene bisacrylamide.
  • said matrix is crosslinked dextran or crosslinked copolymer of allyl dextran and N,N'-methylene bisacrylamide.
  • said matrix is crosslinked dextran.
  • said matrix is crosslinked copolymer of allyl dextran and N,N'-methylene bisacrylamide.
  • said matrix is crosslinked agarose.
  • Cross-linking increases the mechanical strength of the resin.
  • crosslinked agarose can endure a higher flow pressure compared to other polysaccharides such as dextran or copolymers comprising dextran, as well as compared to for instance polyacrylamide-based matrices.
  • dextran-based matrices may swell or shrink depending on which solvent is used, whereas crosslinked agarose remains stable.
  • said matrix is crosslinked agarose.
  • Polysaccharide beads such as agarose beads, useful in the present invention can be produced by conventional means well known in the art and can be produced e.g. as described in US6602990 Bl, US7396467B2 or US8309709 B2, which are incorporated herein.
  • the agarose beads may comprise 0.5-20 wt.% agarose.
  • the agarose content may be 0.5-20 wt.%, 1.5-20 wt.%, 2-20 wt.%, >2-20 wt.%, 2.5-20 wt.%, 3-20 wt.%, 3.5-20 wt.%, 4-20 wt.%, 4.5-20 wt.%, 5-20 wt.%, 5.5-20 wt.%, 6-20 wt.%, 2-10 wt.%, 4-10 wt.%.
  • said beads have a median particle size D50v in the range of 5-200 pm.
  • D50v is the median particle size of the cumulative volume distribution, and can be measured by an electronic particle counter, such as MultisizerTM3 Coulter Counter (Beckman Coulter) using the electrical sensing zone (ESZ) method.
  • an electronic particle counter such as MultisizerTM3 Coulter Counter (Beckman Coulter) using the electrical sensing zone (ESZ) method.
  • ESZ electrical sensing zone
  • the bead size D50v may be 5-200 pm, 10-200 pm, 20-200 pm, 40- 200 pm, 60-200 pm, 80-200 pm, 100-200 pm, 120-200 pm, 140-200 pm, 160-200 pm, 180- 200 pm, 5-180 pm, 5-160 pm, 5-140 pm, 5-120 pm, 5-100 pm, 5-80 pm, 5-60 pm, 5-40 pm, 5-20 pm, or 10-50 pm.
  • Particles smaller than D50v of 5 pm may result in high pack-pressure and column-nets, i.e. leakage of small beads through the column filter.
  • a non-functionalized matrix lacks reactivity and adsorptive properties. Accordingly, a non-functionalized matrix does not bind biomolecules and therefore, upon contacting a sample comprising a biomolecule with a chromatography medium comprising non-functionalized matrix beads, the chromatography beads do not bind any biomolecules of the sample (although some biomolecules, depending on their size, may be retained to varying degrees).
  • said non-functionalized matrix is inactive and/or inert with regards to any biomolecules of the sample.
  • Typical SEC resins are composed of such non-functionalized matrices.
  • said chromatography medium is a SEC resin comprising a homogeneous porous matrix that is non-functionalized.
  • RNA may be purified according to the herein disclosed method, wherein a chromatography medium comprises a homogeneous porous matrix functionalized with any such ligand.
  • Non-limiting examples of such ligands include affinity ligands directed to target molecules other than RNA, such as protein A based affinity ligands for affinity capture of IgG (for example in case of MabSelect SuReTM resins (Cytiva®) and MabSelect PrismATM resins (Cytiva®)), albumin-binding affinity ligands (for example in case of Blue Sepharose FF resins (Cytiva®)) and affinity ligands for viral vectors such as AAV and AVB Sepharose High Performance resins (Cytiva®).
  • affinity ligands directed to target molecules other than RNA such as protein A based affinity ligands for affinity capture of IgG (for example in case of MabSelect SuReTM resins (Cytiva®) and MabSelect PrismATM resins (Cytiva®)), albumin-binding affinity ligands (for example in case of Blue Sepharose FF resins (Cytiva
  • CIEX cation exchange
  • SP Sepharose FF resins Cytiva®
  • SP Sepharose HP resins Cytiva®
  • metal affinity ligands for IMAC such as in case of HisTrap FF resins (Cytiva®).
  • S sulfonate
  • SP Sepharose HP resins Cytiva®
  • IMAC metal affinity ligands for IMAC, such as in case of HisTrap FF resins (Cytiva®).
  • the Blue Sepharose FF, SP Sepharose FF and HisTrap FF resins All use a typical SEC matrix, Sepharose 6 FF (Cytiva®), as the base matrix to which the respective ligands are coupled.
  • AVB Sepharose High Performance and SP Sepharose HP resins have the ligands coupled onto a base matrix which is another typical SEC matrix, Sepharose HP (Cytiva®).
  • a base matrix which is another typical SEC matrix
  • Sepharose HP Sepharose HP
  • the matrix is non-functionalized or is functionalized with a ligand that does not bind RNA.
  • the matrix is functionalized with a ligand that does not bind RNA.
  • a functionalized matrix may potentially provide a selective purification of the RNA in that it may bind a specific component other than RNA that needs to be removed from the RNA sample.
  • the chromatography medium is packed in a column.
  • various column formats are suitable for the method as disclosed herein.
  • said chromatography medium may be packed in any column format and size.
  • said column has a diameter of between about 25 mm and about 50 mm and a bed height of between about 100 mm and about 250 mm.
  • the height/diameter ratio is 3.85.
  • higher ratios as well as lower ratios e.g. in the range of 0.5-30, such as 0.5-10, such as 2-5, may be suitable as long as the load volume (% of sample volume loaded in relation to the column volume) is adjusted accordingly.
  • said column has a column volume and a volume loading capacity, wherein said volume loading capacity is less than said column volume.
  • Column volume is known as the geometrical volume of a column interior (also known as the chromatography bed).
  • volume loading capacity corresponds to the maximum sample volume that can be loaded onto a column without compromising the purity and yield of the purified product, in this case the purified RNA.
  • the volume loading capacity may be expressed in percentage of the CV and the sample-to- column volume ratio influences resolution.
  • sample volume is one important parameter in SEC which influences high-resolution separation. The inventors have found that a sample loading volume that is lower than the CV is beneficial in the method as disclosed herein.
  • the volume loading capacity is equal to or less than about 50% of the CV, such as equal to or less than about 40% of the CV, such as equal to or less than about 35% of the CV, such as equal to or less than about 30% of the CV, such as equal to or less than about 25% of the CV, such as equal to or less than about 20% of the CV, such as equal to or less than about 15% of the CV, such as equal to or less than about 10% of the CV.
  • a sample loading volume that is about 20% or less of the CV is particularly advantageous for the method as disclosed herein.
  • the volume loading capacity is from about 1% to about 25% of the CV, such as from about 1% to about 20% of the CV, such as from about 2% to about 20% of the CV, such as from about 2% to about 15% of the CV. In one particular embodiment, the volume loading capacity is about 20% of the CV. In yet another particular embodiment, the volume loading capacity is about 10% of the CV.
  • the method may comprise a chromatography medium which is packed in a column to provide a column volume, wherein the method may comprise a step of loading a volume of the sample onto the column, wherein said volume of the sample is equal to or less than 50% of the column volume, such as 1 - 25 % of the column volume as detailed above.
  • the sample does not need to be diluted.
  • a range of different sample volumes can be used depending on the initial concentration. The same is valid for flow velocities, which will be limited mainly by (but not exclusively) column format and system used.
  • RNA may be purified from a sample in high purity according to the method as disclosed herein. As demonstrated in the Examples below (e.g. in Example 1) by a re-injection of an RNA sample purified according to the disclosed method, near 100% purity may be achieved.
  • RNA is purified from said sample in a purity of at least about 90%, such as at least about 91%, such as at least about 92%, such as at least about 93%, such as at least about 94%, such as at least about 95%, such as at least about 96%, such as at least about 97%, such as at least about 98%, such as at least about 99%, such as about 100%.
  • RNA is purified from said sample in a purity of at least about 95%, such as at least about 96%, such as at least about 97%, such as at least about 98%, such as at least about 99%, such as about 100%. In one embodiment, RNA is purified from said sample in a purity of from at least about 95% to about 100%. In one embodiment, RNA is purified from said sample in a purity of at least about 95%. In one embodiment, RNA is purified from said sample in a purity of about 100%. As illustrated in the appended Examples, the inventors have surprisingly found that said purity may be achieved by a single step of SEC according to the disclosed method, since by reinjection of an RNA sample purified according to the method disclosed herein, said purity may be confirmed.
  • said purity is achieved by step b) of the method as disclosed herein without further steps of separating RNA of said DNase-treated sample from proteins and residual DNA fragments present in said DNase-treated sample.
  • the terms used herein relating to RNA purity are to be understood in relation to all other non-desired components present in said sample, in particular to residual DNA fragments and residual proteins present in said sample, which has been subjected to the method as disclosed herein.
  • the RNA concentration of the sample is dependent on the volume loaded on the column.
  • the RNA concentration of the sample may be 10 mg/mL or less, such as 5 mg/mL or less.
  • the sample concentration of the RNA is 1.0-1.5 mg/mL using a 10% column volume load.
  • the RNA concentration of the sample is 4-6 mg/mL using a 5% column volume load. Accordingly, sample loading volumes may be adapted according to the concentration of the RNA in the sample.
  • RNA is purified from said sample in a recovery yield of at least 90%, such as at least about 91%, such as at least about 92%, such as at least about 93%, such as at least about 94%, such as at least about 95%, such as at least about 96%, such as at least about 97%, such as at least about 98%, such as at least about 99%, such as about 100%.
  • RNA is purified from said sample in a recovery yield of at least about 95%, such as at least about 96%, such as at least about 97%, such as at least about 98%, such as at least about 99%, such as about 100%. In one embodiment, RNA is purified from said sample in a recovery yield of from at least about 95% to about 100%. In one embodiment, RNA is purified from said sample in a recovery yield of at least about 95%. In one embodiment, RNA is purified from said sample in a recovery yield of about 100%. The terms used herein relating to RNA recovery yield are to be understood in relation to the initial amount of RNA present in said sample, which has been subjected to the method as disclosed herein.
  • said RNA, said proteins and said residual DNA fragments are not bound to said matrix that is not functionalized and/or functionalized with a ligand that does not bind and/or is inactive and/or inert with regards to these sample components.
  • said RNA, said proteins and said residual DNA fragments are eluted from the chromatography medium using a single buffer composition, such as a single buffer composition of at least about 1.3 CV, such as at least about 1.5 CV.
  • a column packed with such chromatography medium may be reused for a new run, without implementing any method steps, such as elution, cleaning, regeneration and/or re-equilibration, for removing retained components of the sample.
  • said RNA, said proteins and said residual DNA fragments are eluted from the chromatography medium used in step b) using the same buffer.
  • the volume of said buffer may be at least about 1.3 CV, such as at least about 1.5 CV.
  • the RNA may be purified from the sample in a volume of said buffer corresponding to 0.5 CV or less.
  • the method as disclosed herein allows collection of the purified RNA in less than 10% of the time required for TFF.
  • buffers examples include 10m M Tris-HCI pH 7.5-8 and compositions thereof such as in the range of 10-100 mM Tris pH 7.0-8.0, 10-100 mM Phosphate pH 6.5 -8.0, with or without the presence of salts such as NaCI or KCI or other types of additives such as, but not restricted to, detergents, EDTA, reducing agents, and poloxamers.
  • a chaotropic agent such as urea or guanidine hydrochloride may also be present, in such cases typically added after IVT but before or during SEC. Virtually any pH and buffer composition could be used, as long as the stability of RNA is maintained.
  • Example 9 shows that resolution for the separation is not affected by different buffer compositions and/or pH of said compositions.
  • the RNA is stable and the elution profile the same regardless of pH and composition, within the normal working range for RNA.
  • the present method is surprisingly beneficial for RNA purification, as the chromatography medium used in SEC according to step b) of the method may be reused sequentially in the method as disclosed herein without a need for removing retained components of the sample.
  • the inventors illustrate in Example 7 that the chromatography medium used in SEC according to step b) of the method may be reused at least ten times without any compromise on the performance of said medium for separating RNA from said proteins and said residual DNA fragments.
  • the chromatography medium used in SEC according to step b) of the method may be reused at least about 50 to about 100 times, or more, without any compromise on the performance of said medium for separating RNA from said proteins and said residual DNA fragments.
  • CIP cleaning-in-place
  • CIP cleaning-in-place
  • the single buffer may comprise a reducing agent. This will improve the separation of T7 RNA polymerase from the mRNA during the chromatography step b) of the method disclosed above.
  • Suitable reducing agents are known to the skilled person, and one example of reducing agents is for instance TCEP.
  • the improved separation of T7 RNA polymerase from mRNA when including a reducing agent is shown in Example 8.
  • the single buffer being both an equilibration buffer as well as an elution buffer, is sometimes referred to as a running buffer.
  • the two terms "single buffer” and “running buffer” may be used interchangeably.
  • the method as disclosed herein may further comprise a step prior to step b), wherein said chromatography medium is equilibrated.
  • equilibration may be performed by using a buffer used in said SEC in step b) of the method in a volume of at least about 1.3 CV, such as at least about 1.5 CV.
  • said chromatography medium used in said SEC in step b) of the method may be equilibrated using a buffer used in said SEC in step b) of the method in a volume of at least about 1.3 CV, such as at least about 1.5 CV, prior to subjecting said DNase-treated sample to said SEC.
  • the present method is advantageous for conditioning the RNA present in said sample.
  • said method comprises conditioning of said RNA.
  • said method comprises conditioning of said RNA during said SEC as defined in step b) of the method as disclosed herein; and/or said method comprises a further step of SEC, wherein said conditioning of said RNA occurs during said further step of SEC.
  • said conditioning occurs during said SEC as defined in step b) of the method as disclosed herein.
  • the method as disclosed herein comprises a further step of SEC, wherein said conditioning occurs during said further step of SEC.
  • a chromatography medium as defined above in relation to said SEC in step b) is used in said further step of SEC.
  • the present inventors have surprisingly found that the method as disclosed herein is suitable for determining concentration of the RNA separated in step b) of the method.
  • the concentration of the RNA separated in step b) is determined using the elution profile obtained by said SEC in step b); and/or the method as disclosed herein comprises a further step of SEC, wherein the concentration of the RNA separated in step b) is determined using the elution profile obtained by said further step of SEC.
  • the concentration of the RNA separated in step b) is determined using the elution profile obtained by said SEC in step b).
  • the method as disclosed herein comprises a further step of SEC, wherein the concentration of the RNA separated in step b) is determined using the elution profile obtained by said further step of SEC.
  • a chromatography medium as defined above in relation to said SEC in step b) is used in said further step of SEC.
  • Said further step of SEC for determining said concentration of the RNA may be an analytical SEC, as further explained below.
  • said conditioning of the RNA and said determination of the concentration of the RNA separated in step b) may occur simultaneously.
  • said RNA is conditioned during step b) and the concentration of the RNA separated in step b) is determined using the elution profile obtained by said SEC in step b).
  • said method comprises a further step of SEC, wherein said RNA is conditioned during said further step of SEC and the concentration of the RNA separated in step b) is determined using the elution profile obtained by said further step of SEC.
  • said RNA is conditioned during step b) and the concentration of the RNA separated in step b) is determined using the elution profile obtained by said further step of SEC. It will be appreciated that said further step of SEC is not performed with the purpose of separating RNA of said DNase-treated sample from proteins and residual DNA fragments present in said DNase-treated sample in the embodiments discussed immediately above.
  • said RNA is a single stranded RNA, such as an mRNA.
  • Said RNA may be a synthetic RNA.
  • said sample is obtained by an RNA manufacturing method, such as by in vitro transcription (IVT).
  • the RNA comprises a sequence of at least about 500 nucleotides, such as at least about 750 nucleotides, such as at least about 1000 nucleotides, such as at least about 1500 nucleotides, such as at least about 2000 nucleotides, such as at least about 3000 nucleotides, such as at least about 4000 nucleotides.
  • said RNA comprises a sequence of at least about 750 nucleotides, such as at least about 800 nucleotides, such as at least about 850 nucleotides, such as at least about 900 nucleotides, such as at least about 950 nucleotides, such as at least about 1000 nucleotides, such as at least about 1500 nucleotides, such as at least about 2000 nucleotides, such as at least about 3000 nucleotides, such as at least about 4000 nucleotides.
  • said RNA comprises a sequence of at least about 750 nucleotides, such as at least about 800 nucleotides, such as at least about 850 nucleotides, such as at least about 900 nucleotides, such as at least about 950 nucleotides, such as at least about 1000 nucleotides or more.
  • said RNA comprises a sequence of from about 1000 nucleotides to about 5000 nucleotides, such as from about 1000 nucleotides to about 4500 nucleotides.
  • the RNA comprises a sequence of at least about 20000 nucleotides, or more. According to some embodiments, the RNA is a linear sequence.
  • the RNA is a circular RNA.
  • capping of the 5' end may be important for both stability of an mRNA as well as improving protein translation. Such capping may be performed during IVT.
  • said mRNA is capped.
  • modified nucleotides may be applied for mRNA production and may be useful in nucleic acid therapeutics. These are known to alter subsequent usability of mRNA, for example by affecting translation, stability and/or splicing.
  • said mRNA comprises natural nucleotides and/or modified nucleotides.
  • said mRNA comprises natural nucleotides.
  • said mRNA comprises modified nucleotides.
  • RNA with sequences smaller than 400 nucleotides will enter the beads of the SEC resin. However, this may be dependent on the flow rate. A slower flow rate leads to interaction of the smaller RNA molecules with the resin, whereas larger RNA molecules are less prone to enter the resin, regardless of flow rate. Thus, with the herein disclosed method a separation of mRNA larger than 400 nucleotides is achieved, regardless of flow rate. For smaller mRNA, it is more dependent on flow rate and there is a risk that mRNA may be lost to the second peak comprising proteins, residual DNA fragments present in said DNase-treated sample, as well as salts and other buffer components and/or NTPs, resulting in a lower recovery for the mRNA.
  • the RNA may include a poly(A) sequence, e.g. at the 3' end of a linear mRNA.
  • the method as disclosed herein further comprises at least one additional method step.
  • This at least one additional method step could be included prior to or subsequent to the method as disclosed herein.
  • the step can be a chromatographic capture step, i.e. a chromatography step that binds the target RNA, which is subsequently eluted.
  • said additional method step is selected from the group consisting of affinity chromatography, hydrophobic interaction chromatography, desalting and buffer exchange, anion exchange, chromatography with multimodal/mixed chromatography resins, and any combination thereof.
  • the method as disclosed herein does not comprise an additional method, such as TFF, before and/or after the SEC step.
  • SEC according to step b) of the method as disclosed herein may be used to simultaneously condition RNA in an appropriate buffer system as well as to remove unwanted residual reagents from the sample, which conditioning allows direct load onto a second column in an additional method step, such as affinity capture by use of a oligo dT ligand (e.g. in the form of membrane chromatography for example as described in WO 2022/162018 or CIMmultus® Oligo dT Monolithic Column (Sartorius)), and/or any other mode of capture, such as hydrophobic interaction chromatography (HIC) and/or anion exchange (Al EX) purification, aimed at capturing RNA.
  • oligo dT ligand e.g. in the form of membrane chromatography for example as described in WO 2022/162018 or CIMmultus® Oligo dT Monolithic Column (Sartorius)
  • any other mode of capture such as hydrophobic interaction chromatography (HIC) and/or anion exchange (A
  • Said additional method step may be a further step of SEC for conditioning of said RNA and/or for removal of buffer and salt components.
  • Said additional method step may be preceding and/or subsequent to step b) of the method as disclosed herein. In some embodiments, said additional method step is subsequent to step b).
  • the present method particularly useful for purifying RNA, wherein a single step of SEC according to step b) of the method may be sufficient to obtain a RNA sample with high purity. Accordingly, in some embodiments, said method does not comprise an additional method step preceding and/or subsequent to step a) and/or step b) for separating RNA of said DNase-treated sample from proteins and/or residual DNA fragments present in said DNase-treated sample.
  • said RNA is purified on a preparative scale, semi-preparative scale or on an analytical scale.
  • said RNA is purified on a preparative scale.
  • said RNA is purified on a semi-preparative scale.
  • said RNA is purified on an analytical scale.
  • said SEC in step b) may be preparative SEC or analytical SEC.
  • preparative SEC typically refers to a high-resolution size-based separation of biomolecules with fractionation and analytical SEC refers to a high-resolution size-based separation without fractionation. The two types are not mutually exclusive.
  • Preparative SEC is normally performed to isolate one or more components of a sample and the separated components obtained in preparative SEC can directly be transferred to a suitable buffer for assay or storage.
  • Analytical SEC is typically performed to check the quality of the sample, to establish the presence or study the properties of a biomolecule or to measure the relative proportions of analytes in a mixture.
  • analytical SEC may be applied for determining concentration of the RNA separated in step b) of the method as disclosed herein.
  • preparative SEC may be used for purifying RNA on a preparative scale (i.e. large-scale purification) or on a semi-preparative scale (i.e.
  • small-scale purification depending on the type of sample subjected to the method as disclosed herein.
  • small-scale purification may be beneficial for samples having a small sample volume, e.g. as applicable in precision medicine.
  • Large-scale purification may be on the other hand beneficial for purifying mass components often utilized for manufacturing purposes, such as for example in preparation for mRNA vaccines.
  • the method as disclosed herein is beneficial in both applications. This is shown in Example 10, where different formats of chromatography columns are compared.
  • a size exclusion chromatography medium is unexpectedly advantageous in the method for purifying RNA according to the present disclosure.
  • a size exclusion chromatography medium for separating RNA present in a DNase-treated sample from proteins and residual DNA fragments present in said DNase-treated sample.
  • said use further comprises determining the concentration of the RNA separated from said proteins and said residual DNA fragments.
  • said use further comprises conditioning of said RNA.
  • said use further comprises conditioning of said RNA and determining the concentration the RNA separated from said proteins and said residual DNA fragments.
  • said RNA is separated from said proteins and said residual DNA fragments simultaneously.
  • said size exclusion chromatography medium is used simultaneously for separating said RNA from said proteins and said residual DNA fragments and for determining concentration of the RNA separated from said proteins and said residual DNA fragments.
  • said size exclusion chromatography medium is used simultaneously for separating said RNA from said proteins and said residual DNA fragments, for determining concentration of the RNA separated from said proteins and said residual DNA fragments and for conditioning said RNA.
  • said size exclusion chromatography medium is used simultaneously for separating said RNA from said proteins and said residual DNA fragments and for conditioning said RNA.
  • said matrix is non-functionalized or functionalized with a ligand that does not retain RNA.
  • the matrix is nonfunctionalized.
  • said matrix selected from the group consisting of crosslinked agarose, crosslinked dextran and crosslinked copolymer of allyl dextran and N,N'-methylene bisacrylamide.
  • said matrix is crosslinked agarose or crosslinked dextran.
  • said matrix is crosslinked agarose or crosslinked copolymer of allyl dextran and N,N'-methylene bisacrylamide.
  • said matrix is crosslinked dextran or crosslinked copolymer of allyl dextran and N,N'-methylene bisacrylamide.
  • said matrix is crosslinked dextran.
  • said matrix is crosslinked copolymer of allyl dextran and N,N'-methylene bisacrylamide.
  • said matrix is crosslinked agarose.
  • said size exclusion chromatography medium is packed in a column.
  • said column has a diameter of from about 25 mm to about 50 mm and a bed height of from about 100 mm to about 250 mm.
  • said column has a column volume and a volume loading capacity, wherein said volume loading capacity is less than said column volume.
  • the volume loading capacity is equal to or less than about 50% of the column volume, such as equal to or less than about 40% of the column volume, such as equal to or less than about 35% of the column volume, such as equal to or less than about 30% of the column volume, such as equal to or less than about 25% of the column volume, such as equal to or less than about 20% of the column volume, such as equal to or less than about 15% of the column volume, such as equal to or less than about 10% of the column volume.
  • the volume loading capacity is from about 1% to about 25% of the column volume, such as from about 1% to about 20% of the column volume, such as from about 2% to about 20% of the column volume, such as from about 2% to about 15% of the column volume. In one embodiment, the volume loading capacity is about 20% of the column volume, preferably about 10% of the column volume.
  • said RNA is purified from said DNase-treated sample in a purity of at least about 90%, such as at least about 91%, such as at least about 92%, such as at least about 93%, such as at least about 94%, such as at least about 95%, during said use.
  • the RNA is purified from said DNase-treated sample in a purity of at least about 96%, such as at least about 97%, such as at least about 98%, such as at least about 99%, during said use.
  • said RNA is purified from said DNase-treated sample in a recovery yield of at least 90%, such as at least about 91%, such as at least about 92%, such as at least about 93%, such as at least about 94%, such as at least about 95%, during said use.
  • the RNA is purified from said DNase-treated sample in a recovery yield of at least about 96%, such as at least about 97%, such as at least about 98%, such as at least about 99%, during said use.
  • the RNA is a single stranded RNA, such as an mRNA.
  • said sample is obtained by in vitro transcription.
  • the RNA comprises a linear sequence of at least about 500 nucleotides, such as at least about 750 nucleotides, such as at least about 1000 nucleotides, such as at least about 1500 nucleotides, such as at least about 2000 nucleotides, such as at least about 3000 nucleotides, such as at least about 4000 nucleotides.
  • the inventors illustrate in Example 7 that the chromatography medium used in SEC according to the present disclosure may be reused at least ten times without any compromise on the performance of said medium for separating RNA from said proteins and said residual DNA fragments.
  • the inventors in fact envision that the use of the chromatography medium as disclosed herein may be repeated at least about 50 to about 100 times, or more, without any compromise on the performance of said medium for separating RNA from said proteins and said residual DNA fragments.
  • said use of the chromatography medium as disclosed herein is a repeated use, such as wherein said chromatography medium is used according to the present disclosure at least about 10 times, such as at least about 50 to about 100 times, or more.
  • said repeated use of the chromatography medium does not require a harsh cleaning step in between uses according to the present disclosure.
  • said repeated use may comprise a standard CIP of the chromatography medium in between every 5 uses or less frequently, such as between every 10 uses or less frequently, according to the present disclosure.
  • the value is in fact in the range of from 9 to 11, such as in the range of from 9.9 to 10.9, such as in the range of from 9.8 to 10.8, such as in the range of from 9.7 to 10.7, such as in the range of from 9.6 to 10.6, such as in the range of from 9.5 to 10.5, such as in the range of from 9.4 to 10.4, such as in the range of from 9.3 to 10.3, such as in the range of from 9.2 to 10.2, such as in the range of from 9.1 to 10.1.
  • Figure 1 shows a chromatogram from the experiment wherein mRNA is purified from a DNase-treated crude IVT sample using SEC (Example 1).
  • Curve (A) corresponds to the elution profile of the first run recorded at 260 nm.
  • Curve (B) corresponds to the elution profile of the second run recorded at 260 nm.
  • Curve (C) corresponds to the conductivity measurement during the first run.
  • Curve (D) corresponds to the conductivity measurement during the second run.
  • Figure 2 shows chromatograms from the experiments wherein mRNAs of various lengths are purified from DNase-treated crude IVT reaction samples using SEC (Example 2).
  • Figure 2A shows separation of a sample comprising 1042 nucleotide-long mRNA product from residual components.
  • Figure 2B shows separation of a sample comprising 1975 nucleotide-long mRNA product from residual components.
  • Figure 2C shows separation of a sample comprising 4525 nucleotide-long mRNA product from residual components.
  • curve (E) represents measurement of absorbance at 260 nm.
  • Curve (F) represents measurement of absorbance at 280 nm.
  • Curve (G) represents measurement of conductivity.
  • Figure 3 shows elution volumes in SEC of individual nucleotides used in IVT reaction samples and of reference proteins (Example 3).
  • Figure 3A shows a chromatogram with the relative elution position and absorbance maximum for all nucleotides used in IVT reaction samples: ATP (H), GTP (I), UTP (J) and CTP (K). Absorbance is measured at 260 nm.
  • Figure 3B shows a chromatogram with elution profiles of various reference proteins thyroglobulin (L), ferritin (M) and blue dextran (N) obtained in SEC and superimposed to the elution profile (O) and conductivity curve (P) of a DNase-treated crude IVT sample comprising mRNA (4525 nucleotide-long) obtained in SEC under the same conditions as used for the reference proteins. Absorbance was measured at 260 nm.
  • Figure 4 shows the chromatograms recorded at 280 nm from experiments wherein the volume loading capacity for purifying mRNA using SEC is investigated (Example 4).
  • Figure 4 shows superimposed chromatograms of four separate runs with different sample loading volumes. Elution profiles using a loading volume of 21.2% of CV (S), a loading volume of 10.6% of CV (Q), a loading volume of 5.3% of CV (R) and a loading volume of 2.1% of CV (T) are shown.
  • Figure 5 shows the results from the experiments wherein the determination of mRNA concentration using SEC is investigated (Example 5).
  • Figure 5A shows superimposed chromatograms recorder at 260 nm according to Example 5, wherein eluted mRNA from a first injection (U) was re-injected. Re-injection was repeated four times. The corresponding elution profiles for each injection in descending order are: first injection (U), second injection (V), third injection (W), fourth injection (X) and fifth injection (Y).
  • Figure 5B shows a calibration curve of recorded absorbance data for both peak heights and peak areas for each re-injection obtained in figure 5A.
  • Figure 6 shows the chromatograms from the experiments wherein different base matrices are tested for use in SEC for purifying mRNA of various lengths from a IVT reaction samples (Example 6).
  • Figure 6A shows the elution profiles recorded at 260 nm in the first run after injection of a DNase-treated crude IVT reaction sample comprising a 1975 nucleotide-long mRNA product to Sepharose 6FF, Sepharose 4FF and Sepharose HP, respectively.
  • Figure 6B shows the elution profiles recorded at 260 nm in the second run, wherein the two eluted peaks from the first run were collected, pooled and thereafter run on the same columns and base matrices.
  • Figure 6C shows chromatograms recorded at 260 nm, wherein DNase-treated crude IVT reaction samples comprising mRNA of different lengths (1042, 1975 and 4525 nucleotide-long mRNA, respectively) according to Example 6 were injected to three different base matrices (Sepharose 6FF, Sepharose 4FF and Sepharose HP, respectively).
  • Figure 6D shows chromatograms recorded at 260 nm, wherein pooled elution peaks (2nd run) and purified mRNA (3rd run) obtained from purifying DNase- treated IVT reaction samples comprising mRNA of different lengths (1042, 1975 and 4525 nucleotide-long mRNA, respectively) according to Example 6 were injected to two different base matrices.
  • Figure 7 shows superimposed chromatograms of 10 consecutive SEC runs of a sample containing purified mRNA according to Example 7. Absorbance was measured at 260 nm.
  • FIG 8 shows elution profiles of purified T7 RNA polymerase (abbreviated as T7 RNAP in figure) on Sepharose 6 FF.
  • Figure 8A shows the SDS-PAGE of purified T7-RNA polymerase, indicating 99% purity.
  • Curve (AA) in Figure 8B shows the chromatogram of purified T7 RNAP recorded at 280 nm (left y-axis) where the Sepharose 6FF column is equilibrated and eluted with Tris 10 mM, EDTA ImM, tris(2-carboxyethyl) phosphine (TCEP) 5 mM, pH 7.5.
  • Tris 10 mM, EDTA ImM tris(2-carboxyethyl) phosphine (TCEP) 5 mM, pH 7.5.
  • Curve (AB) corresponds to identical injection with Tris 10 mM, EDTA ImM pH 7.5 as running buffer.
  • Curve (AC) shows a chromatogram at 260 nm (right y-axis) of purified 1975 nucleotide-long mRNA on identical Sepharose 6 FF column with Tris 10 mM, EDTA ImM, TCEP 5mM, pH 7.5 as running buffer, to illustrate the elution volumes of mRNA in relation to T7 RNA polymerase.
  • Figure 9 shows a comparison of using different running buffers in the method according to the invention.
  • Fig. 9A shows the superimposed UV260 traces of Sepharose 6FF purification of crude IVT comprising of 1975-nucleotides mRNA in various of buffer pH.
  • Curve (AD) corresponds to running buffer of Tris 10 mM, EDTA ImM, pH 7.5 (the default condition).
  • Curve (AE) corresponds to running buffer of sodium acetate 50 mM, pH 4.5.
  • Curve (AF) corresponds to running buffer of sodium acetate 50 mM, pH 5.5.
  • Curve (AG) corresponds to running buffer of sodium citrate 50 mM, pH 6.5.
  • Curve (AH) corresponds to running buffer of HEPES 50 mM, pH 7.5.
  • Curve (Al) corresponds to running buffer of HEPES 50 mM, pH 8.5. Both mRNA fractions (Peak 1, abbreviated Pl in Figure 9B) and impurity fractions (Peak2, abbreviated P2 in Figure 9B) are analyzed by Fragment Analyzer to visualize presence of mRNA and mRNA integrity in respective buffer pH.
  • FIG. 10 shows the results from the experiments illustrating different column formats and scalability.
  • Fig. 10A shows the superimposed UV 260 curves of preparative purification of DNase-treated crude IVT comprising of 1975-nucleotides mRNA. All three columns were with 20 cm bed height, crude IVT sample of 15% CV was injected to equilibrated columns.
  • Curve (AJ) corresponds to the run performed on 2xHiScreen column.
  • Curve (AK) corresponds to the run performed on 2xHiPrep column.
  • Curve (AL) corresponds to the run performed on XK50/30 column.
  • Peak 1 (abbreviated for Pl) for mRNA and peak 2 (abbreviated for P2) for impurities were analysed on Fragment Analyzer for mRNA integrity analysis.
  • Figure 11 shows a superimposed chromatogram of UV260 of crude IVT comprising of RNA in size range of 100 to 400 nucleotides on Sepharose 6FF.
  • a linear flow rate of 67 cm/h is applied whereas in Figure 11B the flow rate is 200 cm/h.
  • Curve (AS) in Fig. 11A and curve (AV) in Fig. 11B correspond to crude IVT comprising of 100-nucleotides RNA.
  • Curve (AT) in Fig. 11A and curve (AW) in Fig. 11B correspond to crude IVT comprising of 200- nucleotides RNA.
  • Curve (AU) in Fig. 11A and curve (AX) in Fig. 11B correspond to crude IVT comprising of 400-nucleotides RNA.
  • Figure 12 illustrate the UV260 traces on Sepharose 6FF runs with crude IVT comprising of either 200 or 400 nucleotides mRNA together with their respective conductivity curve and fractionation information.
  • curve (AW) shows UV260 trace of crude IVT comprising of 200 nucleotides in overlay with its conductivity curve (AZ) and the reference line (BA) for middle of conductivity peak.
  • curve (AX) shows UV260 trace of crude IVT comprising of 400 nt nucleotides in overlay with its conductivity curve (BB) and the reference line (BC) for middle of conductivity peak.
  • FIG. 12A 400 nucleotides mRNA reinjected on Sepharose 6FF.
  • the peak 1 fractions isolated for both 200 and 400 nucleotides contain only mRNA, free from impurities such as free NTPs.
  • Figure 12C CGE analysis of fractions collected from P2 in Figure 12A (white arrows) and 12B (grey arrows) are performed by Fragment Analyzer, indicating 400 nucleotides mRNA does not enter the pore of Sepharose 6FF whereas 200 nucleotides mRNA does.
  • Figure 13 shows chromatograms of the respective elution profiles of the analysed samples using SEC base matrices coupled with a sulphopropyl CIEX ligand (Fig.
  • a first elution peak with a peak maximum approximately between 1.7 and 2.1 mL, was recorded at 260 nm which corresponds to the elution of mRNA.
  • a second elution peak with a peak maximum approximately between 3.3 and 4.1 mL, was recorded.
  • the second peak corresponds to residual components of the analysed crude IVT samples, such as fragmented DNA, enzymes and free NTPs.
  • RNA of a DNase- treated sample is separated from proteins and residual DNA fragments present in said sample using SEC.
  • the Examples showcase obtaining purified RNA samples in a recovery yield of at least 95% or higher, and/or with a purity of at least 95% or higher.
  • the Examples demonstrate measuring concentration of RNA in a sample using SEC as well as applicability of various base matrices for the method as disclosed herein. Robustness of the method and remarkable reusability of a SEC chromatography medium for the disclosed method are also illustrated.
  • Example 1 Purification of mRNA from DNase treated crude IVT samples using SEC
  • RNA of a DNase-treated sample is separated from proteins and residual DNA fragments present in said sample using SEC.
  • a DNase-treated crude IVT reaction sample comprising a 1975 nucleotide-long mRNA product was subjected to SEC, by which mRNA was separated from residual components, such as nucleotides, enzymes, proteins, salts and DNA fragments present in said sample following nuclease digestion.
  • a crude IVT reaction sample comprising a 1975 nucleotide-long mRNA product, free NTPs, DNA template for said mRNA and RNA polymerase (T7 RNA polymerase) was obtained from a reaction mixture containing 40 mM Tris-HCI pH 8, 50 mM MgAc, 10 mM DTT, 0.02% TritonX-100 and 7.5 mM of respectively, ATP, CTP, GTP, UTP, 1 U/pL Murine RNase inhibitor, 0.002 U/pL Inorganic Pyrophosphatase, 4 U/pL T7 RNA Polymerase and 150 pg/mL DNA template.
  • mRNA product refers to mRNA transcripts obtained in an IVT reaction based on a DNA template.
  • DNase digestion of the template was performed according to standard laboratory procedures. Briefly, template digestion was performed on the IVT reaction by the addition of CaCI2 to 2.5 mM and DNase I followed by incubation at 37.7°C.
  • the DNase I was inactivated by addition of EDTA to a final concentration of 6 mM. Following all steps, the material was diluted to a final dilution factor of 5x, i.e. the mRNA concentration is 5x lower than directly after the IVT reaction.
  • Sepharose 6FF Fast Flow
  • d 50 v particle size of approximately 90 pm
  • exclusion limits [M] for globular proteins and dextran is ⁇ 4 x 10 6 respectively ⁇ 2 x 10 6 .
  • sample loading volume 4 mL buffer: 10 mM Tris pH 7.5, 1 mM EDTA flow velocity: 5 mL/min (equal to 57 cm/h). running time: approximately 20 minutes
  • the initial sample concentration of crude diluted IVT was 0.25 mg/mL of which the eluted fractions with purified RNA with the highest absorbance were selected and pooled, resulting in a concentration of 0.15 mg/mL which was injected in the following experiment.
  • the buffer used for equilibration and elution was 10 mM Tris pH 7.5, 1 mM EDTA.
  • sample volumes up to 5 mL, approximately 10% of the CV, has been used, as well as higher flow velocities, up to 15 mL/min, equivalent of 170 cm/h.
  • the experiment was performed by an initial injection of 4 mL of the crude DNase- treated IVT reaction sample to the SEC column and run as described above.
  • curve (A) in the chromatogram in Fig. 1 two elution peaks were recorded.
  • the first elution peak approximately between 17 and 25 mL corresponds to the elution of mRNA and a second elution peak starting at about 35 mL corresponds to the elution of residual components present in the crude DNase-treated IVT reaction sample, respectively.
  • Salts present in the sample buffer are eluted last, as can be seen by the increase in conductivity with a peak maximum between 53-54 mL (curve (C) in Fig. 1).
  • the amount of salts in the collected mRNA sample used in the second injection is significantly less, near zero, in comparison to that in the sample of the first run. This is indicative of that components of the crude DNase-treated IVT reaction sample other than mRNA are successfully separated from the mRNA, and the collected mRNA sample after the first run is a purified mRNA sample. As apparent from the comparison of the elution profiles of the first and the second injections, mRNA has been purified at least with about 95% purity, in fact close to about 100% purity.
  • mRNA of the crude DNase-treated IVT reaction sample was successfully separated from other components of the sample (such as proteins, DNA fragments, salts and other buffer components) in a single run of SEC, as apparent from the comparison of the chromatograms obtained in the first and second runs.
  • the method as described herein is surprisingly beneficial in purifying mRNA from a sample.
  • Example 2 Purification of mRNAs of various lengths from DNase treated crude IVT reaction samples
  • Example 1042 nucleotide-long mRNA product a 1975 nucleotide-long mRNA product or a 4525 nucleotide- long mRNA product are subjected to SEC, by which mRNA was separated from residual components, such as nucleotides, enzymes, proteins, salts and DNA fragments present in said sample following nuclease digestion.
  • the Experiment was prepared and performed as described in Example 1 but in the present case three different samples were obtained, comprising an mRNA product of one of the three different sizes (1042 nucleotide-long, 1975 nucleotide-long or 4525 nucleotide- long), free NTPs, DNA template for said mRNAs respectively, and RNA polymerase (T7 RNA polymerase). DNase treatment of the samples was performed prior to loading as described above.
  • the samples were diluted 5x and thereafter injected and run individually on a
  • chromatograms presented in Fig. 2 correspond to the respective elution profiles of the analysed samples: the sample comprising the 1042 base-long mRNA product (2A), the sample comprising the 1975 base-long mRNA product (2B) and the sample comprising the 4525 nucleotide-long mRNA product (2C).
  • curve (E) represents the absorbance at 260 nm
  • curve (F) represents the absorbance at 280 nm
  • curve (G) represents the conductivity.
  • a first elution peak approximately between 13 and 24 mL, was recorded at both 260 and 280 nm which corresponds to the elution of mRNA.
  • a second elution peak approximately between 33 and 60 mL, was also recorded at both wavelengths. This second peak corresponds to residual components of the analysed crude IVT samples, such as fragmented DNA, enzymes and free NTPs.
  • salts present in the sample buffer were eluted last.
  • the method as disclosed herein is suitable for purifying a wide range of different RNAs, such as single-stranded RNAs having a length of at least about 1000 nucleotides, since mRNA products of various lengths within an extended range could be efficiently separated from remaining components of a crude DNase-treated IVT reaction sample in a single run of SEC.
  • mRNA products which comprise a mix of mRNA transcripts of various lengths such as mRNA transcripts having a length of at least about 1000 nucleotides, can also be efficiently purified from a sample using SEC.
  • Example 3 Elution volumes of individual nucleotides and reference proteins used in IVT reactions
  • This Example describes control experiments which demonstrate the elution profile of free NTPs as well as reference proteins in SEC.
  • the presented results are useful in evaluating relative elution volumes corresponding to proteins and free NTPs present in a RNA-containing sample subjected to SEC, for Example a DNase-treated crude IVT reaction sample, and demonstrate the performance of the herein disclosed method for the removal of these from said sample.
  • Free NTPs Four samples, each comprising one of the nucleotides , ATP, GTP, UTP or CTP were prepared to evaluate elution profile of free NTPs in SEC. The samples were individually tested using a Prepacked Prep 26/10 Sepharose 6 FF column (26 mm x 100 mm) with a CV of 53 ml (Cytiva). Equilibration was performed with 10 mM Tris pH 7.5, 1 mM EDTA. SEC method was performed as described in Example 1, with a flow rate of 15 mL/min and totally 75 pl of 100 nM NTP was injected. Absorbance was measured at 260 nm.
  • Reference proteins Samples comprising individual model proteins of different sizes were prepared using reference protein stock solutions of the Gel Filtration HMW Calibration Kit (Cytiva). In particular, samples comprising Thyroglobulin (669 kDa), Ferritin (440 kDa) or Blue Dextran (2000 kDa) were prepared and analysed in SEC using a Prepacked Prep 26/10 Sepharose 6FF column (26 mm x 100 mm) with a CV of 53 ml (Cytiva). SEC was performed as described in Example 1, and absorbance was measured at 260 nm.
  • Fig. 3A The absorbance at 260 nm of each nucleotide, in descending order, is ATP (H), GTP (I), UTP (J) and CTP (K). As can be seen in the chromatogram, the relative elution position and absorbance maximum for all nucleotide occurs around 42-44 mL.
  • free NTPs present in a sample are expected to have an elution volume with an absorbance maximum at 42-43 mL when a Prepacked Prep 26/10 Sepharose 6 FF column (26 mm x 100 mm) with a CV of 53 ml is used for SEC.
  • a Prepacked Prep 26/10 Sepharose 6 FF column 26 mm x 100 mm
  • the second peak corresponding to residual components of the analysed samples has an elution volume with an absorbance maximum at 42-43 mL.
  • 1 and 2 encompass free NTPs present in the analysed sample.
  • mRNA of the analysed samples which has been separated from the residual components, and which corresponds to the first peak of the presented chromatograms in Examples 1 and 2 is free from any free NTPs.
  • mRNA of crude DNase-treated IVT reaction samples as demonstrated in Examples 1 and 2 is successfully separated from each type of residual free nucleotides present in said sample using the method as disclosed herein.
  • the present data support that free NTPs are expected to elute subsequently to mRNA present in a sample using a SEC matrix for purifying mRNA when relative elution volumes of these are compared, thus the herein demonstrated separation of these occurs independently of the column dimensions. Consequently, it is concluded that in each of the herein described Examples, free NTPs are eluted separately (in the corresponding second peaks) from the RNA present in the analysed samples, when RNA is purified according to the disclosed method.
  • the largest expected entity that needs to be removed following an IVT reaction is the T7 RNA polymerase at 99 kDa.
  • the largest molecule in the kit Blue Dextran 2000, elutes at a similar position as the 4525 nucleotide-long mRNA construct (N).
  • the largest proteins Thyroglobulin (669 kDa) and Ferritin (440 kDa) also elute at a similar elution volume ((L) and (M), respectively).
  • T7 RNA polymerase has a significantly lower molecular weight than that of these reference proteins and is thus expected to elute at a higher elution volume than the elution volume observed for Blue Dextran 2000, Thyroglobulin and Ferritin. Accordingly, T7 RNA polymerase is expected to be eluted separately from the elution peak corresponding to the mRNA construct present in the IVT reaction sample (the first elution peak (O) shown in Fig. 3B). Thereby, mRNA free from residual proteins may be purified according to the method as disclosed herein.
  • mRNA of crude DNase-treated IVT reaction samples as demonstrated in for example, Examples 1 and 2 is successfully separated from each type of residual free nucleotides as well as residual proteins present in said sample using the method as disclosed herein.
  • Example 4 Volume loading capacity for purifying mRNA
  • sample loading volumes were tested in the method as disclosed herein using a DNase-treated IVT reaction sample comprising mRNA for loading in different volumes.
  • sample loading volume in relation to the CV was evaluated for achieving sufficient separation of mRNA from residual components present in a DNase- treated IVT reaction sample using SEC.
  • a crude IVT reaction sample comprising a 1927 nucleotide-long mRNA product, free NTPs, DNA template for said mRNA and RNA polymerase (T7 RNA polymerase) was obtained. DNase treatment of the sample was performed prior to loading, as described in Example 1.
  • Fig. 4 shows chromatograms recorded for the tested sample loading volumes, namely 21.2% CV load (S), 10.6% CV load (Q), 5.3% CV load (R) and 2.1% CV load (T), using the HiScreen column packed with Sepharose 6 FF and having a CV of 4.7 mL and a bed height of 100 mm and a diameter of 7.7 mm.
  • Two elution peaks were recorded for each sample, wherein, as demonstrated before, the first elution peak corresponds to the elution of mRNA and the second peak corresponds to the elution of residual components present in the crude DNase-treated IVT reaction sample.
  • the absolute elution volumes differ from those described in the above Examples due to the difference in the column dimensions, however, the relative elution volumes of the peaks are indicative of the separation of the mRNA from the residual components.
  • all of the tested sample loading volumes enable a separation between the peaks, i.e. are applicable for the separation of mRNA from residual components present in the initial DNase-treated crude IVT reaction sample using SEC. Accordingly, the use of a sample loading volume that is less than the CV is demonstrated for RNA purification according to the present method. In particular, it is demonstrated that using a sample loading volume that is about 20% of the CV or less can be used in the method disclosed herein. Thus, a SEC column having a volume loading capacity of about 20% or below can be efficiently used.
  • a sample loading volume of about 10% of the CV or less is advantageous for obtaining a better resolution of the peaks, and thus, of the separation of mRNA from residual components present in the sample. This may be considered beneficial in obtaining a higher recovery yield of a purified RNA sample with a higher purity.
  • This Example demonstrates the method as disclosed herein, wherein the concentration of RNA present in a sample is obtained with high accuracy using SEC.
  • the present Example demonstrates that the concentration of RNA may be obtained during purifying RNA from a sample using SEC as well as by analysing a purified RNA sample using SEC.
  • a crude IVT reaction sample comprising a 1975 nucleotide-long mRNA product, free NTPs, DNA template for said mRNA and RNA polymerase (T7 RNA polymerase) was obtained.
  • T7 RNA polymerase DNA template for said mRNA and RNA polymerase
  • the sample was subjected to a DNase-treatment and was diluted 5x, and an additional 5x dilution with 10 mM Tris pH 7.5 and 1 mM EDTA, prior to loading onto and run on a Prepacked Prep 26/10 Sepharose 6 FF column (26 mm x 100 mm) with a CV of 53 ml (Cytiva).
  • the experiment was performed by an initial injection of 4 mL of the crude DNase- treated IVT reaction sample to the SEC column. 4 mL of the resulting mRNA eluate was thereafter re-injected. 4 mL sequential re-injection of each eluate was performed for a total of four times in addition to the first injection with the crude DNase-treated IVT material. Absorbance was recorded at 260 nm during the runs.
  • the concentration in the injected samples was determined from the measured absorbance values at 260 nm in the chromatogram both by the peak height and the peak area.
  • the corresponding mRNA mass (in mg) in the injected volume was calculated using the mass extinction coefficient of single-stranded RNA (mg-1 mL cm-1) with a 0.2 cm path length UV cell.
  • the data from the peak height and peak area, respectively, was used to construct calibration curves, which was compared to separate analysis with an UV spectrometer (NanoDrop) according to standard protocols.
  • the initial injection of 4 mL of the crude DNase-treated IVT reaction sample to the SEC column is represented by curve (U) (first injection) in the chromatogram in Fig. 5A.
  • the curve (U) shows two elution peaks, wherein the first elution peak, approximately between 17 and 25 mL, corresponds to the elution of mRNA and the second elution peak, starting at about 35 mL corresponds, to the elution of residual components present in the crude DNase-treated IVT sample.
  • the mRNA eluate i.e. the first peak elute
  • was then re-injected to the column curve (V); second injection).
  • RNA concentration measured by the method as described herein using SEC corresponds to the concentrations measured by the conventional method using Nanodrop with high accuracy.
  • each reinjection corresponds to a lower absorbance maximum of the elution peak between 17 and 25 mL, which indicates that the mRNA concentration of the injected sample was gradually decreased by re-injection of the same volume (4 mL) of the eluates collected in the previous SEC runs, which corresponds to a gradual dilution of the samples during these consecutive runs.
  • the recorded absorbance data for both peak heights and peak areas for each re- injection (obtained from the second to the fifth injections) of this serial dilution were thus presented as calibration curves as shown in Fig. 5B.
  • both type of data i.e. peak hight and peak area, obtained from the elution profile during SEC of a sample comprising RNA are suitable for determining the concentration of said RNA.
  • the present Example demonstrates that the concentration of RNA in a sample, such as a DNase-treated crude IVT reaction sample and/or a purified RNA sample, can be measured with high accuracy using SEC according to the method as disclosed herein, based on the elution profile obtained during the run of said sample, such as by recording absorbance at 260 nm.
  • concentration determination of single-stranded RNA in a sample but it is to be appreciated the herein disclosed method also enables concentration determination of double-stranded RNA by using the corresponding extinction coefficient for the above calculation.
  • Example 6 Testing of different base matrices used in SEC for purifying mRNA of various lengths from DNase treated crude IVT reaction samples
  • RNA of various lengths is demonstrated according to the method as disclosed herein using different SEC matrix alternatives, wherein RNA of a DNase-treated sample is separated from proteins and residual DNA fragments present in said sample using SEC.
  • a crude IVT reaction sample comprising free NTPs, RNA polymerase (T7 RNA polymerase) and a 1975 nucleotide-long mRNA product together with the corresponding DNA template was obtained.
  • the sample was subjected to DNase treatment, as described in Example 1. 100 pl of the sample was injected at 0.5 ml/min flow rate and run on different HiScreen columns (7.7 mm x 100 mm) having a CV of 4.7 mL packed with the following base cross-linked agarose matrices: 1) Sepharose 6 FF (Cytiva), 2) Sepharose 4 FF (Cytiva), and 3) Sepharose High Performance (Cytiva).
  • Sepharose 6FF is based on spherical cross-linked 6% agarose and has a particle size d 5 ov of approximately 90 pm, and the exclusion limits [M ] for globular proteins and dextran is ⁇ 4 x 10 6 Da and ⁇ 2 x 10 6 Da, respectively.
  • Sepharose 4FF is based on spherical cross-linked 4% agarose and has a particle size d 5 ov of approximately 90 pm, and the exclusion limits [M] for globular proteins and dextran is ⁇ 3 x 10 7 Da and ⁇ 6 x 10 6 Da, respectively.
  • Sepharose High Performance it is also based on cross-linked 6% agarose with a mean bead size of 34 pm, and an exclusion limit for globular proteins of ⁇ 4 x 10 6 Da. Further product specifications can be found on cytivalifesciences.com. Chromatograms recorded during these runs are presented in Fig. 6A. b) In a second set of experiments, eluted peaks (both the first and second peaks corresponding to the mRNA eluate and the eluate of the remaining components of a crude IVT reaction sample) from runs as described in a) were collected and pooled for a second run using the same columns and base matrices as indicated above.
  • Each sample comprised free NTPs, RNA polymerase (T7 RNA polymerase) and one of 1) a 1042 nucleotide-long mRNA product, 2) a 1975 nucleotide-long mRNA product or 3) a 4525 nucleotide-long mRNA product together with the corresponding DNA template for the respective mRNAs.
  • the samples were subjected to DNase treatment, as described in Example 1 prior to the first run. 100 pl of the collected mRNA peak samples (having an mRNA concentration of 0.1 pg/pl) was injected at 0.5 ml/min flow rate and run using the above specified SEC method as in Example 1. Chromatograms of runs from the third set of experiments are presented in Fig. 6C.
  • Each sample comprised free NTPs, RNA polymerase (T7 RNA polymerase) and one of 1) a 1042 nucleotide-long mRNA product, 2) a 1975 nucleotide-long mRNA product or 3) a 4525 nucleotide-long mRNA product together with the corresponding DNA template for the respective mRNAs.
  • the samples were subjected to DNase treatment, as described in Example 1 prior to the first run. Eluted peaks (both the first and second peaks corresponding to the mRNA eluate and the eluate of the remaining components of a crude IVT reaction sample) from the first runs as described immediately above were collected and pooled for a second run using the same columns and base matrices as indicated above.
  • absorbance was measured at 260 nm.
  • the columns were equilibrated between each run using a buffer containing 10 mM Tris (pH 7.5) and 1 mM EDTA with a volume corresponding to at least one column volume.
  • the obtained chromatograms are also informative on the shape of the mRNA elution peaks which may be characterized by the asymmetry value thereof, as known in the art.
  • the asymmetry value is dependent on the matrix used as well as on the nature of the sample analyzed. It is ideally near 1, which means that the peak has a sharp symmetrical shape.
  • a value near 1 may be beneficial in the separation of components of a sample using SEC, since a better resolution may be obtained between components having different elution volumes.
  • a peak having an asymmetry value greater than 1 is indicative of a "tailing effect" which means that the elution of the component is prolonged. When this happens, the purity and/or recovery yield of the purified component may be compromised. As demonstrated in Fig.
  • the asymmetry value obtained using the same matrix varies depending on the size of the purified mRNA, moreover varies between different matrices when purified mRNA of the same length is analyzed. Consequently, while each of the tested SEC matrices is suitable for purifying RNA according to the method as disclosed herein, it is envisioned that depending on the size of the RNA, the most suitable SEC matrix may be chosen accordingly. Such selection may be beneficial for optimizing purity and/or RNA recovery yield. Based on the values of asymmetry presented in Fig. 6C, the Sepharose 6 FF resin (Cytiva), appears to perform particularly well among the tested alternatives, independently of the size of the mRNA.
  • each tested alternative SEC matrix is suitable for purifying RNA according to the method as disclosed herein.
  • mRNA of the DNase-treated crude IVT samples was separated from residual components present in said samples using each matrix.
  • a purified mRNA sample is obtained using each tested matrix and independently of the size of the mRNA present in the initial sample. Since the second elution peaks are absent in chromatograms of the third runs, it is also concluded that the mRNA sample obtained by collecting the first elution peak from the second runs can be characterized with near 100%, such as at least 95% purity.
  • Example 7 Testing of reusability of a SEC column in a method as disclosed herein
  • This Example demonstrates reusability capacity of a column packed with a SEC matrix in a method as disclosed herein.
  • sequential injection and run of a purified RNA sample comprising a 1975 nucleotide-long mRNA product has been carried out according to the present method without performing any cleaning steps in between the runs.
  • the below presented results indicate that a column packed with a SEC matrix in a method as disclosed herein can be reused and/or recycled for the method rapidly and without any decline in its performance.
  • a purified mRNA sample comprising a 1975 nucleotide-long mRNA product has been obtained.
  • 100 pl of the sample (having an mRNA concentration of 0.125 mg/mL) was injected and run consecutively 10 times at a flow rate of 0.5 mL/min on a HiScreen column (7.7 mm x 100 mm) having a CV of 4.7 mL packed with Sepharose 6 FF (Cytiva). No cleaning steps in between the runs have been performed. No separate equilibration was performed between the runs, since the buffer volumes used before and after sample injection is sufficient for equilibration between the injections. Absorbance at 260 nm was measured.
  • Fig. 7 shows superimposed chromatograms of the 10 consecutive SEC runs, wherein one elution peak corresponding to the elution of mRNA present in the purified mRNA sample appears near identical between each individual run.
  • the peak area of these elution peaks is presented in Table 2. Accordingly, measured peak areas between the superimposed elution peaks are near identical with a standard deviation of 0.6214 (and 0.260965 with excluding the peak area of the first run). Table 2. Peak area of the elution peak ofmRNA in 10 consecutive SEC analyses of a purified mRNA sample
  • this Example shows that a column packed with a SEC matrix can be used with reliable performance and high accuracy in consecutive runs of samples comprising RNA without implementing a cleaning step of the column in between the runs.
  • the present Example concerns re-injection of a purified mRNA sample, it is to be understood that the herein demonstrated accuracy, reproducibility and reusability apply to consecutive runs of any samples comprising RNA according to the method as disclosed herein.
  • the disclosed method is expected to efficiently separate mRNA from a sample with high accuracy and steady performance also after a repeated number of runs.
  • the method as disclosed herein enables a repeated number of SEC runs for purifying mRNA, wherein a harsh cleaning step between individual runs is omitted. This is advantageous for numerous reasons.
  • a chromatography medium such as a column, packed with a SEC matrix may be reused at least 50-100 times, such as 100 times or more in a method as disclosed herein, without implementing any harsh cleaning step in between the runs.
  • Example 8 T7 RNAP alone elutes in peak 2 when reducing agent is included in running buffer
  • T7 RNA polymerase is the key component for mRNA synthesis during IVT but also a major impurity to be separated from mRNA during downstream purification.
  • T7 RNAP was purified to high purity and injected to Sepharose 6FF under similar running conditions as used for mRNA purification.
  • This Example demonstrates that when including reducing agent in running buffer, pure T7 RNA polymerase (T7 RNAP) likely is preserved in its monomeric state and elutes at retention volume similar or later than peak 2 as seen for free NTPs and example proteins shown in Example 1 and 3, thereby separated from the mRNA peak (peak 1).
  • pure T7 RNA polymerase elutes partially in peak 1, likely due to formation of higher oligomer or aggregates.
  • T7 RNAP When purified T7 RNAP of at least 10-fold concentrated as in IVT reaction was injected onto Sepharose 6 FF, it was clear that the inclusion of reducing agent (in this case TCEP 5mM) in running buffer was important to maintain T7 RNAP eluting as a single peak starting at around 0.875 CV, as seen in curve (AA) in Fig 8B.
  • reducing agent in this case TCEP 5mM
  • T7 RNAP shows elution profile consisting of 2 peaks (curve (AB) in Fig. 8B). The first peak ranging from approximately 0.31 to 0.625 CV therefore overlaps with that of pure mRNA, whereas the majority of T7 RNAP elutes at a second peak with retention volume starting at 0.875 CV.
  • Example 9 S6FF separation of mRNA and impurities works across pH range of 4.5 to 8.5 Material and methods
  • DNase-treated crude IVT comprising of 1975-nucleotides long mRNA with concentration of 3.3 mg/ml was injected at 15% CV as load to 2xHiScreen Sepharose 6 FF column (bed height 20 cm, 9.3 mL CV) .
  • the buffers used include Tris 10 mM, EDTA ImM, pH 7.5; sodium acetate 50 mM, pH 4.5, sodium acetate 50 mM, pH 5.5, sodium citrate 50 mM, pH 6.5; HEPES 50 mM, pH 7.5 and HEPES 50 mM, pH 8.5.
  • Peak 1 for mRNA and peak 2 for impurities were analyzed on Fragment Analyzer: mRNA peak 1 fractions were diluted to final concentration of 0.2 mg/ml before analysis and peak 2 fractions were kept undiluted.
  • HEPES 50 mM, pH 7.5 corresponds to curve (AH) in Fig 9A and HEPES 7.5 in Fig 9B; HEPES 50 mM, pH 8.5, corresponds curve (Al) in Fig. 9A and HEPES 8.5 in Fig. 9B.
  • Fig. 9A the separation offered by Sepharose 6FF for mRNA from other impurities is functional across the tested buffer pH range, with two clear and distinct peaks around 0.5 CV (Pl) and 1 CV (P2), respectively.
  • Fig. 9B shows that mRNA of 1975-nucleotides size is found only in Pl fractions, while P2 is free from mRNA, regardless of tested buffer. The mRNA integrity was regarded unchanged in all the buffer pH tested. pH range lower than 4.5 or higher than 8.5 were not tested in this experiment, out of concern of the mRNA stability in those conditions.
  • Sepharose 6FF offers scalable, preparative purification of mRNA from crude IVT
  • a crude IVT reaction sample comprising free NTPs, RNA polymerase (T7 RNA polymerase) and a 1975 nucleotide-long mRNA product together with the corresponding DNA template was obtained as described in Example 1.
  • the sample was subjected to DNase treatment, also as described in Example 1.
  • the crude IVT originating from the same batch was applied to three sizes of Sepharose 6FF columns including 2xHiScreen (diameter 0.77 cm), 2xHiPrep (diameter 2.6 cm ) and XK50/30 (diameter 5 cm).
  • a UV cell with 0.5 mm path length was used for all three runs. Column format information is listed in Table 3 below.
  • Peak 1 (Pl) for mRNA and peak 2 (P2) for impurities were analysed on Fragment Analyzer for mRNA integrity analysis, as described in Example 9.
  • Sepharose 6FF could be used for preparative purification of mRNA with scalable processable sample volume and productivity when appropriate column size is chosen for the input scale. As shown in Fig. 10B, three Sepharose 6FF columns of increasing sizes exhibit similar elution profiles when purifying DNase-treated crude IVT sample comprising of 1975- nucleotide mRNA, with the mRNA successfully extracted in Peak 1 only whereas Peak 2 is free from mRNA.
  • Sepharose 6FF packed in column formats starting from 2xHiPrep upwards offers a productivity of at least 236 mg of mRNA per hour, estimated by amount of mRNA purified within 2CV of elution.
  • the separation of mRNA from DNAse- treated crude IVT is similar for different column formats with a common bed height of 20 cm and a common sample load of 15% CV (see Fig. 10A). Peak 1 (Pl) is at approx. 0.4-0.7 CV, and peak 2 (P2) is at approx.1-1.3 CV. Due to the chemical and structural characteristics of mRNA, especially long constructs, the crude IVT sample as well as the eluted mRNA exhibits high viscosity.
  • delta-column pressure during mRNA purification by Sepharose 6FF, since high pressure contributed by mRNA is expected during sample application and mRNA elution in Peak 1.
  • the delta-column pressure is influenced by many factors, including but not limited to mRNA concentration in sample, percent of CV loaded and size of the mRNA.
  • the actual reachable productivity by Sepharose 6FF is subject to the practical tolerance by the specific mRNA of interest.
  • Crude IVT reactions comprising free NTPs, RNA polymerase (T7 RNA polymerase) and either as 100, 200 or 400 nucleotide-long mRNA product together with the corresponding DNA template was obtained as described in Example 1.
  • the crude IVT samples were subjected to DNase treatment, as described in Example 1 before being injected individually on Sepharose 6FF in 2xHiScreen column format (CV 9.3 mL). The sample load was 4.3% of CV for all runs.
  • the chromatography was performed with 67 cm/h or 200 cm/h linear flow rate on an AKTA Pure 25 system with an UV cell of 2 mm path length. Unified running buffer of Tris 10 mM, EDTA ImM pH 7.5 was used for all runs.
  • mRNA of 400 nucleotides size (curve (AU) in Fig. 11A, curve (AX) in Fig. 11B) has similar elution profile regardless of linear flow rate applied.
  • Performing Sepharose 6FF purification on crude IVT comprising of mRNA that are smaller than 400 nucleotides likely require faster linear flow rate to maximize the separation from the impurities.
  • Example 12 mRNA size cut-off for successful separation from crude IVT by Sepharose 6FF
  • DNase treated crude IVTs comprising of either 200 or 400 nucleotides mRNA are individually injected to Sepharose 6FF packed in 2xHiScreen column format (20 cm bed height). Chromatography was performed using Tris 10 mM, EDTA ImM, pH 7.5 as running buffer on an AKTA Pure 25 system coupled with an UV cell of 2 mm path length. The linear flow rate was set to be 200 cm/h for both runs. The fractions from Sepharose 6FF crude mRNA purification were pooled for peak 1 for re-injection onto the same column. In addition, peak 2 fractions of crude purification as well as new fractions obtained from reinjecting peak 1 pool were further analysed for presence of mRNA on CGE by Fragment Analyzer.
  • Example 13 Testing of different functionalized base matrices
  • RNA is demonstrated according to the method as disclosed herein using two SEC base matrix alternatives coupled with two different ligands, one being a cation exchange (CIEX) ligand and one being a hydrophobic interaction (HIC) ligand. None of the ligands bind RNA under conditions used for SEC with mRNA from DNase treated crude IVT reaction samples Material and methods
  • a crude IVT reaction sample comprising free NTPs, RNA polymerase (T7 RNA polymerase) and a 1975 nucleotide-long mRNA product together with the corresponding DNA template was obtained as described in Example 1.
  • the sample was subjected to DNase treatment, also as described in Example 1.
  • Resin 1 has the same base matrix as Sepharose 6 FF functionalized with a sulphopropyl cation exchange (CIEX) ligand and resin 2 same base matrix as Sepharose HP functionalized with a butyl ligand.
  • CIEX sulphopropyl cation exchange
  • the columns were equilibrated between each run using a buffer containing 10 mM Tris (pH 7.5) and 1 mM EDTA with a volume corresponding to at least one column volume.
  • Fig. 13A Chromatograms from the CIEX testing is presented in Fig. 13A and from the HIC testing in Fig. 13B.
  • the elution profile is substantially the same as for a non-functionalized resin as in the Examples above.
  • the presented results indicate that a column packed with a SEC base matrix coupled with a ligand that does not bind RNA when using SEC conditions can be used for separation of mRNA from proteins and residual DNA fragments present in crude DNase-treated IVT samples.
  • a method for purifying RNA from a sample comprising the RNA, DNA, protein and optionally further components comprising a) enzymatically digesting DNA in said sample to obtain a DNase-treated sample, and b) separating RNA of said DNase-treated sample from proteins and residual DNA fragments present in said DNase-treated sample by size exclusion chromatography (SEC) using a chromatography medium.
  • SEC size exclusion chromatography
  • volume loading capacity is equal to or less than about 50% of the column volume (CV), such as equal to or less than about 40% of the CV, equal to or less than about 35% of the CV, equal to or less than about 30% of the CV, equal to or less than about 25% of the CV, equal to or less than about 20% of the CV, equal to or less than about 15% of the CV, or equal to or less than about 10% of the CV.
  • CV column volume
  • volume loading capacity is about 1% to about 25% of the CV, such as about 1% to about 20% of the CV, such as about 2% to about 20% of the CV, such as about 2% to about 15% of the CV.
  • volume loading capacity is about 20% of the column volume, preferably about 10% of the column volume.
  • RNA is purified from said sample in a purity of at least about 90%, such as at least about 91%, such as at least about 92%, such as at least about 93%, such as at least about 94%, such as at least about 95%.
  • step b) The method according to item 10, wherein said purity is achieved by step b) without further steps of separating RNA of said DNase-treated sample from proteins and residual DNA fragments present in said DNase-treated sample.
  • RNA is purified from said sample in a recovery yield of at least 90%, such as at least about 91%, such as at least about 92%, such as at least about 93%, such as at least about 94%, such as at least about 95%.
  • said RNA, said proteins and said residual DNA fragments are eluted from the chromatography medium used in step b) using the same buffer.
  • step b further comprises, subsequent to step b, one or more chromatography steps, such as an ion exchange chromatography, an affinity chromatography step, a hydrophobic interaction chromatography step or a multimodal chromatography step.
  • chromatography steps such as an ion exchange chromatography, an affinity chromatography step, a hydrophobic interaction chromatography step or a multimodal chromatography step.
  • RNA is a single stranded RNA, such as an mRNA.
  • RNA comprises a linear sequence of at least about 500 nucleotides (nt), such as at least about 750 nt, such as at least about 1000 nt, such as at least about 1500 nt, such as at least about 2000 nt, such as at least about 3000 nt, such as at least about 4000 nt.
  • nt nucleotides
  • volume loading capacity is equal to or less than about 50% of the CV, such as equal to or less than about 40% of the CV, such as equal to or less than about 35% of the CV, such as equal to or less than about 30% of the CV, such as equal to or less than about 25% of the CV, such as equal to or less than about 20% of the CV, such as equal to or less than about 15% of the CV, such as equal to or less than about 10% of the CV.
  • volume loading capacity is from about 1% to about 25% of the CV, such as from about 1% to about 20% of the CV, about 2% to about 20% of the CV, or about 2% to about 15% of the CV.
  • volume loading capacity is about 20% of the column volume, preferably about 10% of the column volume.
  • RNA is purified from said DNase-treated sample in a purity of at least about 90%, such as at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, during said use.
  • RNA is purified from said DNase-treated sample in a recovery yield of at least 90%, such as at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, during said use.
  • RNA is a single stranded RNA, such as an mRNA.
  • RNA comprises a linear sequence of at least about 500 nucleotides (nt), such as at least about 750 nt, at least about 1000 nt, at least about 1500 nt, at least about 2000 nt, at least about 3000 nt, or at least about 4000 nt.
  • nt nucleotides
  • said matrix comprises beads having a median particle size D50v, such as a bead size D50v of 5- 200 pm, 10-200 pm, 20-200 pm, 40-200 pm, 60-200 pm, 80-200 pm, 100-200 pm, 120-200 pm, 140-200 pm, 160-200 pm, 180-200 pm, 5-180 pm, 5-160 pm, 5-140 pm, 5-120 pm, 5- 100 pm, 5-80 pm, 5-60 pm, 5-40 pm, 5-20 pm, or 10-50 pm.
  • a median particle size D50v such as a bead size D50v of 5- 200 pm, 10-200 pm, 20-200 pm, 40-200 pm, 60-200 pm, 80-200 pm, 100-200 pm, 120-200 pm, 140-200 pm, 160-200 pm, 180-200 pm, 5-180 pm, 5-160 pm, 5-140 pm, 5-120 pm, 5- 100 pm, 5-80 pm, 5-60 pm, 5-40 pm, 5-20 pm, or 10-50 pm.
  • step b) is performed using a single buffer, said single buffer comprising a reducing agent.

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Abstract

La divulgation concerne des procédés de purification d'ARN à partir d'un échantillon à l'aide d'une chromatographie d'exclusion stérique (SEC). L'invention concerne également des utilisations d'un milieu SEC pour purifier l'ARN à partir d'un échantillon.
PCT/EP2025/055779 2024-03-04 2025-03-04 Purification et analyse d'arn Pending WO2025186218A1 (fr)

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