Methods and chromatographic materials for chromatographic recovery of nucleic acid molecules

Abstract

The present disclosure relates to a method for recovering a nucleic acid product from a composition. The method (100) comprises: (i) contacting (110) the composition with a chromatography material functionalized with a ligand. The chromatographic material comprises nanofibers; (ii) optionally washing (120) the functionalized chromatography material with a washing liquid phase; (iii) selectively eluting (130) the product by contacting the functionalized chromatography material with an eluent phase; (iv) cleaning in place (140) comprising regenerating the chromatographic material by contact with a cleaning liquid; (v) repeating steps (i)-(iii) for at least 15 cycles, wherein step (iv) is performed in at least one of said cycles; and (vi) collecting (150) the recovered nucleic acid product. The chromatographic material is capable of maintaining a dynamic binding capacity with 10% penetration for the product after 50 cycles, which is at least 80% of the corresponding dynamic binding capacity of the first cycle.

Description

Technical Field

[0001] The present invention relates to a method for purifying nucleic acid molecules such as polyadenylation products such as mRNA from a biological composition. The method involves contacting the composition with a functionalized chromatography material comprising a convection-based chromatography material. Background Art

[0002] Messenger RNA (mRNA) is a key medium in the central dogma of molecular biology. Single-stranded mRNA is transcribed from one of the DNA chains of the gene and is complementary to it, and its protein coding region specifies the amino acid sequence of the protein. Before it serves as a protein coding template, the precursor mRNA is processed into mRNA through a series of events, which mainly occur in the nucleus, either after transcription or simultaneously with transcription from the DNA gene template. These key 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 used for their own purposes and are crucial to the regulation of overall mRNA stability and translation efficiency. Both the 5' cap and the polyA tail are unique features of mRNA.

[0003] Technological advances in the field of synthetic mRNA have brought synthetic mRNA into the spotlight and are 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 key components 1) a DNA template, 2) ribonucleotides, and 3) an RNA polymerase. The UTR sequence and protein coding sequence are defined by the DNA template. The 3' polyA tail may or may not be designed into the DNA template. If included in the DNA template, the length of the polyA tail is more controllable, while when not designed into the DNA template, post-IVT polyA tailing with a polyA polymerase can be used. There are several different 5'-capping analogs and strategies, including both co-transcriptional and post-transcriptional approaches.

[0005] Oligo(dT) products are often used in "open purification systems" such as magnetic particles or spin centrifuge columns. During the hybridization (binding phase), the mRNA polyA tail hybridizes with the oligo(dT) ligand in a high salt buffer. The high conductivity limits the electrostatic repulsion of the negatively charged backbone of polyA and the oligo(dT) ligand. This is generally followed by washing and gentle elution using a low conductivity buffer or water to destabilize the TA pair and allow elution. During the hybridization or washing phase, undesirable contaminants such as proteins, unreacted ribonucleotides, DNA, CAP analogs, and partial transcripts lacking the polyA portion will not remain on the solid support.

[0006] The length of the oligo(dT) ligands used for the magnetic products is typically between 14-30 nucleotides. The small particle size provides a large particle surface area per mL, and the size of the particles is typically between 1-5 μm. The protocol length for small-scale mRNA purification is typically less than 1 hour. It is worth noting that these products are designed and are typically used to purify mRNA libraries from cell lysates rather than to purify mRNA from IVT reactions. In addition, the known products are designed to run in microcentrifuge tubes, and given their small size and therefore low magnetic properties, it is unlikely that any of these products will be scalable to handle larger sample quantities. In addition, such small particles filled as chromatographic materials will significantly impair flow properties compared to more conventional chromatographic resins.

[0007] Furthermore, chromatographic mRNA separations using known adsorption materials present several disadvantages.

[0008] Separations involving membranes and monoliths can be run at flow rates much higher than systems based on porous beads, with typical residence times of about 0.2-0.5 minutes. However, under dynamic flow, the typical binding capacity of monoliths and membranes at a target penetration of 10% is lower than that of porous beads. The difference in binding capacity between monoliths and membrane materials (compared to materials based on porous beads) can be offset by using higher flow rates. In membrane adsorption chromatography, in contrast to gel permeation chromatography, components of the fluid, such as single molecules, associates or particles, bind to solid surfaces in contact with the fluid without being transported in pores by diffusion, and the molecules can enter the active surface of the solid phase by convective transport. The advantage of membrane adsorbers over packed chromatographic columns is that they are suitable for running at much higher flow rates.

[0009] This is also known as convection-based chromatography. Convection-based chromatography matrices include any matrix in which a hydraulic differential applied between the inflow and outflow of the matrix forces perfusion of the matrix, achieving significant convective transport of species into or out of the matrix, which is achieved 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] Improved solutions are needed to isolate nucleic acids, such as mRNA from in vitro transcription (IVT) reactions, to enable recovery of therapeutic products on an industrial scale. Summary of the invention

[0012] It is an object of the present invention to provide an improved solution for recovering nucleic acid products from compositions.

[0013] According to the present disclosure, this has been achieved by a method for recovering a nucleic acid product from a composition comprising the product. The method comprises the following steps:

[0014] (i) contacting the composition with a chromatography material under conditions that allow nucleic acid base pairing between at least a portion of the nucleic acid product and the ligand, wherein the chromatography material comprises one or more nanofibers, and wherein the chromatography material is functionalized with the ligand;

[0015] (ii) optionally washing the functionalized chromatographic material with a washing liquid phase;

[0016] (iii) selectively eluting the product by contacting the functionalized chromatographic material with an eluent;

[0017] (iv) cleaning in place involving regeneration of the chromatographic material by contact with a cleaning solution;

[0018] (v) repeating steps (i)-(iii) for at least 15 cycles, wherein step (iv) is performed in at least one of said cycles; and

[0019] (vi) collecting the recovered nucleic acid product.

[0020] The chromatographic material is capable of maintaining a dynamic binding capacity for said nucleic acid product after 50 cycles at 10% breakthrough which is at least 80% of the corresponding dynamic binding capacity of the first cycle.

[0021] Like this have the advantage of allowing to use the chromatographic material with high binding capacity and being suitable for higher flow rate.Even if this further has the advantage of allowing large nucleic acid molecules such as mRNA to also have relatively high probability to enter the binding area of ​​chromatographic material during short residence time.Compared with traditional chromatographic solid support, short residence time reduces the time of sample loading, allows to purify the nucleic acid of expectation more quickly, and this is particularly important for sensitive biomolecules such as mRNA.This, and even further combine with the high capacity of chromatographic material, will improve overall process productivity and economy, and accelerate process development.In addition, this method is particularly suitable for continuous chromatography system setting.

[0022] In some embodiments, steps (i)-(iii) are repeated for at least 25 cycles or at least 50 cycles.

[0023] 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.

[0024] The ligand may be a nucleic acid capable of complementary base pairing with a portion of the target nucleic acid product. In an embodiment, the ligand is arranged as a base pair with at least 10 bases of the nucleic acid product. The ligand may be attached to a nanofiber of a chromatographic material.

[0025] In some embodiments, the chromatographic material is functionalized with an oligo(dT) ligand, and wherein the oligo(dT) ligand is (dT) 10-50 Ligand, preferably (dT) 12-30 ligand, and wherein the nucleic acid product comprises a polyA labeled product.

[0026] In some embodiments, the chromatographic material comprises a nonwoven material comprising polymer nanofibers.

[0027] This has the advantage of allowing convection and direct mass transfer of the nucleic acid product to the ligand, resulting in faster kinetics of the target-ligand interaction. Thus, shorter contact times are required for the processing composition compared to typical porous resins.

[0028] The present disclosure further relates to a chromatographic material. The material is convection-based and functionalized with a ligand comprising an oligonucleotide. The material comprises polymer nanofibers and may be in the form of one or more nonwoven membranes and / or sheets. The ligand may be attached to the nanofibers and arranged to base pair with at least a portion of a nucleic acid product.

[0029] In some embodiments, the chromatography material is capable of maintaining a dynamic binding capacity for the nucleic acid product at 10% breakthrough after 50 repeated nucleic acid product binding and elution events that is at least 80% of the corresponding dynamic binding capacity of the first cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A method for repetitive recovery of nucleic acid products from a composition is schematically depicted.

[0031] Figure 2 Graph showing the dynamic binding capacity (DBC) of mRNAs of different lengths on a chromatography material (Fibro) functionalized with oligo(dT) ligands.

[0032] Figure 3 Two different ligand lengths (dT) are shown. 30 and (dT) 20 Plot of dynamic binding capacity and ligand density.

[0033] Figure 4 To display the use of Figure 3 Graph showing the effect of two different linkers, C12 and C6, on the dynamic binding capacity for the same ligand length.

[0034] Figure 5 is a graph showing the pressure profile for 10 consecutive cycles without cleaning in place (CIP), as described in the Examples section.

[0035] Figure 6 Chromatogram depicting conductivity / absorbance and kinetic binding results at different residence times for a series of cycles.

[0036] Figure 7 Depiction Figure 6 Dynamic binding capacity versus retention time for a series of cycles.

[0037] Fig. 8A -B shows a chromatogram depicting the conductivity / absorbance and kinetic binding results of subsequent cycles.

[0038] Fig. 9A -C depicts the experimental and estimated dynamic binding capacity changes after multiple cycles.

[0039] Fig.10 Figure 3 is a graph showing the dynamic binding capacity (DBC) of mRNAs of different sizes during binding at different KCl concentrations.

[0040] Fig.11 is a graph showing the dynamic binding capacity after 200 cleaning-in-place cycles. DETAILED DESCRIPTION

[0041] The invention will now be described in more detail with reference to some non-limiting examples and to the accompanying drawings.

[0042] Throughout the drawings, the same reference numerals refer to the same parts, concepts and / or elements. Therefore, unless otherwise expressly specified, the description of reference numerals in one figure is also applicable to the same reference numerals in other figures.

[0043] Figure 1 An exemplary method for recovering a nucleic acid product from a composition comprising the product is schematically depicted, the method 100 comprising the following steps:

[0044] (i) contacting 110 the composition with a chromatography material under conditions allowing nucleic acid base pairing between at least a portion of the nucleic acid product and the ligand, wherein the chromatography material comprises one or more nanofibers, preferably cellulose nanofibers, wherein the chromatography material is functionalized with the ligand;

[0045] (ii) optionally washing 120 the functionalized chromatography material with a wash phase;

[0046] (iii) selectively eluting 130 the product by contacting the functionalized chromatography material with an eluent; and

[0047] (iv) in situ cleaning 140 including regeneration of the chromatography material with a clean liquid phase;

[0048] (v) repeating steps (i) to (iii) for at least 15 cycles, wherein step (iv) is performed in at least one of said cycles;

[0049] (vi) collecting 150 the recovered nucleic acid product,

[0050] Wherein the chromatographic material is capable of maintaining a dynamic binding capacity for said nucleic acid product after 50 cycles at 10% breakthrough which is at least 80% of the corresponding dynamic binding capacity of the first cycle.

[0051] exist Figure 1 In the embodiment of the present invention, the dotted line connecting the elution 130 step and the in-place cleaning step 140 and the contacting 110 step indicates the start of a new cycle, and in some cycles, the in-place cleaning 140 step is optionally omitted. Collecting 150 the recovered nucleic acid product is independent of the cycle in which the in-place cleaning 140 step is performed. The step of collecting 150 the recovered nucleic acid product involves collecting the recovered nucleic acid product for the entire process. For example, collecting 150 the recovered nucleic acid product may include collecting the eluate from the selective elution step 130 step of all cycles.

[0052] It should be understood that the expression "repeating steps (i)-(iii)" requires the implementation of the contacting step 110 and the optional eluting step 130, and the washing step 120 is optional.

[0053] In some examples, at least one criterion includes the dynamic binding capacity of the nucleic acid product at the last cycle of the at least 50 cycles being at least 80% of the dynamic binding capacity at the first cycle.

[0054] The expression “washing the functionalized chromatography material” relates to washing 120 the chromatography material between contacting 110 and eluting 130 .

[0055] In some examples, the cleaning-in-place step 140 is an optional step, rather than performing step (iv) in at least one of the cycles. In some examples, the cleaning-in-place step 140 is performed between each cycle. Generally, the cleaning-in-place 140 is performed after the selective elution 130.

[0056] The term "regeneration" of a chromatography material is herein intended to mean a process by which the chromatography material is restored or attempts to be restored substantially to its original strength or properties.

[0057] It will be appreciated that cleaning in place (CIP) can be performed in a variety of ways, using different combinations of buffers, chemicals, solvents, temperatures and durations.The expression "cleaning in place" of the chromatography material relates to cleaning and / or regenerating the chromatography material with a cleaning liquid phase.

[0058] In some examples, contacting 110 the composition with the chromatographic material comprises contacting the composition with the chromatographic material for a residence time of 10-15 seconds, for a period of 5-20 seconds, for a period of 3-25 seconds, for a period of 1-35 seconds, or for a period of 8-45 seconds.

[0059] In some instances, the composition is contacted with the chromatographic material 110 for a residence time between 5s-300s. In some of these instances, the residence time is between 15s-120sec. In some instances, the composition is contacted with the chromatographic material 110 and implemented at ambient temperature. In some instances, the composition is contacted with the chromatographic material 110 and implemented during a salt concentration between 100mM-1000mM. In some of these instances, the electrolyte includes KCl, NaCl or other salts with equivalent / equivalent conductivity, such as NaCl. In some instances, the salt concentration is less than 500mM. In some of these instances, the salt concentration is less than 100mM, less than 150mM, less than 200mM, less than 250mM, less than 300mM, less than 350mM, less than 400mM, less than 450mM or less than 600mM.

[0060] In some examples, contacting 110 the composition with a chromatographic material comprises utilizing a composition having a concentration of the product ranging between 0.1-10 mg / mL, In some of these examples, between 1-5 mg / mL or between 2-20 mg / mL.

[0061] In some examples, contacting 110 the composition with a chromatographic material includes treating the composition with DNase I and / or proteinase K prior to contacting the composition with the chromatographic material. In some examples, the composition includes a chelating agent, such as ethylenediaminetetraacetic acid, EDTA.

[0062] In some examples, the composition comprises KCl 200 mM, Tris 10 mM, and EDTA 1 mM, and the pH is about 7.5.

[0063] In some examples, the composition comprises a nucleic acid product from an in vitro transcription (IVT) reaction. In some of these examples, the composition comprises a solution derived from or obtainable from a cell culture, such as a cell lysate, a cell culture supernatant. In some examples, contacting 110 the composition comprises pre-separating and / or purifying the solution derived from or obtainable from a cell culture to obtain the nucleic acid product contained in the composition or a portion thereof.

[0064] In some instances, the composition has enzymatic, synthetic and / or biological origin. It should be understood that the source will affect the method in terms of impurities, pollutants and output. Generally, the method of the present invention allows the use of a relatively short residence time to reclaim the corresponding product, regardless of the composition source, however, the method can benefit from adjustments based on the composition source.

[0065] In some examples, the chromatographic material comprises one or more polymer nanofibers.

[0066] In some examples, the chromatographic material comprises a nonwoven material comprising polymer nanofibers. In some of these examples, the polymer nanofibers comprise cellulose nanofibers.

[0067] The term nanofiber relates to fibers having a diameter in the nanometer range. Typically, nanofibers have a diameter between 1 nm and 1 μm.

[0068] In some instances, the chromatographic material comprises an open structure and is configured to allow convection of the composition through the open structure. Convection allows direct mass transfer of the product to the ligand, thereby providing faster kinetics of the target-ligand interaction. Therefore, in contrast to typical porous resins, the open structure results in a shorter contact time required for processing the composition.

[0069] The mean flow pore size of the chromatographic media is 0.1-2.0 μm. The mean flow pore (MFP) size is an indicator of the flow properties of the material and is measured by capillary flow porometry, which is based on the displacement of a wetting liquid with a known surface tension from the sample pores by the application of a gas under increasing pressure, for example as described in Example 2 below. The larger the MFP size, the greater the liquid flow through the material at a given pressure. The mean flow pore size is calculated from the point at which 50% of the flow through the sample. Therefore, the mean flow pore size corresponds to the pore size calculated at the pressure where the wet curve and the semi-dry curve intersect.

[0070] In another definition, the mean flow pore size of the present matrix material can be considered the effective pore size, which is defined as the size of the largest sphere that can pass through the pores.

[0071] The mean flow pore size of the matrix material may be 0.1-2.0 μm, 0.1-1.8 μm, 0.1-1.6 μm, 0.1-1.4 μm, 0.1-1.2 μm, 0.1-1.0 μm, 0.1-0.8 μm, 0.1-0.6 μm, 0.1-0.4 μm, 0.1-0.2 μm, 0.2-2.0 μm, 0.4-2.0 μm, 0.6-2.0 μm, 0.8-2.0 μm, 1.0-2.0 μm, 1.2-2.0 μm, 1.4-2.0 μm, 1.6-2.0 μm, 1.8-2.0 μm, or 0.5-1.5 μm.

[0072] In some examples, the chromatographic material is functionalized with a ligand having a ligand density of 3-20 μmol / g. In some examples, the ligand density is 1-10 μmol / g, 0.5-5 μmol / g, or 2-30 μmol / g.

[0073] In some instances, the ligand is arranged to base pair with the nucleic acid product through at least 5 bases. In some of these instances, the ligand is arranged to base pair with 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 instances, the ligand is arranged to base pair with 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.

[0074] In some examples, the ligand comprises an oligonucleotide arranged to base pair with at least a portion of the nucleic acid product.

[0075] In some examples, the ligand is coupled to the chromatographic material via a linker such as a C6 or C12 alkyl linker comprising a carbon skeleton. In some of these examples, the C6 linker comprises (CH 2 ) 6 spacer, and accordingly the C12 linker comprises (CH 2 ) 12 Spacer. In some examples, the ligand is coupled to the chromatographic material via a linker comprising a carbon skeleton between 3-18 carbon atoms in length. In some examples, the length of the linker corresponds to the length of a carbon skeleton having 6-12 carbon atoms.

[0076] In some examples, the salt concentration of the eluent phase is between 0-50 mM. In some of these examples, the salt concentration is between 0-10 mM, 0-20 mM, 0-30 mM or 0-40 mM. Generally, the salt concentration is preferably close to 0 mM.

[0077] In some examples, the pH value of the eluent phase is in the range of pH 5.5-pH 9. In some examples, the eluent phase is a buffer and / or an aqueous solution, such as pure water or any buffer in the range of pH 5.5-9.

[0078] In some instances, the eluent phase comprises Tris, Hepes, sodium phosphate and / or sodium citrate. In some instances, the eluent phase comprises a chelating agent, such as EDTA. In some instances, the eluent phase comprises Tris 10 mM and EDTA 1 mM @ pH 7.5.

[0079] Typically, during elution 130, the salt concentration and / or total ion concentration in the liquid phase at the chromatography material is significantly reduced.

[0080] In some examples, washing 120 the functionalized chromatography material is performed at ambient temperature.

[0081] In some examples, washing 120 is performed in at least one third of the cycles or at least half of the cycles. In some of these examples, washing 120 is performed in each cycle.

[0082] In some examples, the salt concentration of the composition is less than 500 mM. In some examples, the salt concentration of the composition is less than 400 mM, less than 300 mM, or less than 200 mM. In some of these examples, the composition comprises KCl, NaCl, or other salts with equivalent / comparable conductivity.

[0083] In some of these examples, the wash phase comprises KCl, NaCl, or other salts of equivalent / comparable conductivity.

[0084] It should be understood that the wash liquid phase utilized during wash 120 may be the same buffer system used during contacting 110 the composition with the chromatography material.

[0085] In some examples, during contacting 110, the wash phase has a lower salt concentration than the composition, thereby facilitating a subsequent elution step.

[0086] In some examples, the conductivity of the wash liquid phase is lower than the buffer system used during contacting 110 .

[0087] In some examples, steps (i)-(iii) are repeated for at least 20 cycles. In some of these examples, the number of repeated cycles 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.

[0088] In some examples, according to the method for determining dynamic binding capacity as described herein, the chromatographic material is capable of maintaining a dynamic binding capacity for the nucleic acid product at 10% breakthrough after 50 cycles that is at least 80% of the corresponding dynamic binding capacity of the first cycle. In some of these examples, the dynamic binding capacity ratio relative to the first cycle is at least 70%, at least 75%, at least 85%, at least 90%, or at least 95%.

[0089] In some examples, the chromatographic material is capable of maintaining a dynamic binding capacity for the nucleic acid product at 10% breakthrough after 60 cycles that is at least 80% of the corresponding dynamic binding capacity at the first cycle. In some of these examples, the dynamic binding capacity for the nucleic acid product at 10% breakthrough at 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.

[0090] It should be understood that the examples of the above dynamic binding capacity standards and the examples of the cycles for comparison can be freely combined. For example, an example chromatography material is disclosed that is capable of maintaining a dynamic binding capacity for the nucleic acid product at 10% breakthrough after 80 cycles that is at least 75% of the corresponding dynamic binding capacity of the first cycle.

[0091] In some examples, clean-in-place 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 twenty cycles, once every thirty cycles, and / or once every fifty cycles. Generally, the optimal frequency of clean-in-place depends on materials and costs, as the clean-in-place 140 step increases the duration of the cycle.

[0092] In some examples, clean-in-place 140 is performed at most once every other cycle, once every other cycle, once every three cycles, 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 twenty cycles, once every thirty cycles, and / or once every fifty cycles.

[0093] In some examples, clean-in-place 140 is performed every 3-9 cycles.

[0094] Cleaning in place involves cleaning steps that can be performed without disassembling the device, such as the chromatography system or the chromatography material holder. Cleaning in place involves soaking, filling and / or flowing solvents, buffers and / or chemicals into contact with the solid phase to be cleaned and / or regenerated, such as the chromatography material, for a period of time that continuously ensures that the functionalized solid phase is sufficiently cleaned to limit inter-cycle contamination and restore its functionality.

[0095] The expression "regenerating the chromatography material" is to be understood as carrying out an operation which attempts to restore the original functionality of said chromatography material, however generally such operation does not lead to a complete restoration of the functionality.

[0096] For example, the in-place cleaning may include NaOH and / or a chaotropic agent such as guanidine hydrochloride or urea. In some examples, the in-place cleaning utilizes a cleaning liquid phase comprising NaOH and / or an isopropanol solution. In some examples, the in-place cleaning uses peracetic acid.

[0097] In some examples, the cleaning liquid phase comprises NaOH 100 mM. In some examples, the cleaning liquid phase comprises NaOH between 10 mM and 1 M.

[0098] In some examples, the cleaning liquid phase comprises sodium hydroxide, nitric acid, phosphoric acid, and / or sulfuric acid.

[0099] In some examples, the cleaning liquid phase has at least pH 8. In some examples, the cleaning liquid phase has at least pH 9, at least pH 10, at least pH 11, at least pH 12, or at least pH 13.

[0100] In some examples, collecting 150 the recovered nucleic acid product comprises collecting at least a portion of the contacted eluate phase. Generally, the step of collecting 150 the recovered nucleic acid product follows standard procedures for the chromatography system used.

[0101] An example method 100 for recovering a polyA-labeled product from a composition comprising the product. The method 100 comprises the following steps:

[0102] (i) contacting 110 the composition with a chromatography material of one or more nonwoven materials comprising polymer nanofibers, preferably cellulose nanofibers, the chromatography material being functionalized with an oligo(dT) ligand, wherein the oligo(dT) ligand is a (dT)10-50 ligand, preferably a (dT)12-30 ligand;

[0103] (ii) optionally washing 120 the functionalized chromatography material with a wash phase;

[0104] (ii) selectively eluting the 130 polyA labeled product by contacting the functionalized chromatography material with an eluent having a salt concentration significantly lower than that of the composition; and

[0105] (iv) repeating steps (i)-(iii) for at least 30 cycles, wherein step (ii) is performed in at least one cycle, and wherein the chromatographic material performance of the last cycle of the at least 30 cycles and the dynamic binding capacity of the polyA-labeled product of the last cycle of the at least 30 cycles are at least 80% of the dynamic binding capacity of the first cycle.

[0106] Another example method for recovering a polyA-labeled product from a composition comprising the product, method 100 comprises the following steps:

[0107] (i) contacting the composition with a chromatography material under conditions that allow base pairing between the polyA tag and the oligo(dT) 110, wherein the chromatography material comprises one or more polymer nanofibers, preferably cellulose nanofibers, wherein the chromatography material is functionalized with an oligo(dT) ligand, and wherein the oligo(dT) ligand is (dT) 10-50 Ligand, preferably (dT) 12-30 Ligand;

[0108] (ii) optionally washing 120 the functionalized chromatography material with a wash phase;

[0109] (iii) selectively eluting 130 the polyA-labeled product by contacting the functionalized chromatography material with an eluent;

[0110] (iv) in situ cleaning 140 including regeneration of the chromatography material with a cleaning liquid; and

[0111] (iv) repeating steps (i) to (iii) for at least 50 cycles, wherein step (iv) is performed in at least one cycle,

[0112] (vi) collecting 150 the recovered polyA-labeled product, and

[0113] Wherein the chromatography material is capable of maintaining a dynamic binding capacity for said polyA-labeled product at 10% breakthrough after 50 cycles which is 80% of the corresponding dynamic binding capacity of at least the first cycle.

[0114] The chromatographic material used in the inventive method comprises a chromatographic material based on convection. The chromatographic material based on convection can be, for example, an adsorption film, wherein the flow through this material is convection rather than diffusion. In some instances, the adsorption film is a polymer nanofiber membrane or comprises a polymer nanofiber membrane, such as cellulose, cellulose acetate and / or cellulose fiber, which have been processed as an adsorber. In some instances, the adsorption film is a monolithic material and / or a conventional membrane made by emulsification or comprises a monolithic material and / or a conventional membrane made by emulsification. Another selection is a 3D printing material.

[0115] In some instances, the adsorption film comprises polymer nanofibers. Generally, polymer nanofibers are in the form of one or more nonwoven sheets, and each sheet comprises one or more of the polymer nanofibers. The nonwoven sheet comprising one or more polymer nanofibers is a mat of the one or more polymer nanofibers, wherein each fiber is oriented substantially randomly, i.e., it is not made so that one or more fibers adopt a specific pattern. Generally, a nonwoven sheet comprising polymer nanofibers is provided by known methods. In some cases, the nonwoven sheet can be composed of a single polymer nanofiber. Alternatively, the nonwoven sheet can comprise two or more polymer nanofibers, such as 2,3,4,5,6,7,8,9 or 10 polymer nanofibers.

[0116] In some examples, the chromatographic material comprises one or more polymer nanofibers. In some examples, the chromatographic material comprises a nonwoven material comprising polymer nanofibers.

[0117] Preferably, the chromatography material used is in the form of one or more membranes or sheets and the composition is passed through a support comprising one or more of said membranes or sheets and optionally one or more frits or other spacer materials.

[0118] Optionally, a heatable metal structure is arranged between the membrane or sheet of chromatography material to facilitate elution of the polyA-labelled product upon heating of the device.

[0119] In some examples, the polymer nanofibers include electrostatically spun polymer nanofibers. Such electrostatically spun polymer nanofibers are well known to those skilled in the art. Alternative methods for producing polymer nanofibers, such as wire drawing, may also be used.

[0120] The average diameter of the polymer nanofibers used in the present invention is generally 10 nm to 1000 nm. For some applications, polymer nanofibers with an average diameter of 200 nm to 100 nm are suitable. Polymer nanofibers with an average diameter of 200 nm to 400 nm may be suitable for certain applications.

[0121] 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 a length of hundreds of meters or even kilometers. However, generally, the length of one or more polymer nanofibers is at most 10 km, preferably 10 m-10 km.

[0122] The area density of nonwoven sheets is generally 1-40 g / m 2 In some embodiments, the area density is 5-25 g / m 2 In some embodiments, the area density is 1-20 g / m 2 or 5-15g / m 2 .

[0123] The thickness of the nonwoven sheet is generally 5-120 μm. In some examples, the thickness is 10-100 μm. In some examples, the thickness is 50-90 μm, 5-40 μm, 10-30 μm or 15-25 μm.

[0124] The polymer for producing the nanofiber used in the inventive method is not particularly limited, provided that the polymer is suitable for chromatographic applications. Therefore, generally, polymer is a polymer suitable for use as a chromatographic material (i.e., adsorber) in chromatography. Suitable polymer includes polyamide, such as nylon, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, polysulfone (e.g., 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.

[0125] In some examples, the polymer used to produce the nanofibers used in the method includes or consists of nylon, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, polysulfone (e.g., polyethersulfone (PES)), polycaprolactone, collagen, chitosan, polyethylene oxide, agarose, agarose acetate, cellulose, cellulose acetate, and / or combinations thereof.

[0126] Typically, the functionalized chromatographic material used in the method is a functionalized cellulose chromatographic material. Preferably, the functionalized chromatographic material is formed by one or more nonwoven sheets, each comprising one or more cellulose or cellulose acetate nanofibers. Cellulose acetate is easy to form nanofibers, such as by electrostatic spinning, and can be easily converted into cellulose after electrostatic spinning.

[0127] In a preferred embodiment, the functionalized chromatographic material used in the method comprises one or more polymer nanofibers. In another embodiment, the functionalized chromatographic material used in the method comprises one or more of any type of polymer fiber. The polymer fiber may have any or all of the same properties as the above-mentioned nanofibers. Generally, the average diameter of the polymer fiber may be 10nm-1000μm, preferably 10nm-750μm, more preferably 10nm-500μm, even more preferably 10nm-400μm, even more preferably 10nm-300μm, even more preferably 10nm-200μm, even more preferably 10nm-100μm, even more preferably 10nm-75μm, even more preferably 10nm-50μm, even more preferably 10nm-40μm, even more preferably 10nm-30μm, even more preferably 10nm-20μm, even more preferably 10nm-10μm, even more preferably 10nm-5μm, even more preferably 10nm-4μm, even more preferably 10nm-3μm, even more preferably 10nm-2μm, even more preferably 10nm-1μm (1000nm).

[0128] In some examples, the ligand arranged to bind a nucleic acid product from the composition is an oligo(dT) ligand arranged to bind to polyadenylated (polyA-labeled) mRNA.

[0129] In some embodiments, the method utilizes nanofibers functionalized with oligo(dT) ligands, such as (dT) 10-50 Ligand, or preferably (dT) 12-30 Ligand.

[0130] The use of multiple nonwoven sheets of polymer nanofibers allows the preparation of thicker materials with greater adsorption capacity. Thus, functionalized chromatography materials are generally formed by providing two or more nonwoven sheets stacked one on top of another, each of the sheets comprising one or more polymer nanofibers, and simultaneously heating and pressing the stacked sheets to fuse the contact points between the nanofibers of adjacent sheets.

[0131] Preferred chromatography material production conditions for pressing and heating the polymer nanofiber / nonwoven sheet can be found in WO-A-2015 / 052460 and WO-A-2015 / 052465, which are incorporated herein by reference in their entirety.

[0132] The functionalized chromatography material used in the methods has a dynamic binding capacity (DBC) that depends on the size of the mRNA and specific examples are given in the examples below.The DBC for 10% breakthrough can be determined according to standard methods, for example using an AKTA Pure system or an equivalent FPLC system.

[0133] Generally, the nucleic acid product (e.g., polyA-labeled product) contained in the composition is a nucleic acid with a size ranging between 200-50000 bases. For example, for a polyA-labeled RNA of 4000-500 nucleotides, the DBC is expected to be 4-16 mg / mL, such as Figure 2 shown.

[0134] The DBC at 10% penetration is generally determined according to the following measurement method:

[0135] 1) Loading material through the functionalized material contained in the AKTA Pure system (Cytiva) stent;

[0136] 2) loading the material at a determined matrix volume / minute flow rate (mV / min) until the concentration after the stent exit exceeds 10% of the loaded concentration as determined by the UV flow cell;

[0137] 3) The total amount of protein loaded onto the disc at 10% breakthrough was determined by analyzing the chromatograms with Unicorn software (Cytiva), taking into account the dead volume in the system and the holder assembly.

[0138] Whether a chromatography material is capable of maintaining at least 80% of the DBC at 10% breakthrough after 50 cycles can be determined by: 1) subjecting a fresh (previously unused) chromatography material to a first binding cycle and measuring the DBC at 10% breakthrough, 2) subjecting 50 chromatography cycles as described herein, and 3) measuring the DBC at 10% breakthrough. If the DBC value determined in step 3) is at least 80% of the DBC value obtained in step 1), then the material is considered capable of maintaining at least 80% of the DBC at 10% breakthrough after 50 cycles.

[0139] As long as the process parameters are the same for the DBC determinations of steps 1) and 3), the actual values ​​of the parameters are not critical and may be selected by one skilled in the art within the normal operating range.

[0140] In some examples, the method utilizes a functionalized chromatographic material mounted in a chromatographic column and / or a support. The column generally comprises one or more functionalized chromatographic media used in the present invention. The column is generally cylindrical.

[0141] Typically, the chromatography columns used comprise one or more functionalized chromatography media stacked and / or wound within a typically cylindrical support. In some examples, the chromatography columns used are designed to operate under axial and / or radial flow.

[0142] The method of the present invention can be run at a high flow rate. In some examples of the method of the present invention, the composition comprising the product to be recovered is contacted 110 with the functionalized chromatographic material for a period of 1 minute or less, or a period of 30 seconds or less, or a period of 20 seconds or less. In some of these examples, the period of time is preferably 10 seconds or less.

[0143] In mRNA purification, the composition is generally introduced into a column capture chromatography system, such as the functionalized chromatography material used in the present invention, configured to cyclically circulate the purification process to extract the target product. The cyclic process generally includes loading 110 the feed onto the device, washing 120 the device, eluting 130 the target product and then cleaning 140 the device before loading 110 the device with new feed. It is desirable to be able to run the device for several cycles before cleaning 140 is required.

[0144] In some examples, the method for recovering the mRNA product comprises the following steps:

[0145] (i) contacting 110 a composition as defined herein with a functionalized chromatography material as defined herein;

[0146] (ii) optionally washing 120 the functionalized chromatography material with a wash phase, preferably with the same or lower salt concentration than the composition;

[0147] (iii) selectively eluting the 120 mRNA product by contacting the functionalized chromatography material with an eluent, such as water, having a significantly lower salt concentration than the composition.

[0148] After the elution step, the method may further include the step of cleaning and regenerating the functionalized chromatographic material. Typically, this is achieved by contacting the functionalized chromatographic material from which the mRNA product and / or product-related impurities are eluted with a buffer. This can be implemented according to conventional methods known for the regeneration phase of such chromatographic methods.

[0149] Generally, the method for reclaiming mRNA product according to the present invention comprises a single binding-elution step or a single flow-through step. Alternatively, the method according to the present invention may comprise more than one binding-elution step in series. In some of these examples, there are 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 comprise more than one flow-through step in series. In some of these examples, there are 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 comprise a combination of binding-elution and flow-through steps in series. In some of these examples, there are two or more, three or more, four or more and / or five or more steps in total.

[0150] Example

[0151] The data generated and presented herein were performed on a prototype device in which oligo(dT) 30 Ligand or oligo(dT) 20 The ligand is immobilized on a convection-based chromatography material. Prototype A is oligo(dT) with an aminated C12 linker 20 The ligand was immobilized on a Fibro VS (vinyl sulfone) membrane.

[0152] Example 1: Preparation of prototype

[0153] Synthesis of oligo-dT ligand

[0154] All oligo(dT) ligands were synthesized using the following standard cycles: 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 equal volumes 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) were performed on oligopilot plus 100 using 5G UnyLinker polystyrene support and β-cyanoethyl phosphoramidite monomers. The phosphoramidite monomers were dissolved in anhydrous acetonitrile to a concentration of 0.150 M and used in the presence of molecular sieves (1.6 mm of 0.3 nm rods). The recycle time for unmodified phosphoramidites was 3 min (with 1.8 molar equivalents) and 5 min (with 2.5 molar equivalents) for amine spacer phosphoramidites. The step-wise coupling efficiency was found to be >99.0%. After oligo ligand synthesis, cleavage and removal of protecting groups from the solid support was performed by treating the resin with 25% aqueous NH3 at 55°C for 12-16 hours. Further, the supernatant was collected and the support was washed with 50% EtOH in water. The collected fractions were evaporated on a rotary evaporator. The crude ligand particles were dissolved in water and the concentration was measured at 260 nm with a UV-VIS spectrophotometer. The purity of the ligand was analyzed on an IEX-UPLC using tris and sodium perchlorate as running buffer.

[0155] Preferably, aminated ligands are used instead of thiolated ligands due to the preparation of the ligands for immobilization, although either can be used. Thiolated ligands are provided with a linker as a dimer, which then requires a reduction and desalting step before reacting with the fiber. Aminated ligands are provided with a terminal amine group that can react with the fiber without any prior reaction. There are slight differences in the synthesis of the two ligands, such as the molar equivalents of thiol or amide (5-10), the cycle time (10-40 min), the iodine concentration used for oxidation (20-50 mM), and the oxidation time (2-4 min).

[0156] Preparation of Glycidyl Vinyl Sulfone Cellulose Membrane (Fibro-VS)

[0157] 50 cellulose acetate disks were washed with distilled water (4 x 600 ml). The washings were removed and replaced with 350 ml of 0.5 M KOH solution. The disks were treated with KOH solution for 10 minutes with stirring before adding 100 ml of glycidol. The reaction medium was stirred vigorously on the disks for 2 hours. After this, the supernatant was removed and the disks were washed with distilled water (4 x 600 ml) to obtain a clean intermediate which was used in the next step without further modification.

[0158] Afterwards, 25 disks were taken out from the glycidol step and suspended in 500 ml H2O, which contained 37.5 g Na2CO3 and 150 ml MeCN. The mixture was stirred vigorously while 100 ml divinyl sulfone was added dropwise over 60 minutes. The reaction mixture was then stirred vigorously for 16 h. After this, the supernatant was decanted and the disks were washed 3 times with 600 ml acetone:H2O (1:1) and washed with distilled H2O (1 x 600 ml). The clean intermediate was used for the next step without further modification.

[0159] Functionalization of Fibro VS membrane with thiolated oligo(dT) ligands

[0160] exist Desalt the thiolated oligo(dT) solution into 150 mM NaCl using a 50 mL desalting column on a pure column. Use 25 mM DTT, 0.1 M NaHCO 3 , 0.01M Na 2 CO 3 The resulting solution was reduced for 1 hour and then further desalted as previously described. The resulting solution was concentrated using a 20mL VivaSpin column MWCO 5kDa. The solution was then diluted to 5.9mg / mL and added to the Fibro VS sheet in a sealable container, followed by sodium sulfate (~3g). The container was sealed and placed on an orbital shaker for 16 hours. After this, the supernatant was discarded and DI water (50mL) was added to each tray. Before implementing any further steps, it was repeated 5x in total.

[0161] Functionalization of Fibro VS membrane with aminated oligo(dT) ligand

[0162] The aminated oligo (dT) sample was dissolved in 150mM NaCl buffer (50mL). By adding DI water (43mL) to the oligo (dT) solution (7mL), the solution was diluted to a concentration of 6.2mg / mL and a volume of 50mL. Sodium sulfate (7.1g) was added and the pH was measured. The solution was added to a Fibro VS T1 sheet in a sealable container and placed on an orbital shaker for 16 hours. After this, the supernatant was discarded and DI water (50mL) was added to the tray and then placed back on the orbital shaker. Before implementing any further steps, the process was repeated 4x.

[0163] Blocking of reactive groups of divinyl sulfone

[0164] To block any residual vinyl sulfone groups on Fibro VS functionalized with oligo(dT), a phosphate 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.

[0165] The sheet of functionalized material was placed in a sealable container and submerged in 25 mL of buffered thioglycerol solution before being placed on an orbital shaker for at least 16 h. After this, the thioglycerol solution was discarded and DI water (50 mL) was added to the sheet before being placed back on the orbital shaker for at least 15 minutes. This washing process was repeated an additional 3 times. The final wash was replaced with glycerol: ethanol: water (50 mL, 20:20:60 v / v%) and soaked for 1 hour before removing the wet overmolding into the desired device.

[0166] Example 2: Aperture Measurement Method

[0167] Mean flow pore size can be measured using capillary flow analysis using commercially available equipment. In one example, the equipment used was a POROLUX™ 100 porometer (IB-FT GmbH, Berlin, Germany) according to the manufacturer's manual, and the method is shown in Table 1.

[0168] Table 1: Capillary flow porometry

[0169]

[0170] Example 3: Binding capacity analysis of Fibro Oligo dT20 prototype A

[0171] Materials and methods

[0172] mRNA-uncapped FlucV01 (1975 nt, including 100 nt polyA tail) was generated by in vitro transcription and pre-purified by oligo-dT purification to achieve 94% or higher purity. mRNA was eluted with Tris-EDTA buffer or RNAse-free water and stored at a concentration of more than 300 μg / mL at -20 degrees Celsius until used in chromatography experiments (quantification of DBC).

[0173] Chromatography system - equipped with 2mm UV cell Pure 25.

[0174] Chromatographic columns - Fibro Oligo dT20 (Prototype A) was packed in a HiTrap device (Cytiva, Sweden) with 2 membrane layers, with a final membrane volume of 0.4 ml.

[0175] Stock solution used:

[0176] KCl 3M prepared in RNase-free water

[0177] EDTA 500mM, pH 8.0 (RNase-free)

[0178] Tris 1M, pH 7.5 (RNase-free)

[0179] Binding buffer (port A1): KCl 200mM, Tris 10mM, EDTA 1mM pH 7.5

[0180] Elution buffer (inlet B1): RNase-free water

[0181] Cleaning-in-place buffer (Inlet A2): NaOH 0.1M

[0182] Example method for determining DBC 10% value: FlucV01 mRNA was diluted to 0.2 mg / mL by diluting stock mRNA with RNase-free water and adjusting KCl, Tris and EDTA concentrations using stock solutions so that the mRNA sample contained KCl 200 mM, Tris 10 mM, EDTA 1 mM pH 7.5. Before determining the DBC value, the sample was first injected through a bypass using a super loop to measure Amax (or 100% penetration) by monitoring UV 260 nm. To measure the dynamic binding capacity (DBC), the mRNA sample was then injected into a HiTrap device containing 0.4 ml Fibro Oligo dT20 (prototype A) while monitoring UV at 260 nm. Unless otherwise specified, a flow rate of 2 ml / min was used in all stages, except for the sample application stage, where the flow rate was set to a variable to achieve different residence times (e.g., the flow rate was set to 0.8 ml / min to achieve a residence time of 30 s).

[0183] The following phases were used to bind and elute mRNA and quantify DBC 10%.

[0184] - Equilibration: 8 ml binding buffer from inlet A1

[0185] - Sample application: interrupt sample application at 50% of Amax or until the superloop is exhausted

[0186] - Unbound wash: 2 ml binding buffer from inlet A1

[0187] - Elution: 8 ml RNase-free water from inlet B1

[0188] Wash the column with the following phases and prepare it for the next experiment

[0189] - Column wash: 8 ml RNase-free water from inlet B1

[0190] -CIP: 8 ml NaOH 0.1M from inlet A2

[0191] - Equilibration: 8 ml binding buffer from inlet A1

[0192] DBC is calculated by the following equation or by using the DBC calculation extension in Unicorn Evaluation Classic:

[0193] DBC = mass of mRNA bound (mg) / column volume (ml)

[0194] =C×(VBT-Vdelay) / column volume (ml)

[0195] Where C = concentration of mRNA in the feed expressed in mg / ml

[0196] VBT = volume corresponding to the desired penetration (e.g. 10% in this experiment)

[0197] Vdelay = system and column void volume = conductivity equal to the volume at the midpoint between running buffer and sample input (after injection).

[0198] Example Results of Dynamic Binding Capacity Method

[0199] Figure 2 The method shows that mRNA molecules of different lengths can be successfully purified using functionalized chromatography materials. Prototype A is oligo(dT) with an aminated C6 linker 20 The ligand is fixed on the Fibro VS membrane. The length of mRNA is between about 400nt and 4100nt.

[0200] Figure 3 The effect of oligo(dT) ligand length on dynamic binding capacity and ligand density is shown. 30Oligonucleotides (mRNA surrogates) were diluted in binding buffer until 10% breakthrough (residence time 24 seconds) to determine dynamic binding capacity. Binding buffer consisted of 10 mM Tris, 400 mM NaCl, 1 mM EDTA, pH 7.4. Ligand density was determined by phosphorus ICP-SFMS.

[0201] Figure 4 The effect of the ligand linker on the dynamic binding capacity is shown. Figure 3 The assay was performed as shown.

[0202] Figure 5 shows a consistent pressure distribution over 10 consecutive cycles without CIP. Figure 3 As shown, using poly(dA) 30 Oligonucleotides were used as mRNA surrogates for these runs.

[0203] The excellent flow properties of the prototype are preferably used in a larger device. A 50 mL device should likely provide acceptable capacity and a reduced loading time of 20 min for 1 liter of feed compared to 2500 min for a 0.4 mL device.

[0204] Example 4: Dynamic Binding Capacity (DBC) Experiment

[0205] In two independent experiments, homemade mRNA was used to The dynamic binding capacity of Fibro Oligod T20 (prototype A) was tested on pure chromatography systems.

[0206] Method for assessing changes in binding capacity over multiple cycles

[0207] Oligo(dT) purified polyA-terminated mRNA (1975 nt) will be used to measure DBC over time, including CIP with 100 mM NaOH Fibro(oligodT).

[0208] In summary, mRNA will be diluted to between 200-250 μg / mL in Tris-EDTA (TE) buffer (pH 7.5) and supplemented with potassium chloride (final concentration 200 mM). Before the DBC run begins, a small amount of mRNA sample is injected in the bypass and the maximum absorbance at 260 nm measured with a 2 mm UV cell is recorded. The method of use is detailed in Table 2 below. CV represents column volume.

[0209] Table 2: Sample loading. For the remaining cycle numbers where no mRNA sample was injected, 10 CV of equilibration buffer was applied to the column at the same flow rate as indicated for sample loading (ie, 0.8 mL / min).

[0210]

[0211] for Figure 5 The data shown in Figure 8 were obtained using the following liquid flow rates and volumes:

[0212] - Equilibrate at 10mL / min for 20CV

[0213] - Sample application. 0.8 mL / min until 50% of Amax

[0214] - Wash. 20CV at 5mL / min

[0215] - Elution. 30CV at 2mL / min

[0216] -CIP. 20CV 100 mM NaOH at 10 mL / min

[0217] - Equilibrate at 10 mL / min for 20 CV

[0218] Figure 6 A chromatogram is depicted depicting the absorbance at 260 nm and the DBC values ​​for a series of cycles at different retention times. Sample application was interrupted at 50% of the Amax value measured at 260 nm (500 mAU). The ligand used was oligo(dT) with a C12 linker 20 And the composition contains 200mM KCl and 1975nt long mRNA product. The results of 30 seconds residence time RT are the average of the first, second and seventh cycles. 15s RT is cycle 3. 5s RT is cycle 4. 60s RT is cycle 5. 120s RT is cycle 6.

[0219] Figure 7 Depiction Figure 6 Dynamic binding capacity versus retention time for a series of cycles.

[0220] Fig. 8A -B depicts the measured chromatogram showing the absorbance at 260 nm and the dynamic binding capacity results for three cycles. Fig. 8A Displays the chromatogram separated with a 5% Y offset, and Figure 8B Overlay of chromatograms is shown. The chromatograms depict only the sample loading phases of the first cycle C1, the second cycle C2, and the seventh cycle C7 and the calculated dynamic binding capacity. These three chromatograms correspond to Figure 6 Average 30 sec RT chromatogram in .

[0221] Fig. 9A -C depicts experimental and estimated dynamic binding capacity changes after multiple cycles. Fig. 9AThe dynamic binding capacity at cycle 50 and cycle 200 at 10% breakthrough based on experimental values ​​is depicted. Fig. 9B Depicted from Fig. 9A The dynamic binding capacity at cycle 200 is calculated as a percentage of the corresponding dynamic binding capacity at cycle 50. In general, settings with values ​​above 80% of the initial dynamic binding capacity at 10% breakthrough are considered reusable.

[0222] For each cycle between 50 and 200, a clean-in-place solution was used. Cycles 50 and 200 were run to determine the dynamic binding capacity, and runs between 50 and 200 were run to recover nucleic acid products from the composition comprising the in vitro transcribed sample. For the dynamic binding capacity runs at cycles 50 and 200, a contact composition comprising 200 mM KCl was used.

[0223] Fig. 9C Description Based on the parameters given in Table 2, the estimated reduction in dynamic binding capacity of a chromatography material comprising polymer nanofibers (closed cartridge) compared to a conventional less reusable chromatography resin (open cartridge) for repeated cycles.

[0224] Example 5: Dynamic Binding Capacity of mRNAs of Various Sizes and Binding Conditions

[0225] Materials and methods

[0226] Poly(A) tail positive mRNA (including polyA) with a theoretical length of 1042, 1942, 1975, 3580 or 4525 nt 100 Tail) was used to measure DBC on the above prototype A at various KCl concentrations during binding. All mRNAs used here were pre-purified by oligo(dT) affinity chromatography to obtain high levels of polyA positive material, which was detected by A max The measurement allows reliable concentration estimation and minimizes biased signals. Briefly, pre-purified mRNA was diluted in Tris 10 mM pH 7.5, EDTA 1 mM and 200-500 mM KCl to a final concentration of 0.2-0.25 mg / ml. max The total absorbance at 260nm contributed by the input mRNA sample is defined, and the single combination of mRNA and binding buffer is measured before DBC runs. Use the binding buffer with three different KCl concentrations (200mM, 300mM and 500mM) to measure DBC. In all experiments, the residence time of 30s is used, which corresponds to the flow velocity of 0.8ml / min during sample application. During the binding period, the data points of KCl 200mM are implemented to nonlinear regression fitting and as shown in the dotted line in the figure.

[0227] result

[0228] Fig.10 The DBC of Prototype A for different mRNAs at 200 mM (circle solid dots), 300 mM (open squares) and 500 mM (solid triangles) KCl concentrations during binding are shown. At all used binding KCl concentrations, the DBC is negatively correlated with the size of the mRNA. In contrast, the DBC is directly correlated with the salt concentration in the tested range of 200-500 mM.

[0229] Example 6: Dynamic binding capacity over 200 cycles using 0.1 M NaOH cleaning in place.

[0230] This experiment was performed to demonstrate performance stability over repeated use, including cleaning in place (CIP) using 100 mM NaOH.

[0231] Materials and methods

[0232] DBC was measured at intervals of up to 200 cycles on the prototype A device using pre-purified mRNA (1975 nt). Briefly, the same batch of pre-purified mRNA (corresponding to Fig.10 mRNA 3) was diluted to 0.25 mg / ml. A residence time of 30 s was used in all runs, corresponding to a flow rate of 0.8 ml / min during sample application. mRNA was injected with a fixed volume only when the number of cycles was 1, 2, 3, 5, 10, 20, 40, 60, 80, 81, 100, 120, 140, 160, 180 and 200 to ensure complete penetration and accurate calculation of DBC. In non-DBC runs, an equal volume of binding buffer (without mRNA) was used as sample. All 200 cycles included the following stages: equilibration at 10 mL / min in binding buffer for 20 CV, sample application at 0.8 mL / min (for DBC runs) or 10 mL / min (non-DBC runs) for 35 CV, column wash at 10 mL / min for 10 CV in binding buffer, elution at 10 mL / min for 20 CV in Tris 10 Mm, EDTA 1 mM, and CIP at 10 ml / min for 15 CV in NaOH 100 mM. DBC (QB10) is derived from a single chromatographic run and is plotted as a round solid point versus cycle number.

[0233] result

[0234] Fig.11The dynamic binding capacity (DBC) of the prototype was shown to be maintained for at least 200 cycles in CIP with 100 mM NaOH. This indicates that this method allows cleaning and regeneration of the chromatography material for more than 200 repetitions of the cycle while maintaining a high binding capacity.

[0235] in conclusion

[0236] The high reusability of the method may allow the user to repeat over 200 cycles without significant loss of performance. The method may allow the use of one chromatographic device for multiple batches or even different molecules without significant impact on performance differences between cycles. The flexibility of the reusable method may allow for accelerated development time.

[0237] Measurement data associated with Fibro-oligo(dT) demonstrates improved capacity and reusability while maintaining purification performance. As the field of therapeutic mRNA rapidly advances, there is increasing interest in process intensification to improve productivity and cost-effectiveness. Upstream IVT mRNA titers are expected to increase and require increased downstream purification efficiency. Efficient and rapid kinetics prevent downstream purification using oligo(dT) capture steps from becoming rate limiting in the manufacturing process.

[0238] The predictions for scale-up using polymer nanofibers compared to a reference resin are shown in Table 3. The reference resin is typically a traditional agarose bead-type chromatography resin.

[0239] Table 3: Estimated performance comparison of 2000 base mRNA purification from IVT feed mRNA.

[0240]

[0241]

[0242] References US20140296464A1 US20160288089A1 US2019308169A1 US2019234914A1.

Claims

1. A method for recovering a nucleic acid product from a composition comprising the product, the method (100) comprising the following steps: (i) contacting the composition with a chromatography material under conditions that allow nucleic acid base pairing between at least a portion of the nucleic acid product and a ligand (110), wherein the chromatography material comprises one or more nanofibers, and wherein the chromatography material is functionalized with the ligand; (ii) optionally washing (120) the functionalized chromatographic material with a washing liquid phase; (iii) selectively eluting (130) the product by contacting the functionalized chromatographic material with an eluent; (iv) in-situ cleaning (140) comprising regenerating the chromatography material by contact with a cleaning solution; (v) repeating steps (i)-(iii) for at least 15 cycles, wherein step (iv) is performed in at least one of said cycles; and (vi) collecting (150) the recovered nucleic acid product, The chromatography material is capable of maintaining a dynamic binding capacity for the nucleic acid product after 50 cycles with 10% breakthrough which is at least 80% of the corresponding dynamic binding capacity of the first cycle.

2. The method of claim 1, wherein steps (i)-(iii) are repeated for at least 25 cycles, or at least 50 cycles.

3. The method according to claim 1 or 2, wherein contacting the composition with the chromatographic material (110) is performed under conditions where the salt concentration is less than 500 mM.

4. The method according to any one of the preceding claims, wherein the nucleic acid product is single-stranded RNA, double-stranded RNA, mRNA, pre-mRNA, single-stranded DNA and / or double-stranded DNA.

5. A method according to any one of the preceding claims, wherein the chromatography material comprises a nonwoven material comprising polymer nanofibers.

6. A method according to any one of the preceding claims, wherein the composition has a salt concentration of at most 250 mM.

7. A method according to any one of the preceding claims, wherein the eluent phase has a salt concentration of at most 50 mM.

8. The method according to any one of the preceding claims, wherein the cleaning liquid phase is above pH 9.

9. A method according to any one of the preceding claims, wherein the chromatography material has a ligand density of the ligand in the range of 3-20 μmole of the ligand per gram of chromatography material.

10. The method of any one of the preceding claims, wherein the ligand is arranged to base pair with the nucleic acid product having at least 10 bases.

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

12. A chromatographic material, wherein the material is convection-based and functionalized with ligands comprising oligonucleotides, wherein the material comprises polymer nanofibers and is in the form of one or more nonwoven membranes and / or sheets, and wherein the ligands are arranged to base pair with at least a portion of a nucleic acid product.

13. The chromatographic material of claim 12, wherein the ligand density is 3-20 μmole / g.

14. The chromatographic material according to claim 12 or 13, wherein the ligand is (dT) 10-50 Ligand, preferably (dT) 12-30 The ligand is arranged to form base pairing with the polyA-labeled product.

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

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