Methods and chromatographic materials for chromatographic recovery of nucleic acids

Abstract

The present disclosure relates to a method for recovering a nucleic acid product from a composition. The method (100) comprises the following steps: (i) contacting the composition with a chromatography material functionalized with a ligand (110). The chromatography material comprises nanofibers. Step; (ii) optionally, washing the functionalized chromatography material with a washing liquid phase (120); (iii) selectively eluting the product by contacting the functionalized chromatography material with an elution liquid phase (130); (iv) a stationary washing step comprising regenerating the chromatography material by contacting it with a cleaning liquid phase (140); (v) repeating steps (i) to (iii) at least 15 cycles, wherein step (iv) is carried out in at least 1 cycle of the cycles; and (vi) collecting the recovered nucleic acid product (150). The chromatography material has the ability to retain the dynamic binding capacity at 10% breakthrough for the nucleic acid product, which is at least 80% of the corresponding dynamic binding capacity of the first cycle after 50 cycles.

Description

Technical Field

[0001] The present invention relates to a method for purifying nucleic acid molecules such as polyadenylated products like mRNA from biological compositions. The method includes the step of contacting the composition with a functionalized chromatography material comprising a convective chromatography material.

Background Art

[0002] Messenger RNA (mRNA) is a key mediator in the central dogma of molecular biology. Single-stranded mRNA is transcribed from one of the DNA strands of a gene and is complementary to it, and its protein-coding region specifies the amino acid sequence of the protein. Before its role as a template for encoding proteins, pre-mRNA is processed into mRNA through a series of events that mainly occur in the nucleus either post-transcriptionally or simultaneously with transcription from the DNA gene template. These important events include 5'-capping, intron splicing, polyadenylation of the 3' end, and shuttling from the nucleus to the cytoplasm. All of these features and events are for their own purposes and are important for the regulation of overall mRNA stability and translation efficiency. The 5' cap and poly-A tail are both inherent features of mRNA.

[0003] Advances in technology in the field of synthetic mRNA have brought synthetic mRNA into the spotlight and it is currently being evaluated in several preclinical and clinical studies for various diseases.

[0004] The production of synthetic mRNA by in vitro transcription (IVT) involves the main components: 1) a DNA template, 2) ribonucleotides, and 3) an RNA polymerase. The UTR sequences and protein coding sequences are defined by the DNA template. The 3' polyA tail may or may not be designed within the DNA template. When included in the DNA template, the length of the polyA tail is more controllable, but when not designed within the DNA template, polyA tailing after IVT with polyA polymerase can be used. There are several different 5'-capping analogs and strategies, both co-transcriptional and post-transcriptional approaches.

[0005] Oligo(dT) products are commonly used in "open purification systems" such as magnetic particles or spin columns. During hybridization (the binding step), the mRNA polyA tail hybridizes with the oligo(dT) ligand in a high-salt buffer. The high electrical conductivity limits the electrostatic repulsion between the negatively charged backbone of polyA and the oligo(dT) ligand. Subsequently, washing and gentle elution are typically performed using a low-electrical conductivity buffer or water that destabilizes the TA pairs to enable elution. Unwanted contaminants such as proteins, unreacted ribonucleotides, DNA, CAP analogs, and partial transcripts lacking the polyA portion are not retained on the solid support during the hybridization or washing steps.

[0006] The length of the oligo(dT) ligand for magnetic products is typically in the range of 14 to 30 nucleotides. Small particle sizes provide a large particle surface area per mL, and the particle size generally ranges from 1 to 5 μm. The length of the procedure for small-scale mRNA purification is generally shorter than 1 hour. Notably, these products are designed for and commonly used for the purification of mRNA pools from cell lysates, not for the purification of mRNA from IVT reactions. In addition, known products are designed to be operated in microcentrifuge tubes, and given their small size and consequently low magnetism, it is highly unlikely that any of these products can be scaled up to process larger sample volumes. Additionally, such small particles packed as chromatography materials would significantly impair fluidity compared to more conventional chromatography resins.

[0007] Furthermore, there are several drawbacks to using known adsorbent media materials for chromatographic mRNA separation.

[0008] Separations involving membranes and monoliths can be carried out at much higher flow rates than systems based on porous beads, with typical residence times on the order of 0.2 to 0.5 minutes. However, the typical binding capacity at 10% breakthrough of the target for monoliths and membranes under dynamic flow is lower than that of porous beads. The difference in binding capacity between monolith and membrane materials (compared to materials based on porous beads) may be offset by utilizing higher flow rates. In membrane adsorption chromatography, in contrast to gel permeation chromatography, there is binding of components of the fluid, such as individual molecules, aggregates, or particles, to the surface of the solid in contact with that fluid without the need for transport through pores by diffusion, and the active surface of the solid phase is accessible to molecules by convective transport. The advantage of membrane adsorbers over packed chromatography columns is their compatibility with being operated at much higher flow rates.

[0009] This is also referred to as convection-based chromatography. Convection-based chromatography matrices include any matrix in which the application of a hydrostatic pressure difference between the inflow and outflow of the matrix forces perfusion of the matrix to achieve substantial convective transport of substances into or out of the matrix, which is brought about very rapidly at high flow rates.

[0010] Convection-based chromatography and membrane adsorbers are described, for example, in US20140296464A1, US20160288089A1, US2019308169A1, and US2019234914A1, which are hereby incorporated by reference in their entirety.

[0011] There is a need for improved solutions for separating nucleic acids, for example, for separating mRNA from in vitro transcription (IVT) reactants to enable the recovery of therapeutic products on an industrial scale.

Prior Art Documents

Patent Documents

[0012]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Summary of the Invention

Means for Solving the Problems

[0013] One object of the present invention is to provide an improved solution for recovering nucleic acid products from a composition.

[0014] This is achieved using a method for recovering a nucleic acid product from a composition comprising the nucleic acid product according to the present disclosure. The method comprises the following steps: (i) contacting the composition, under conditions enabling base pairing between at least a portion of the nucleic acid product and a ligand, with a chromatography material comprising one or more nanofibers and functionalized with the ligand; (ii) optionally, washing the functionalized chromatography material with a washing liquid phase; (iii) selectively eluting the product by contacting the functionalized chromatography material with an elution liquid phase; (iv) a cleaning-in-place step comprising regenerating the chromatography material by contacting it with a cleaning liquid phase; (v) repeating steps (i) to (iii) at least 15 cycles, wherein step (iv) is carried out in at least one of the cycles; and (vi) collecting the recovered nucleic acid product. The chromatography material has the ability to retain the dynamic binding capacity at 10% breakthrough for the nucleic acid product, which is at least 80% of the corresponding dynamic binding capacity of the first cycle after 50 cycles.

[0015] This has the advantage of enabling the use of chromatography materials having a high binding capacity and being suitable for higher flow rates. This further has the advantage of enabling large nucleic acid molecules, such as mRNA, to have a relatively high probability of accessing the binding regions of the chromatography material even during short residence times. The short residence time compared to traditional chromatography solid supports reduces the sample loading time, which enables faster purification of the desired nucleic acids, which is particularly important for sensitive biomolecules such as mRNA. When combined with the high capacity of the chromatography material, this will further improve the overall productivity and economy of the method and accelerate method development. In addition, such methods are particularly well-suited in continuous chromatography system settings.

[0016] In some embodiments, steps (i)-(iii) are repeated at least 25 cycles or at least 50 cycles.

[0017] In some embodiments, the nucleic acid product is single-stranded RNA, double-stranded RNA, mRNA, pre-mRNA, single-stranded DNA, and / or double-stranded DNA.

[0018] The ligand can be a nucleic acid capable of complementary base pairing via a portion of the nucleic acid product of interest. In an embodiment, the ligand is arranged to base pair via at least 10 bases of the nucleic acid product. The ligand can attach to the nanofibers of the chromatography material.

[0019] In some embodiments, the chromatography material is functionalized with an oligo(dT) ligand, and the oligo(dT) ligand is (dT) 10~50 ligand, preferably (dT) 12~30 ligand, and the nucleic acid product comprises a polyA-tagged product.

[0020] In some embodiments, the chromatography material comprises a non-woven material comprising polymer nanofibers.

[0021] This enables convective and direct mass transfer of nucleic acid products to the ligand, and thus has the advantage of resulting in faster kinetics of target-ligand interaction. Consequently, the contact time required for processing the composition is shorter compared to typical porous resins.

[0022] The present disclosure further relates to a chromatographic material. The material is convective and is functionalized with a ligand comprising an oligonucleotide. The material comprises polymer nanofibers and can be in the form of one or more non-woven membranes and / or sheets. The ligand that can adhere to the nanofibers is arranged to base pair with at least a portion of the nucleic acid product.

[0023] In some embodiments, the chromatographic material has the ability to retain the dynamic binding capacity at 10% breakthrough for the nucleic acid product, which is at least 80% of the corresponding dynamic binding capacity of the first cycle after 50 repeated nucleic acid product binding and elution events.

Brief Description of the Drawings

[0024]

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[0025] Here, the present invention will be described in more detail with respect to some non-limiting examples and the accompanying figures.

[0026] Throughout the figures, the same reference numerals refer to the same parts, concepts, and / or elements. Consequently, what is said with respect to a reference numeral in one figure is equally applicable to the same reference numeral in other figures, unless otherwise specifically indicated.

[0027] FIG. 1 schematically depicts an example of a method for recovering a nucleic acid product from a composition containing the nucleic acid product, and the method 100 includes the following steps: (i) Under conditions that enable nucleic acid base pairing between at least a portion of the nucleic acid product and the ligand, contacting the composition with a chromatography material comprising one or more nanofibers and functionalized with the ligand in step 110; (ii) Optionally, washing the functionalized chromatography material with a washing liquid phase in step 120; (iii) Selectively eluting the product by contacting the functionalized chromatography material with an elution liquid phase in step 130; (iv) A step of stationary washing comprising regenerating the chromatography material with a cleaning liquid phase in step 140; (v) A step of repeating steps (i) to (iii) at least 15 cycles, wherein step (iv) is carried out in at least one cycle of said cycles; and (vi) A step of collecting the recovered nucleic acid product in step 150 comprising, wherein the chromatography material has the ability to retain the dynamic binding capacity at 10% breakthrough for said nucleic acid product, which is at least 80% of the corresponding dynamic binding capacity of the first cycle after 50 cycles.

[0028] In Figure 1, the dashed line connecting the elution step 130 and the stationary washing step 140 to the contacting step 110 represents starting a new cycle, including the option of omitting the stationary washing step 140 in some cycles. The step 150 of collecting the recovered nucleic acid product is independent of the cycle in which the stationary washing step 140 is carried out. The step 150 of collecting the recovered nucleic acid product is related to collecting the recovered nucleic acid product for the whole method. For example, the step 150 of collecting the recovered nucleic acid product may include collecting the eluate of the selectively eluting step 130 from all cycles.

[0029] The expression "repeating steps (i) to (iii)" involves carrying out the contacting step 110 and the selectively eluting step 130 while the washing step 120 is optional.

[0030] In some examples, at least one criterion includes that the dynamic binding capacity for the nucleic acid product in the last cycle of the at least 50 cycles is at least 80% of the dynamic binding capacity of the first cycle.

[0031] The expression "washing the functionalized chromatography material" relates to a step 120 of washing the chromatography material between the contacting step 110 and the eluting step 130.

[0032] In some examples, instead of step (iv) being performed in at least one cycle of the cycles, the stationary wash step 140 is an optional step. In some examples, the stationary wash step 140 is performed between each cycle. Typically, the stationary wash step 140 is performed after the selectively eluting step 130.

[0033] The term "regeneration" of the chromatography material, as used herein, means a method of substantially restoring or attempting to restore the chromatography material to its original strength or properties.

[0034] Stationary washing, CIP, can be performed in a number of modes with different combinations of buffer, chemicals, solvents, temperature, and duration. The expression "stationary washing" of the chromatography material relates to cleaning and / or regenerating the chromatography material in a cleaning liquid phase.

[0035] In some examples, the step 110 of contacting the composition with the chromatography material includes that the composition has a residence time of contacting the chromatography material for 10 to 15 seconds. In some of these examples, for 5 to 20 seconds, for 3 to 25 seconds, for 1 to 35 seconds, or for 8 to 45 seconds.

[0036] In some examples, the step 110 of contacting the composition with the chromatographic material has a residence time of 5 seconds to 300 seconds. In some of these examples, the residence time is 15 seconds to 120 seconds. In some examples, the step 110 of contacting the composition with the chromatographic material is carried out at room temperature. In some examples, the step 110 of contacting the composition with the chromatographic material is carried out in a salt concentration of 100 mM to 1000 mM. In some of these examples, the electrolyte includes KCl, NaCl, or other salts having an equivalent / comparable electrical conductivity such as NaCl. In some examples, the salt concentration is less than 500 mM. In some of these examples, the salt concentration is less than 100 mM, less than 150 mM, less than 200 mM, less than 250 mM, less than 300 mM, less than 350 mM, less than 400 mM, less than 450 mM, or less than 600 mM.

[0037] In some examples, the step 110 of contacting the composition with the chromatographic material includes using a composition containing the product in a concentration range of 0.1 to 10 mg / mL. In some of these examples, it is 1 to 5 mg / mL or 2 to 20 mg / mL.

[0038] In some examples, the step 110 of contacting the composition with the chromatographic material includes subjecting the composition to deoxyribonuclease I treatment and / or proteinase K treatment before contacting it with the chromatographic material. In some examples, the composition includes a chelating agent such as ethylenediaminetetraacetic acid, EDTA, etc.

[0039] In some examples, the composition includes 200 mM KCl, 10 mM Tris, and 1 mM EDTA and has a pH of about 7.5.

[0040] In some examples, the composition comprises nucleic acid products from in vitro transcription (IVT) reactions. In some of these examples, the composition comprises a cell lysate, a solution resulting from or obtained from cell culture such as cell culture supernatant. In some examples, the step 110 of contacting the composition comprises prior separation and / or purification of the solution resulting from or obtained from cell culture to obtain the nucleic acid product contained in the composition or a portion thereof.

[0041] In some examples, the composition is of enzymatic, synthetic, and / or biological origin. It should be understood that its origin may affect the method with respect to impurities, contaminants, and yield. Typically, the methods of the present invention allow for a relatively short residence time to recover the corresponding product, regardless of the origin of the composition, although the method may benefit from adjustments based on the origin of the composition.

[0042] In some examples, the chromatography material comprises one or more polymer nanofibers.

[0043] In some examples, the chromatography material comprises a nonwoven material comprising polymer nanofibers. In some of these examples, the polymer nanofibers comprise cellulose nanofibers.

[0044] The term nanofiber relates to fibers having a diameter in the nanometer range. Typically, nanofibers have a diameter of 1 nm to 1 μm.

[0045] In some examples, the chromatography material comprises an open structure and is formed to allow convection of the composition through the open structure. Convection allows for direct mass transfer of the product to the ligand, thereby providing faster kinetics of the target-ligand interaction. As a result, the open structure results in a shorter contact time required for processing the composition, as opposed to typical porous resins.

[0046] The chromatography medium can have an average flow pore diameter of 0.1 to 2.0 μm. The average flow pore (MFP) diameter is an indicator of the flow characteristics of the material and is measured by capillary flow porometry based on the movement of a wetting liquid with a known surface tension from sample pores by applying a gas at an increasing pressure, as described, for example, in Example 2 below. The higher the MFP diameter, the greater the flow rate of the liquid through the material at a given pressure. The average flow pore diameter is calculated from the point at which 50% of the flow rate flows through the sample. Thus, the average flow pore diameter corresponds to the pore diameter calculated at the pressure where the wetting curve and the semi-dry curve meet.

[0047] In an alternative definition, the average flow pore diameter of the matrix material can be regarded as the effective pore diameter defined as the size of the largest sphere that can pass through the pores.

[0048] The average flow pore diameter of the matrix material can be 0.1 to 2.0 μm, 0.1 to 1.8 μm, 0.1 to 1.6 μm, 0.1 to 1.4 μm, 0.1 to 1.2 μm, 0.1 to 1.0 μm, 0.1 to 0.8 μm, 0.1 to 0.6 μm, 0.1 to 0.4 μm, 0.1 to 0.2 μm, 0.2 to 2.0 μm, 0.4 to 2.0 μm, 0.6 to 2.0 μm, 0.8 to 2.0 μm, 1.0 to 2.0 μm, 1.2 to 2.0 μm, 1.4 to 2.0 μm, 1.6 to 2.0 μm, 1.8 to 2.0 μm, or 0.5 to 1.5 μm.

[0049] In some examples, the chromatography material is functionalized with a ligand having a ligand density of 3 to 20 μmol / g. In some examples, the ligand density is 1 to 10 μmol / g, 0.5 to 5 μmol / g, or 2 to 30 μmol / g.

[0050] In some examples, the ligand is arranged to base pair with the nucleic acid product via at least 5 bases. In some of these examples, the ligand is arranged to base pair via at least 10 bases, at least 15 bases, at least 25 bases, at least 50 bases, at least 100 bases, or at least 200 bases. In some of these examples, the ligand is arranged to base pair via at most 10 bases, at most 15 bases, at most 25 bases, at most 50 bases, at most 100 bases, or at most 200 bases.

[0051] In some examples, the ligand comprises an oligonucleotide arranged to base pair with at least a portion of the nucleic acid product.

[0052] In some examples, the ligand is linked to the chromatography material via a linker such as a C6 or C12 alkyl linker containing a carbon skeleton. In some of these examples, the C6 linker contains a (CH2)6 spacer, and correspondingly, the C12 linker contains a (CH2) 12 spacer. In some examples, the ligand is linked to the chromatography material via a linker containing a carbon skeleton with a length of 3 to 18 carbons. In some embodiments, the linker has a length corresponding to a carbon skeleton length having 6 to 12 carbon atoms.

[0053] In some examples, the elution liquid phase has a salt concentration of 0 to 50 mM. In some of these examples, the salt concentration is 0 to 10 mM, 0 to 20 mM, 0 to 30 mM, or 0 to 40 mM. Typically, the salt concentration is preferably approximately 0 mM.

[0054] In some examples, the elution liquid phase has a pH value in the range of pH 5.5 to pH 9. In some examples, the elution liquid phase is pure water or a buffer and / or an aqueous solution such as any buffer within the range of pH 5.5 to 9.

[0055] In some instances, the elution phase comprises Tris, Hepes, sodium phosphate, and / or sodium citrate. In some instances, the elution phase comprises a chelating agent such as EDTA. In some instances, the elution phase comprises 10 mM Tris and 1 mM EDTA at pH 7.5.

[0056] Typically, the salt concentration and / or total ion concentration in the liquid phase of the chromatographic material is significantly reduced during the elution step 130 .

[0057] In some examples, the step 120 of washing the functionalized chromatographic material is performed at room temperature.

[0058] In some examples, washing step 120 is performed in at least one-third of the cycles, or at least half of the cycles. In some of these examples, washing step 120 is performed every cycle.

[0059] In some instances, the composition has a salt concentration of less than 500 mM. In some instances, the composition has a salt concentration of less than 400 mM, less than 300 mM, or less than 200 mM. In some of these instances, the composition includes KCl, NaCl, or other salts with equivalent / comparative electrical conductivities.

[0060] In some of these examples, the wash liquid phase contains KCl, NaCl, or other salts with equivalent / comparative electrical conductivities.

[0061] The wash liquid phase utilized during wash step 120 can be the same buffer system used during step 110 of contacting the composition with the chromatographic material.

[0062] In some instances, the wash solution phase has a lower salt concentration than the composition in contacting step 110, thereby facilitating the subsequent elution step.

[0063] In some examples, the wash liquid phase has a lower electrical conductivity than the buffer system used during contact step 110.

[0064] In some examples, steps (i)-(iii) are repeated at least 20 cycles. In some of these examples, the number of cycles repeated is at least 30, at least 40, at least 50, at least 60, at least 80, at least 100, at least 125, at least 150, at least 175, or at least 200.

[0065] In some examples, the chromatography material has the ability to retain the dynamic binding capacity at 10% breakthrough for the nucleic acid product, which is at least 80% of the corresponding dynamic binding capacity of the first cycle by the method for determining the dynamic binding capacity as described herein after 50 cycles. In some of these examples, the ratio of the dynamic binding capacity to the first cycle is at least 70%, at least 75%, at least 85%, at least 90%, or at least 95%.

[0066] In some examples, the chromatography material has the ability to retain the dynamic binding capacity at 10% breakthrough for the nucleic acid product, which is at least 80% of the corresponding dynamic binding capacity of the first cycle after 60 cycles. In some of these examples, the dynamic binding capacity at 10% breakthrough for the nucleic acid product in the first cycle is compared to the corresponding value after 15 cycles, 20 cycles, 30 cycles, 40 cycles, 70 cycles, 80 cycles, 100 cycles, 125 cycles, 150 cycles, or 200 cycles.

[0067] The above examples for the dynamic binding capacity criteria and the examples of the cycles at which the comparison is made can be freely combined. For example, an example of a chromatography material is disclosed that has the ability to retain the dynamic binding capacity at 10% breakthrough for the nucleic acid product, which is at least 75% of the corresponding dynamic binding capacity of the first cycle after 80 cycles.

[0068] In some examples, the clean-in-place step 140 is performed at least once every other cycle, once every three cycles, once every four cycles, once every five cycles, once every six cycles, once every seven cycles, once every eight cycles, once every nine cycles, once every ten cycles, once every 20 cycles, once every 30 cycles, and / or once every 50 cycles. Typically, the optimal frequency for the clean-in-place step 140 depends on materials and cost, as the clean-in-place step 140 increases the duration of the cycle.

[0069] In some examples, the clean-in-place step 140 is performed at most once every other cycle, once every other cycle, once every third cycle, once every three cycles, once every four cycles, once every five cycles, once every six cycles, once every seven cycles, once every eight cycles, once every nine cycles, once every ten cycles, once every 20 cycles, once every 30 cycles, and / or once every 50 cycles.

[0070] In some instances, the clean-in-place step 140 is performed once every 3-9 cycles.

[0071] Cleaning in place refers to a cleaning process that is performed without the need to disassemble a unit such as a chromatography system or a holder for a chromatography material. Cleaning in place involves immersing, flooding, and / or flushing a solvent, buffer, and / or chemical into contact with the solid phase to be cleaned and / or regenerated, such as a chromatography material, for a duration that ensures that the functionalized solid phase is sufficiently cleaned to limit contaminants from cycle to cycle and restore its functionality.

[0072] The expression "reclaiming a chromatographic material" should be understood as performing an act of attempting to restore the original functionality of the chromatographic material, but typically such an act does not result in a complete restoration of functionality.

[0073] For example, the stationary washing step may include NaOH and / or a chaotropic agent such as guanidine hydrochloride or urea. In some examples, the stationary washing step utilizes a cleaning liquid phase containing NaOH and / or an isopropanol solution. In some examples, the stationary washing step utilizes peracetic acid.

[0074] In some examples, the stationary washing step includes 100 mM of NaOH. In some examples, the cleaning liquid phase contains 10 mM - 1 M of NaOH.

[0075] In some examples, the cleaning liquid phase contains sodium hydroxide, nitric acid, phosphoric acid, and / or sulfuric acid.

[0076] In some examples, the cleaning liquid phase has a pH of at least 8. In some examples, the cleaning liquid phase has a pH of at least 9, at least 10, at least 11, at least 12, or at least 13.

[0077] In some examples, step 150 of collecting the recovered nucleic acid product includes collecting at least a portion of the contacted elution liquid phase. Typically, step 150 of collecting the recovered nucleic acid product follows the standard procedure for the chromatographic system used.

[0078] An example of method 100 for recovering the product from a composition containing a polyA-tagged product. Method 100 includes the following steps: (i) Step 110 of contacting a composition with a chromatography material comprising one or more non-woven materials of polymer nanofibers, preferably cellulose nanofibers, wherein the chromatography material is functionalized with an oligo(dT) ligand, and the oligo(dT) ligand is (dT) 10~50 ligand, preferably (dT) 12~30 ligand; (ii) Optionally, step 120 of washing the functionalized chromatography material with a washing liquid phase; (iii) Step 130 of selectively eluting a polyA-tagged product by contacting the functionalized chromatography material with an elution liquid phase having a salt concentration significantly lower than that of the composition; and (iv) A step of repeating steps (i) to (iii) at least 30 cycles, wherein step (ii) is carried out in at least one cycle, and the performance of the chromatography material for the last cycle of the at least 30 cycles and the dynamic binding capacity for the polyA-tagged product of the last cycle of the at least 30 cycles are at least 80% of the dynamic binding capacity of the first cycle.

[0079] Another example of the method is for recovering the product from a composition containing a polyA-tagged product, and the method 100 comprises the following steps: (i) Step 110 of contacting the composition with a chromatography material under conditions that allow base pair formation between a polyA tag and an oligo(dT), wherein the chromatography material comprises one or more polymer nanofibers, preferably cellulose nanofibers, the chromatography material is functionalized with an oligo(dT) ligand, and the oligo(dT) ligand is (dT) 10~50 ligand, preferably (dT) 12~30 ligand; (ii) Optionally, step 120 of washing the functionalized chromatography material with a washing liquid phase; (iii) Step 130 of selectively eluting the polyA-tagged product by contacting the functionalized chromatography material with an elution liquid phase; (iv) A step 140 of stationary cleaning, including regenerating the chromatographic material with a cleaning liquid phase; (v) A step of repeating steps (i) to (iii) at least 50 cycles, wherein step (iv) is carried out in at least 1 cycle; and (vi) A step of collecting the recovered poly A-tagged product including, the chromatographic material has the ability to retain the dynamic binding capacity at 10% breakthrough for the poly A-tagged product, which is at least 80% of the corresponding dynamic binding capacity of the first cycle after 50 cycles.

[0080] The chromatographic material used in the method of the present invention includes a convective chromatographic material. The convective chromatographic material can be, for example, an adsorptive membrane in which the flow through such a material is more convective than diffusive. In some examples, the adsorptive membrane is, for example, a polymeric nanofiber membrane such as cellulose, cellulose acetate, and / or cellulose fiber that has been treated for use as an adsorbent medium, or includes it. In some examples, the adsorptive membrane is a monolithic material and / or a normal membrane produced by emulsification, or includes it. Another alternative is a 3D printed material.

[0081] In some examples, the adsorbent membrane includes polymer nanofibers. Typically, the polymer nanofibers are in the form of one or more nonwoven sheets, where each sheet includes one or more of the polymer nanofibers. A nonwoven sheet including one or more polymer nanofibers is a mat of the one or more polymer nanofibers where each fiber is essentially randomly oriented, i.e., it is not fabricated such that the fibers take on a specific pattern. Nonwoven sheets including polymer nanofibers are typically supplied by known methods. The nonwoven sheet can, in certain circumstances, consist of a single polymer nanofiber. Alternatively, the nonwoven sheet can include two or more polymer nanofibers, e.g., two, three, four, five, six, seven, eight, nine, or ten polymer nanofibers.

[0082] In some examples, the chromatography material includes one or more polymer nanofibers. In some examples, the chromatography material includes a nonwoven material including polymer nanofibers.

[0083] Preferably, the chromatography material utilized is in the form of one or more membranes or sheets, and the composition is passed through a holder including the one or more membranes or sheets and optionally one or more frits or other spacer materials.

[0084] Optionally, when the device is heated, a heatable metal structure is disposed between the membranes or sheets of the chromatography material that promotes elution of the poly-A tagged product.

[0085] In some examples, the polymer nanofibers include electrospun polymer nanofibers. Such electrospun polymer nanofibers are well known to those skilled in the art. Alternative methods for making polymer nanofibers, such as drawing, can also be used.

[0086] The polymer nanofibers used in the present invention typically have an average diameter ranging from 10 nm to 1000 nm. For some applications, polymer nanofibers having an average diameter from 200 nm to 100 nm are suitable. Polymer nanofibers having an average diameter from 200 nm to 400 nm may be suitable for certain applications.

[0087] The length of the polymer nanofibers used in the present invention is not particularly limited. Thus, conventional methods, such as electrospinning, can produce polymer nanofibers with lengths of hundreds of meters or even several kilometers. Typically, one or more polymer nanofibers have a length of up to 10 km, preferably from 10 m to 10 km.

[0088] The nonwoven sheet typically has a basis weight from 1 g / m 2 to 40 g / m 2 In some examples, the basis weight is from 5 g / m 2 to 25 g / m 2 In some examples, the basis weight is from 1 g / m 2 to 20 g / m 2 or 5 - 15 g / m 2

[0089] The nonwoven sheet typically has a thickness from 5 μm to 120 μm. In some examples, the thickness is from 10 μm to 100 μm. In some examples, the thickness is from 50 μm to 90 μm, 5 - 40 μm, 10 - 30 μm, or 15 - 25 μm.

[0090] ​The polymer used to produce the nanofibers used in the method of the present invention is not particularly limited, provided that the polymer is suitable for use in chromatographic applications. Thus, typically, the polymer is a chromatographic material, i.e., a polymer suitable for use as an adsorbent medium in chromatography. Suitable polymers include polyamides such as nylon, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, polysulfone, such as polyethersulfone (PES), polycaprolactone, collagen, chitosan, polyethylene oxide, agarose, agarose acetate, cellulose, cellulose acetate, and combinations thereof. Polyethersulfone (PES), cellulose, and cellulose acetate are preferred. In some cases, cellulose and cellulose acetate are preferred.

[0091] In some examples, the polymer used to produce the nanofibers used in the method comprises, or consists of, nylon, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, polysulfone, such as polyethersulfone (PES), polycaprolactone, collagen, chitosan, polyethylene oxide, agarose, agarose acetate, cellulose, cellulose acetate, and / or combinations thereof.

[0092] Typically, the functionalized chromatographic material used in the method is a functionalized cellulose chromatographic material. Preferably, the functionalized chromatographic material is formed from one or more nonwoven sheets, each comprising one or more cellulose or cellulose acetate nanofibers. Cellulose acetate can be readily formed into nanofibers, for example, by electrospinning, and can be readily converted to cellulose after electrospinning.

[0093] In a preferred embodiment, the functionalized chromatography material used in the method comprises one or more polymer nanofibers. In another embodiment, the functionalized chromatography material used in the method comprises one or more polymer fibers of any type. The polymer fibers can have any or all of the same properties as the nanofibers described above. Typically, the polymer fibers can have an average diameter from 10 nm to 1000 μm, preferably from 10 nm to 750 μm, more preferably from 10 nm to 500 μm, even more preferably from 10 nm to 400 μm, even more preferably from 10 nm to 300 μm, even more preferably from 10 nm to 200 μm, even more preferably from 10 nm to 100 μm, even more preferably from 10 nm to 75 μm, even more preferably from 10 nm to 50 μm, even more preferably from 10 nm to 40 μm, even more preferably from 10 nm to 30 μm, even more preferably from 10 nm to 20 μm, even more preferably from 10 nm to 10 μm, even more preferably from 10 nm to 5 μm, even more preferably from 10 nm to 4 μm, even more preferably from 10 nm to 3 μm, even more preferably from 10 nm to 2 μm, even more preferably from 10 nm to 1 μm (1000 nm).

[0094] In some examples, the ligand arranged to bind a nucleic acid product from a composition is an oligo(dT) ligand arranged to bind polyadenylated (polyA-tagged) mRNA.

[0095] In some examples, the method is (dT) 10~50 ligand, or preferably (dT) 12~30 utilizes nanofibers functionalized with an oligo(dT) ligand such as a (dT) ligand.

[0096] By using a plurality of non-woven sheets of polymer nanofibers, a thicker material with a higher capacity for adsorption can be prepared. Thus, a functionalized chromatography material is typically formed by supplying non-woven sheets, two or more of which are stacked one on top of the other, each sheet containing one or more polymer nanofibers, and simultaneously heating and pressing the stack of sheets to fuse the contact points between the nanofibers of adjacent sheets.

[0097] Preferred chromatography material preparation conditions for the pressurization and heating of polymer nanofiber / non-woven sheets can be found in WO-A-2015 / 052460 and WO-A-2015 / 052465, which are hereby incorporated by reference in their entirety.

[0098] The functionalized chromatography material used in the method has a dynamic binding capacity (DBC) that depends on the size of the mRNA, and specific examples are shown in the following examples. The DBC for 10% breakthrough can be determined according to standard average values, for example, using an AKTA Pure system or an equivalent FPLC system.

[0099] Typically, the nucleic acid products contained in the composition, such as polyA-tagged products, are nucleic acids having a size in the range of 200 to 50,000 bases. For example, as shown in Figure 2, a DBC of 4 to 16 mg / mL is expected for polyA-tagged RNA of 4000 to 500 nucleotides.

[0100] The DBC for 10% breakthrough is typically determined according to the following assay method: 1) Pass the loading material through the functionalized material contained in a holder in an AKTA Pure system (Cytiva); 2) When the material is determined by the UV flow cell, load it at a flow rate (mV / min) of the determined matrix volume per minute until the concentration after the holder outlet exceeds 10% of the loaded concentration; 3) Considering the dead volume in the system and the holder device, the total amount of protein loaded on the disk at 10% breakthrough was determined through the analysis of the chromatogram in Unicorn software (Cytiva).

[0101] Whether the chromatography material has the ability to retain at least 80% of the DBC at 10% breakthrough after 50 cycles can be determined by: 1) performing the first binding cycle on a fresh (previously unused) chromatography material and measuring the DBC at 10% breakthrough; 2) performing 50 chromatography cycles as described herein; and 3) measuring the DBC at 10% breakthrough. If the value of the DBC determined in step 3) represents at least 80% of the value of the DBC obtained in step 1), the material is considered to have the ability to retain at least 80% of the DBC at 10% breakthrough after 50 cycles.

[0102] As long as the method parameters are the same for the DBC determination in steps 1) and 3), the actual values of the parameters are not decisive and can be selected within the normal operating range by those skilled in the art.

[0103] In some examples, the method utilizes a functionalized chromatography material housed in a chromatography cartridge and / or a holder. The cartridge typically contains one or more functionalized chromatography media utilized in the present invention. The cartridge is typically cylindrical in shape.

[0104] Typically, the chromatography cartridges used contain one or more functionalized chromatography media, typically stacked and / or wound inside a cylindrical holder. In some instances, the chromatography cartridges used are designed to operate under axial and / or radial flow.

[0105] The methods of the invention can be operated at high flow rates. In some examples of the methods of the invention, the composition containing the product to be recovered is contacted with the functionalized chromatographic material for a time period of 1 minute or less, or 30 seconds or less, or 20 seconds or less. 110 In some of these examples, the time period is preferably 10 seconds or less.

[0106] In mRNA purification, a composition is typically introduced into a column capture chromatography system, such as the functionalized chromatography material used in the present invention, configured for a cyclic purification process to extract the target product. The cyclic process typically involves loading a feed onto a unit 110, washing the unit 120, eluting the target product 130, and then cleaning the unit 140 before the unit is loaded 110 with a new feed. It is desirable to be able to operate the unit for several cycles before it needs to be cleaned 140.

[0107] In some examples, the method for recovery of mRNA products comprises the steps of: (i) contacting 110 a composition as defined herein with a functionalized chromatographic material as defined herein; (ii) optionally washing 120 the chromatographic material with a wash liquid phase, preferably having the same or a lower salt concentration than the composition; (iii) selectively eluting 120 the mRNA product by contacting the functionalized chromatographic material with an elution phase, e.g., water, having a salt concentration significantly lower than that of the composition.

[0108] After the elution step, the method may further include a step of cleaning and regenerating the functionalized chromatography material. Typically, this is effected by contacting the functionalized chromatography material from which the mRNA product and / or product-related impurities have been eluted with a buffer. This can be carried out according to the usual methods known for the regeneration stage of such chromatography methods.

[0109] Typically, the method for recovering an mRNA product according to the present invention includes a single binding-elution step or a single flow-through step. Alternatively, the method according to the present invention may include successive, more than one binding-elution step. In some of these examples, two or more, three or more, four or more, and / or five or more binding-elution steps. Alternatively, the method according to the present invention may include successive, more than one flow-through step. In some of these examples, two or more, three or more, four or more, and / or five or more flow-through steps. Alternatively, the method according to the present invention may include successive combinations of binding-elution steps and flow-through steps. In some of these examples, in total, two or more, three or more, four or more, and / or five or more steps.

Examples

[0110] The data generated and presented herein was obtained with a prototype device having an oligo(dT) 30 ligand or oligo(dT) 20 ligand immobilized on a convective chromatography material. Prototype A had an oligo(dT) 20 ligand with an aminated C12 linker immobilized on a Fibro VS (vinyl sulfone) membrane.

[0111] (Example 1) Preparation of the prototype Synthesis of the oligo dT ligand All oligo(dT) ligands were synthesized using the standard cycle of acid-catalyzed detritylation (3%, v / v dichloroacetic acid in toluene), coupling (5-(benzylmercapto)-1H-tetrazole (BMT) as activator, 0.3 M in acetonitrile), capping (Cap A, 20%, v / v, N-methylimidazole / acetonitrile, and an equal amount of B1 (40%, v / v, acetic anhydride in acetonitrile) and B2 (60%, v / v lutidine in acetonitrile) as Cap B, mixed in situ for capping), and iodine-based oxidation (0.05 M iodine in pyridine containing 10% v / v water) using a 5G UnyLinker polystyrene support and β-cyanoethyl phosphoramidite monomers on an automated solid-phase synthesizer (AKTA oligopilot plus 100). The phosphoramidite monomers were dissolved to a concentration of 0.150 M in anhydrous acetonitrile and used in the presence of molecular sieves (0.3 nm rods of 1.6 mm). The recycle time used for unmodified phosphoramidite was 3 minutes (at 1.8 molar equivalents), and for amine spacer phosphoramidite was 5 minutes (at 2.5 molar equivalents). The stepwise coupling efficiency was found to be >99.0%. After synthesis of the oligoligand, cleavage from the solid support and deprotection of the protecting groups were performed by treating the resin with 25% aqueous NH3 at 55 °C for 12 - 16 h. Further, the supernatant solution was collected and the support was washed with water and 50% EtOH in water. The collected fractions were evaporated on a rotary evaporator. The crude ligand pellet was dissolved in water and the concentration was measured at 260 nm in a UV-VIS spectrophotometer. The purity of the ligand was analyzed by IEX-UPLC using tris and sodium perchlorate as running buffer.

[0112] Preferably, the aminated ligand is used in place of the thiolated ligand for the preparation of the ligand for immobilization, although either can be used. The thiolated ligand is supplied as a dimer with a linker and then requires a reduction and desalting step before reacting with the fiber. The aminated ligand is supplied with a terminal amino group that can react with the fiber without any prior reaction. There are slight differences in the synthesis of both ligands, for example, different molar equivalents (5 - 10) of thiol or amidite, recycle times (10 - 40 minutes), iodine concentration (20 - 50 mM) for oxidation, and oxidation times (2 - 4 minutes).

[0113] Preparation of glycidol vinyl sulfone cellulose membrane (Fibro-VS) Fifty cellulose acetate disks were washed with distilled water (4 × 600 ml). The wash solution was removed and replaced with 350 ml of 0.5 M KOH solution. The disks were treated with the KOH solution for 10 minutes with stirring before the addition of 100 ml of glycidol. The reaction medium was stirred vigorously on the disks for 2 hours. After this time, the supernatant was removed and the disks were washed with distilled water (4 × 600 ml) to obtain a clean intermediate that was used in the next step without further modification.

[0114] Thereafter, twenty-five disks were removed from the glycidol step and suspended in 500 ml of H2O containing 37.5 g of Na2CO3 and 150 ml of MeCN. The mixture was stirred vigorously while 100 ml of divinyl sulfone was added dropwise over 60 minutes. Thereafter, the reaction mixture was stirred vigorously for 16 hours. After this time, the supernatant was decanted and the disks were washed 3 times with 600 ml of acetone:H2O (1:1) and once with distilled H2O (1 × 600 ml). The clean intermediate was used in the next step without further modification.

[0115] Functionalization of Fibro VS membrane with thiolated oligo(dT) ligand The thiolated oligo(dT) solution was desalted in AKTA pure using a 50 mL desalting column into 150 mM NaCl. The resulting solution was reduced for 1 hour using 25 mM DTT, 0.1 M NaHCO3, 0.01 M Na2CO3, and subsequently desalted further as previously described. The resulting solution was concentrated using a 20 mL VivaSpin column MWCO 5 kDa. The solution was then diluted to 5.9 mg / mL and added to a Fibro VS sheet in a sealable container, followed by the addition of sodium sulfate (approx. 3 g). The container was sealed and placed on an orbital shaker for 16 hours. After this time, the supernatant was discarded and DI water (50 mL) was added to each tray. This was repeated a total of 5 times before any further steps were carried out.

[0116] Functionalization of the Fibro VS membrane with amino - oligo(dT) ligand The amino - oligo(dT) sample was dissolved in 150 mM NaCl buffer (50 mL). The solution was diluted to a concentration of 6.2 mg / mL and a volume of 50 mL by adding DI water (43 mL) to the oligo(dT) solution (7 mL). Sodium sulfate (7.1 g) was added and the pH was measured. This solution was added to a T1 sheet of Fibro VS in a sealable container and placed on an orbital shaker for 16 hours. After this time, the supernatant was discarded, DI water (50 mL) was added to the tray, and then it was placed back on the orbital shaker. This method was repeated 4 times before any further steps were carried out.

[0117] Blocking of divinylsulfone reactive groups To block any remaining vinylsulfone groups on the Fibro VS functionalized with oligo(dT), a phosphoric acid buffer solution of thioglycerol (2.5 v / v% thioglycerol, pH 8.3) was prepared by dissolving disodium hydrogen phosphate dodecahydrate (3.58 g) and disodium EDTA dihydrate (37 mg) in water (95 mL) with stirring. Thioglycerol (2.5 mL) was added, and the resulting solution was basified to pH 8.3 using saturated NaOH solution and diluted to 100 mL.

[0118] The sheet of the functionalized material was placed in a sealable container, immersed in 25 mL of a buffered thioglycerol solution, and then placed on an orbital shaker for at least 16 hours. After this time, the thioglycerol solution was discarded, DI water (50 mL) was added to the sheet, and then it was returned to the orbital shaker and placed for at least 15 minutes. This washing process was repeated 3 more times. The final wash was exchanged with glycerol:ethanol:water (50 mL, 20:20:60 v / v%), immersed for 1 hour, and then the moisture was removed and overmolded onto the desired unit.

[0119] (Example 2) Pore size measurement method The average flow pore size can be measured using capillary flow analysis with a commercially available device. In the example, the device used was a POROLUXTM 100 porometer (IB-FT GmbH, Berlin, Germany) according to the manufacturer's manual, and the methodology was as shown in Table 1.

[0120] [Table 1]

[0121] (Example 3) Binding capacity analysis of Fibro oligo dT20 prototype A Materials and methods mRNA - capless FlucV01 (1975 nucleotides including a 100 - nucleotide polyA tail) was prepared by in vitro transcription and pre - purified to reach a purity of over 94% by oligo dT purification. The mRNA was eluted in Tris - EDTA buffer or ribonuclease - free water and stored at - 20°C at a concentration above 300 μg / mL until used in chromatography experiments (quantification of DBC).

[0122] Chromatography system - Akta Pure 25 equipped with a 2 mm UV cell The chromatography column - Fibro oligo dT20 (Prototype A) was packed into a HiTrap device (Cytiva, Sweden) having two membrane layers and a final membrane volume of 0.4 ml.

[0123] Stock solutions used: 3 M KCl prepared in ribonuclease - free water 500 mM EDTA, pH 8.0 (ribonuclease - free) 1 M Tris, pH 7.5 (ribonuclease - free) Binding buffer (Inlet A1): 200 mM KCl, 10 mM Tris, 1 mM EDTA pH 7.5 Elution buffer (Inlet B1): ribonuclease - free water Stationary wash buffer (Inlet A2): 0.1 M NaOH

[0124] Example of a method for determining the DBC 10% value: FlucV01 mRNA was diluted to 0.2 mg / mL by diluting the stock mRNA with ribonuclease - free water and adjusting the KCl, Tris, and EDTA concentrations using the stock solutions so that the mRNA sample contained 200 mM KCl, 10 mM Tris, 1 mM EDTA pH 7.5. Before determining the DBC value, the sample was first injected through a bypass using a superloop and the Amax (or 100% breakthrough) was measured by monitoring UV at 260 nm. To measure the dynamic binding capacity (DBC), the mRNA sample was then injected into a HiTrap device containing 0.4 ml of Fibro oligo dT20 (Prototype A) while monitoring the UV at 260 nm. A flow rate of 2 ml / min was used at all stages except the sample application stage, where the flow rate was set as a variable to achieve various residence times (e.g., the flow rate was set to 0.8 ml / min to achieve a residence time of 30 seconds).

[0125] The following steps were used to bind and elute mRNA and to quantify DBC 10%. - Equilibration: 8 ml of binding buffer from inlet A1 - Sample application: Interrupt sample application at 50% of Amax or until depletion of superloop - Washing of unbound fraction: 2 ml of binding buffer from inlet A1 - Elution: 8 ml of ribonuclease-free water from inlet B1

[0126] The column was washed for the next experiment and the following steps were used to prepare it. - Washing of column with water: 8 ml of ribonuclease-free water from inlet B1 - CIP: 8 ml of 0.1 M NaOH from inlet A2 - Equilibration: 8 ml of binding buffer from inlet A1

[0127] DBC is calculated by the following equation or by using the DBC calculation extension in Unicorn Evaluation Classic: DBC = mass of bound mRNA (mg) / column volume (ml) = C × (VBT - Vdelay) / column volume (ml) where C = concentration of mRNA in mg / ml in the feed VBT = volume corresponding to the desired breakthrough (e.g., 10% in this experiment) Vdelay = void volume of the system and column = volume at the point when the electrical conductivity is equal to the midpoint between the running buffer and the sample input (after injection)

[0128] Results of the dynamic binding capacity method example Figure 2 shows that mRNA molecules of various lengths can be successfully purified by a method utilizing a functionalized chromatography material. Prototype A has an oligo(dT) with an aminoated C6 linker immobilized on a Fibro VS membrane 20It was a ligand. The length of the mRNA was about 400 nucleotides to 4100 nucleotides.

[0129] Figure 3 shows the effect on the dynamic binding capacity and ligand density of the length of the oligo(dT) ligand. The dynamic binding capacity was determined by loading poly(dA) 30 oligonucleotide (mRNA substitute) up to 10% breakthrough (24-second residence time). The binding buffer was composed of 10 mM Tris, 400 mM NaCl, 1 mM EDTA, pH 7.4. The ligand density was determined by Phosphor ICP-SFMS.

[0130] Figure 4 shows the effect on the dynamic binding capacity of the ligand linker. The dynamic binding capacity was determined as described in Figure 3.

[0131] Figure 5 shows a consistent pressure profile over 10 consecutive cycles without CIP. These runs were performed using poly(dA) 30 oligonucleotide as a substitute for mRNA as described in Figure 3.

[0132] The excellent flow properties of the prototype are preferably utilized in larger devices. For a 50 mL device, an acceptable capacity is obtained, and presumably, the loading time for a 1 L feed is shortened to 20 minutes compared to 2500 minutes for a 0.4 mL device.

[0133] (Example 4) Dynamic Binding Capacity (DBC) Experiment In two independent experiments, Fibro oligo dT20 (Prototype A) was tested in an AKTA pure chromatography system using in-house mRNA.

[0134] Method for evaluating changes in binding ability over multiple cycles Using oligo(dT)-purified polyA-tailed mRNA (1975 nucleotides), DBC is determined against time course including CIP of Fibro(oligo dT) with 100 mM NaOH.

[0135] Briefly, the mRNA is diluted to 200 - 250 μg / mL in Tris-EDTA (TE) buffer, pH 7.5, and potassium chloride (final concentration 200 mM) is added. The maximum absorbance value at 260 nm measured in a 2 mm UV cell is recorded before the start of DBC execution by injecting a small amount of the mRNA sample in bypass. See the details of the method used in Table 2 below. CV represents the column volume.

[0136]

Table 2

[0137] For the data shown in FIGS. 5 - 8, the following liquid phase flow rates and volumes were used: - Equilibration 20 CV at 10 mL / min - Sample application 0.8 mL / min up to 50% of Amax - Wash 20 CV at 5 mL / min - Elution 30 CV at 2 mL / min - CIP 20 CV at 10 mL / min 100 mM NaOH - Equilibration 20 CV at 10 mL / min

[0138] FIG. 6 depicts a chromatogram showing the absorbance at 260 nm and DBC values for a series of cycles at various retention times. The sample application was interrupted at 50% of the determined Amax value (500 mAU) at 260 nm of the sample. The ligand used was oligo(dT) with a C12 linker. 20and the composition contained 200 mM KCl and an mRNA product 1975 nucleotides in length. The results for RT with a 30-second residence time were the average values for the first, second, and seventh cycles. The RT for 15 seconds was cycle 3. The RT for 5 seconds was cycle 4. The RT for 60 seconds was cycle 5. The RT for 120 seconds was cycle 6.

[0139] Figure 7 depicts a plot of the dynamic binding capacity against the retention time for a series of cycles in Figure 6.

[0140] Figures 8A - 8B depict the measured chromatograms showing the absorbance at 260 nm and the results of the dynamic binding capacity for three cycles. Figure 8A shows the chromatogram separated with a 5% Y offset, and Figure 8B shows the overlaid chromatogram. The chromatograms only depict the sample loading stages for the first cycle C1, the second cycle C2, and the seventh cycle C7 and their calculated dynamic binding capacities. The three chromatograms correspond to the average 30-second RT chromatogram in Figure 6.

[0141] Figures 9A - 9C depict the experimental and estimated changes in the dynamic binding capacity after multiple cycles. Figure 9A depicts the dynamic binding capacity at 10% breakthrough for cycles 50 and 200 based on experimental values. Figure 9B depicts the dynamic binding capacity from Figure 9A for cycle 200 as a percentage of the corresponding dynamic binding capacity for cycle 50. Typically, a setting with a value above 80% of the initial dynamic binding capacity at 10% breakthrough is considered reusable.

[0142] For each cycle between 50 and 200, static washes were performed with 100 mM NaOH solution. Cycles 50 and 200 were run to determine dynamic binding capacity, and the runs between 50 and 200 were for the recovery of nucleic acid products from compositions containing in vitro transcription samples. For the dynamic binding capacity runs in Cycles 50 and 200, a step of contacting a composition containing 200 mM KCl was used.

[0143] Figure 9C depicts the estimated decrease in dynamic binding capacity for a chromatographic material containing polymer nanofibers (black squares) compared to a traditional less-reusable chromatographic resin (white squares) for repeated cycles, according to the parameters shown in Table 2.

[0144] (Example 5) Dynamic Binding Capacity of mRNA of Various Sizes and Binding Conditions Materials and Methods Poly(A)-tailed mRNAs of theoretical lengths of 1042 nucleotides, 1942 nucleotides, 1975 nucleotides, 3580 nucleotides, or 4525 nucleotides (including polyA 100 tails) were used to measure the DBC in the above Prototype A at various KCl concentrations during binding. All mRNAs used here were pre-purified by oligo(dT) affinity chromatography to achieve a high level of polyA-positive material for reliable concentration estimation by measurement and to minimize offset signals. Briefly, the pre-purified mRNA was diluted to a final concentration of 0.2 - 0.25 mg / ml in Tris 10 mM pH 7.5, EDTA 1 mM, and 200 - 500 mM KCl. max maxIt was defined by the total absorption at 260 nm contributed by the input mRNA sample and measured prior to DBC execution for each individual combination of mRNA and binding buffer. DBC was measured using binding buffers with three different KCl concentrations: 200 mM, 300 mM, and 500 mM. A residence time of 30 seconds was used throughout all experiments, which corresponded to a flow rate of 0.8 ml / min during sample application. A non-linear regression fit was performed on the data points for 200 mM KCl during binding and shown as a dotted line in that figure.

[0145] Results Figure 10 shows the DBC of prototype A for various mRNAs at KCl concentrations of 200 mM (black circles), 300 mM (white squares), and 500 mM (black triangles) during binding. The DBC was inversely correlated with the mRNA size across all KCl concentrations used during binding. In contrast, the DBC was directly correlated with the salt concentration in the tested range from 200 mM to 500 mM.

[0146] (Example 6) Dynamic binding capacity over 200 cycles of stationary washing with 0.1 M NaOH This experiment was conducted to demonstrate the performance stability in repeated use, including stationary cleaning-in-place (CIP) with 100 mM NaOH.

[0147] Materials and methods The DBC was measured in the prototype A unit using pre-purified mRNA (1975 nucleotides) in the interval up to 200 cycles. Briefly, the same batch of pre-purified mRNA (corresponding to mRNA 3 in Figure 10) was diluted to 0.25 mg / ml with Tris 10 mM, EDTA 1 mM pH 7.5 buffer and 200 mM KCl equal to the binding buffer (KCl 200 mM, Tris 10 mM, EDTA 1 mM pH 7.5). A residence time of 30 seconds corresponding to a flow rate of 0.8 ml / min during sample application was used throughout all runs. The mRNA was injected only at cycle numbers 1, 2, 3, 5, 10, 20, 40, 60, 80, 81, 100, 120, 140, 160, 180, and 200 in a fixed volume that ensured complete breakthrough of the DBC and accurate calculations. In non-DBC runs, the same volume of the binding buffer (without mRNA) was used as the sample. All 200 cycles included the following steps: equilibration in the binding buffer as 20 CV at 10 mL / min, sample application as 35 CV at 0.8 mL / min (for DBC runs) or 10 mL / min (for non-DBC runs), column washing in the binding buffer as 10 CV at 10 mL / min, elution in Tris 10 mM, EDTA 1 mM as 20 CV at 10 mL / min, and CIP in 100 mM NaOH as 15 CV at 10 mL / min. The DBC (QB10) was derived from individual chromatography runs and plotted as black circles against the cycle number.

[0148] Results Figure 11 shows that the dynamic binding capacity (DBC) of the prototype was maintained for at least 200 cycles of CIP with 100 mM NaOH. This indicates that the method enables cleaning and regeneration of the chromatography material repeated over 200 cycles while retaining high binding capacity.

[0149] Conclusion The high reusability of the method may enable the user to repeat it over 200 cycles without significant loss of performance. The method may also enable the use of one chromatography device for multiple batches or even different molecules without significant impact on the performance difference between cycles. The flexibility of the reusable method may enable acceleration of the development time.

[0150] Measurement data regarding Fibro-oligo(dT) demonstrate improved capacity and reusability while maintaining purification performance. As the area of therapeutic mRNA grows rapidly, much attention has been paid to method enhancement to increase productivity and cost efficiency. An increase in the upstream IVT mRNA titer is expected, and an improvement in efficiency in downstream purification is required. High efficiency and rapid kinetics can prevent the downstream purification using the oligo(dT) capture step from becoming rate-limiting in the manufacturing process.

[0151] An upscaling plan using polymer nanofibers compared to the reference resin is shown in Table 3. The reference resin is typically a normal agarose bead type chromatography resin.

[0152]

Table 3

[0153] (References) US20140296464A1 US20160288089A1 US2019308169A1 US2019234914A1

Explanation of Signs

[0154] A method for recovering the nucleic acid from a composition containing 100 nucleic acid products 110 Contact the composition with the chromatography material 120 Wash the chromatography material Elute the 130 product Clean the 140 chromatography material Collect the 150 product

Claims

**Claim 1** A method for recovering a nucleic acid product from a composition containing the nucleic acid product, the method (100) comprising the following steps: (i) contacting the composition, under conditions enabling nucleic acid base pairing between at least a part of the nucleic acid product and a ligand, with a chromatography material comprising one or more nanofibers and functionalized with the ligand (step 110); (ii) optionally, washing the functionalized chromatography material with a washing liquid phase (step 120); (iii) selectively eluting the product by contacting the functionalized chromatography material with an elution liquid phase (step 130); (iv) a stationary washing step comprising regenerating the chromatography material by contacting it with a cleaning liquid phase (step 140); (v) a step of repeating steps (i) to (iii) at least 15 cycles, wherein step (iv) is carried out in at least one cycle of said cycles; and (vi) a step of collecting the recovered nucleic acid product (step 150) comprising, wherein the chromatography material has the ability to retain the dynamic binding capacity at 10% breakthrough for said nucleic acid product, which is at least 80% of the corresponding dynamic binding capacity of the first cycle after 50 cycles. A method. **Claim 2** The method according to claim 1, wherein steps (i) to (iii) are repeated at least 25 cycles or at least 50 cycles. **Claim 3** The method according to claim 1 or 2, wherein the step (110) of contacting the composition with the chromatography material is carried out under conditions of a salt concentration of less than 500 mM. **Claim 4** The method according to any one of claims 1 to 3, wherein the nucleic acid product is single-stranded RNA, double-stranded RNA, mRNA, pre-mRNA, single-stranded DNA, and / or double-stranded DNA. **Claim 5** The method according to any one of claims 1 to 4, wherein the chromatography material comprises a non-woven material containing polymer nanofibers. **Claim 6** The method according to any one of claims 1 to 5, wherein the composition has a salt concentration of at most 250 mM. **Claim 7** The method according to any one of claims 1 to 6, wherein the elution liquid phase has a salt concentration of at most 50 mM. **Claim 8** The method according to any one of claims 1 to 7, wherein the cleaning liquid phase has a pH above 9. **Claim 9** The method according to any one of claims 1 to 8, wherein the chromatographic material has a ligand density of the ligand in the range of 3 to 20 μmol per gram of the chromatographic material.

10. The method according to any one of claims 1 to 9, wherein the ligand is arranged to form base pairs with at least 10 bases of the nucleic acid product.

11. The chromatographic material is functionalized with an oligo(dT) ligand, and the oligo(dT) ligand is (dT) 10～50 ligand, preferably (dT) 12～30 ligand, and the nucleic acid product comprises a polyA-tagged product. The method according to any one of claims 1 to 10

12. A chromatographic material that is convective and functionalized with a ligand containing an oligonucleotide, contains polymer nanofibers, and is in the form of one or more non-woven membranes and / or sheets, wherein the ligand is arranged to form base pairs with at least a part of the nucleic acid product.

13. The chromatographic material according to claim 12, wherein the density of the ligand is 3 to 20 μmol / g.

14. The ligand is arranged to form base pairs with the polyA-tagged product, (dT) 10～50 A ligand, preferably (dT) 12～30 The chromatography material according to claim 12 or 13, which is a ligand

15. The ability to retain the dynamic binding capacity at 10% breakthrough for the nucleic acid product, which is at least 80% of the corresponding dynamic binding capacity of the first cycle after 50 repeated nucleic acid product binding and elution events, of the chromatographic material according to any one of claims 12 to 14.

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