Systems and methods for electrostatic-based purification of nucleic acids

Abstract

A separation substrate is modified with primary amine ligands, e.g., 2-aminoethyl methacrylate hydrochloride (AEMA), for continuous electrostatic purification of mRNA vaccines from in vitro transcription (IVT) bioreactor effluent. Modified membrane surface charges are responsive to changes in pH, as are charges associated with the mRNA and impurities in the sample. By reducing the pH of mobile phases in contact with the membrane below the isoelectric point (pl) of the peptide impurities in the sample, the peptides become net positive while the mRNA products remain net negative, allowing the mRNA product to bind while the peptides are eluted away. Salt solutions to increase the ionic strength and cationic species to increase the N / P ratio of the mobile phase / membrane further purify the mRNA from other nucleic acid species, e.g., pDNA, dsRNA. Raising mobile phase pH above the pl of the membrane, e.g., about 8.5, can then elute purified mRNA product.

Description

SYSTEMS AND METHODS FOR ELECTROSTATIC-BASED PURIFICATION OF NUCLEIC ACIDSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Nos. 63 / 568,141, filed March 21, 2024; 63 / 635,187, filed April 17, 2024; 63 / 685,534, filed August 21, 2024; and 63 / 712,101, filed October 25, 2024, which are incorporated by reference as if disclosed herein in their entireties.BACKGROUND

[0002] Nucleic acids such as RNA molecules are the new frontier for vaccines and treating genetic and rare diseases. Numerous therapies based on RNA have been developed and include: cell-free messenger RNA (mRNA) for vaccines and other diseases, RNA-involved genome editing technologies, microRNA (miRNA) for regulating gene expression in biological systems primarily by silencing the target mRNA, small interfering RNA (siRNA) that mediates gene activity for treating a rare condition that can impair heart and nerve function, long noncoding RNA (IncRNA) are associated with a variety of genetic disorders ranging from monogenic to complex disorders, and RNA from liquid blood samples for sequencing and disease detection. Nucleic acids such as DNA molecules are intimately involved with many diseases such as Huntingtin and sickle cell anemia.

[0003] In each of these examples, there is a need to synthesize (or isolate from biological fluids like blood plasma, urine, or eye fluids) abundant pure cell free RNA (cfRNA or DNA), and hence purify nucleic acids (RNA) from protein-containing solutions (like in vitro transcription (IVT) bioreactor effluent (broth) during production of mRNA vaccines, blood plasma or other biological-related fluids).

[0004] Celebrated for their global life-saving impact, single strand messenger RNA (mRNA) vaccines, including those developed by Pfizer / BioNtech and Modema, have become integrated into modem medicine. These vaccines use in vitro-transcribed mRNA to illicit an immune response through the production of antigenic molecules in vivo providing a safe and efficacious vaccination platform. Following the success in combating the COVID-19 pandemic, the scope of the mRNA biologic toolbox is rapidly expanding, with growing applications in both prophylactic and therapeutic areas. While identifying the biggest challenge in realizing the full potential of the mRNA technology is subjective, it is widely acknowledgedthat reducing process- and product-related impurities can reduce the immunogenicity of mRNA biologies. Long double stranded RNA (ds-RNA), which can vary significantly in length and structure, presents a challenge.

[0005] Current methods to purify mRNA from the IVT broth suffer from a variety of drawbacks. First, the original DNA template is digested by DNase treatment and the 5 triphosphate group of mRNAs removed to reduce immune response using heat-labile antarctic phosphatase. Then, unreacted nucleotides, short oligonucleotide, enzyme proteins, residual salts and immune-inducing unreacted double stranded RNAs are removed. Since purifying nucleic acids from complex solutions for large-scale purification is less developed than protein purification, protein separation methods from the laboratory like liquid chromatography (LC) are currently favored. Also, gel filtration chromatography and electrostatic separations with anion exchange chromatography have been used. Recently, affinity bead chromatography with oligo- dT strands has been used. Except for possibly gel filtration chromatography, the other chromatography methods rely heavily on diffusion, a very slow process that involves long residence times that challenge the integrity of the mRNA. ft has been proposed to purify eukaryotic mRNA by binding poly-A tails on the 3' end to oligo-dT affixed to a surface. Isostabilizing agents, such as tetramethylammonium (TMA+) and tetraethyl ammonium (TEA+) ions and the amino acid betaine, were used to equalize the hydrogen bonding strength of the G-C and A-T base pairs. Elution has been done with either a low ionic strength buffer or a competitive binding oligonucleotide solution. RNA recoveries of >80% have been claimed with efficient separation from endotoxins, DNA template, and enzymes utilized in production.

[0006] However, these approaches face challenges as the dominant diffusional transport mechanism becomes limiting for large biomolecules such as mRNA with diffusivities orders of magnitude lower than monoclonal antibodies. This limitation can result in low ligand utilization, prolonged process time, and decreased device productivity due to slow binding kinetics and high operational pressure drop caused by the smaller particles to reduce diffusional distances.

[0007] To introduce a degree of convection to ease these diffusional challenges, alternative chromatographic devices predominantly relying on convective transport are proving effective for large mRNA molecules. Convective transport, through pressure driven flow in membranes and monoliths, is present in the reduction of mass transfer limitation as the large biomolecules are brought into close contact with adsorption sites via short diffusional distances for interaction. Polymer membranes can be manufactured in various configurations, e.g. flat sheet stacks, fibers and spiral wound, with short bed lengths to provide modular packing density and allow for lowpressure drop operation. These qualities enhance scalability, reduce process footprint, increase process productivity and product integrity, and support continuous operation.

[0008] To improve mRNA product quality by reducing dsRNA-related impurities, separation techniques such as reverse phase-HPLC (RP-HPLC) and cellulosic chromatography have been employed. Additionally, multimodal ligands, which leverage multiple forms of matrix-solute interactions such as electrostatic, hydrogen bonding, and hydrophobic interactions are being investigated. Primary' amine ligands are of particular interest, as they can enhance separation by combining anion exchange with secondary interactions such as hydrogen bonding and hydrophobic interactions, especially under elevated ionic conditions. Furthermore, unlike quaternary amine anionic exchangers, these weaker interactions are pH dependent allowing for binding and controlled elution. This approach has shown promise in separating double and single stranded nucleic acids due to differences in negative electrostatic potential and hydrogen bonding. From an economic standpoint, electrostatic based purification is attractive as it provides a lower cost modification scheme using less expensive polymeric ligands and faster binding kinetics which increase ligand / surface area utilization, and thus dynamic binding capacity', in comparison to affinity ligand approach. While effective in removing double stranded plasmid DNA (pDNA) from mRNA, current efforts struggle to eliminate larger dsRNA molecules due to their increased electrostatic interactions with the positively charged primary amine surface.

[0009] What is desired, therefore, are systems and methods for the selective, fast, reliable, and efficient purification of large and structurally labile molecules, capable of use with larger charged species like charged proteins (and peptides) and charged nucleic acids.SUMMARY

[0010] Aspects of the present disclosure are directed to system and methods that enable purification of labile mRNA quickly, at high yield and purity7, and in a continuous production mode. These embodiments can replace current slow chromatographic methods with a fast, low- fouling membrane microfiltration approach to allow high speed continuous production of mRNA, reduce degradation of mRNA resulting from long residence times in chromatographic bead columns, and reduce the equipment footprint.

[0011] In some embodiments, the separation of single-stranded mRNA products from double-stranded nucleic acid impurities, e.g., dsRNA, is accomplished by adding the polyamine spermine to reduce the negative zeta potential (charge) of the dsRNA, thereby reducing theelectrostatic interactions between the dsRNA and a positively-charged, primary amine-modified microporous regenerated cellulose membranes. These systems and methods leverage electrostatic interactions and hydrogen bonding with spermine as a charge-tuning additive to enhance the separation

[0012] Aspects of the present disclosure are directed to a method of purifying a nucleic acid sample. In some embodiments, the method includes providing a membrane including a separation substrate and a plurality of charged ligands positioned on the separation substrate. In some embodiments, the method includes contacting a medium with the membrane at or below a pH of about 4.0. In some embodiments, the medium includes an mRNA product and one or more impurities. In some embodiments, the method includes preferentially binding one of the impurities or the mRNA product via the charged ligands to form a concentration of bound product. In some embodiments, the method includes eluting a concentration of the bound product from the membrane at a pH above about 4.0.

[0013] In some embodiments, preferentially binding one of the impurities or the mRNA product via the charged ligands to form a concentration of bound product includes binding the mRNA product via primary amine ligands at or below a pH of about 4.0 and eluting a first effluent including a concentration of protein impurities. In some embodiments, the method includes treating the medium, the membrane, or combinations thereof with a salt solution to adjust a corresponding ionic strength thereof prior to eluting a concentration of the bound product. In some embodiments, the method includes treating the medium, the membrane, or combinations thereof with a solution including one or more cationic species prior to eluting a concentration of the bound product. In some embodiments, contacting a medium with the membrane occurs in a mobile phase having a flowrate between about 0.1 to 20 mL / min.

[0014] In some embodiments, the charged ligands have graft density on the separation substrate of 4,000 nmol / m2to 10,000 nmol / m2In some embodiments, the charged ligands include primary amine ligands, including amino acrylate; 2-aminoethyl methacrylate hydrochloride; 3-aminopropyl 2-methylprop-2-enoate; glycyl methacrylate: 2-propenoic acid; ethylene diamine methacrylate; 1 -aminoethyl 2-methylprop-2-enoate; 2-aminopropyl 2- methylprop-2-enoate; 2,3-diaminopropyl 2-methylprop-2-enoate; 2-aminobutyl 2-methylprop-2- enoate; 1 -aminopropyl 2-methylprop-2-enoate; 2-(2-aminoethoxy)ethyl 2-methylprop-2-enoate; l,3-diaminopropan-2-yl 2-methylprop-2-enoate; 3-(2-aminoethoxy)propyl 2-methylprop-2- enoate; 3-[3-(2-aminoethoxy)propoxy]propyl 2-methylprop-2-enoate; 2-[2-[2-(2- aminoethoxy)ethoxy]ethoxy]ethyl 2-methylprop-2-enoate, or combinations thereof. In someembodiments, the cationic species includes N.N'-Bis(3-ammopropyl)-1.4-diaminobutane (spermine), N,N'-bis(3-aminopropyl)butane-l,4-diamine; N'-(3-aminopropyl)butane-l,4- diamine; N'-[3-(3-aminopropylamino)propyl]propane-l,3-diamine; N-ethyl-N'-[3-[3- (ethylamino)propylamino]propyl]propane-1.3-diamine; 1,4, 8,11-tetrazacyclotetradecane; N'-(4- aminobutyl)butane-l,4-diamine; N'-(3-aminopropyl)pentane-l,5-diamine; N,N'-bis[3- (ethylamino)propyl]butane-l,4-diamine; N'-[3-[3-(3 aminopropylamino)propylamino]propyl]propane-l,3-diamine; 1,5,9-triazacyclododecane; N'-[3- (4-aminobutylamino)propyl] butane- 1,4-diamine; N'-(3-aminopropyl)-N-ethylbutane-l,4- diamine; N'-[3-(3-aminopropylamino)propyl]butane-l,4-diamine; N-ethyl-N'-[3- (ethylamino)propyljbutane- 1,4-diamine; N'-[4-[3-(4- aminobutylamino)propylamino]butyl]butane- 1,4-diamine; N'-[3-[4-(3- aminopropylamino)butylamino]propyl]butane-l,4-diamine; 4-aminobutyl-tris(3- aminopropyl)azanium; N',N'-bis(3-aminopropyl)butane- 1,4-diamine; l-N-[4-(3- aminobutylamino)butyl]butane-l,3-diamine; N'-[3-[3-[3-(3- aminopropylamino)propylamino]propylamino]propyl]propane-l,3-diamine; bisbenzimide, or combinations thereof.

[0015] In some embodiments, spermine is provided at a concentration between ImM and 20mM. In some embodiments, the separation substrate has a mean pore diameter between about 0. 1 - 10 pm. In some embodiments, the concentration of bound mRNA product is about 1-8 mg / m2In some embodiments, the concentration of bound mRNA product is about 1.3 mg / m2

[0016] Aspects of the present disclosure are directed to a method of purifying a nucleic acid sample. In some embodiments, the method includes providing a membrane including a separation substrate and a plurality of primary amine ligands positioned on the separation substrate. In some embodiments, the method includes providing a medium including an mRNA product, a concentration of impurities, and a concentration of a cationic species; contacting the medium w ith the membrane at or below a first pH of about 4.0; binding the mRNA product with the membrane to form a concentration of bound product; eluting a first impurities effluent at the first pH; contacting the bound product a salt solution to elute a second impurities effluent; contacting the bound product with a mobile phase having a second pH above a pl of the membrane; and eluting a product effluent at the second pH, the product effluent including a concentration of the bound product. In some embodiments, the impurities include peptides, pDNA, dsRNA, NTPs, MgCh. proteins, other cofactors, or combinations thereof. In some embodiments, the pl of the membrane is between about 8 and about 9.

[0017] Aspects of the present disclosure are directed to a method of purifying a nucleic acid sample, comprising providing a membrane including a separation substrate and a plurality of primary' amine ligands positioned on the separation substrate; providing a medium including an mRNA product, a concentration of impurities, and a concentration of a cationic species to form an N / P ratio greater than about 450 or equivalent; contacting the medium with the membrane in a mobile phase having a pH below about 4.0; capturing mRNA product from the mobile phase; increasing the ionic strength of the mobile phase to elute a first effluent including nucleic acid impurities; and increasing the pH of the mobile phase above about 8.5 to elute a product effluent including mRNA product.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The drawings show embodiments of the disclosed subject matter for the purpose of illustrating the invention. However, it should be understood that the present application is not limited to the precise arrangements and instrumentalities shown in the drawings, wherein:

[0019] FIG. 1 A is chart of a method of purifying a nucleic acid sample according to some embodiments of the present disclosure;

[0020] FIG. IB is chart of a method of purifying a nucleic acid sample according to some embodiments of the present disclosure;

[0021] FIG. 2 is chart of a method of purifying a nucleic acid sample according to some embodiments of the present disclosure;

[0022] FIG. 3 is chart of a method of purifying a nucleic acid sample according to some embodiments of the present disclosure;

[0023] FIGs. 4A-4B are graphs portraying surface characterization data for modified regenerated cellulose membranes according to some embodiments of the present disclosure;

[0024] FIGs. 5A-5D are graphs portraying high-performance liquid chromatography (HPLC) dynamic characterization of pure ss-mRNA uptake and elution by 2- aminoethyl methacrylate hydrochloride (AEMA)-modified membranes according to some embodiments according to some embodiments of the present disclosure;

[0025] FIGs. 6A-6D are graphs portraying dynamic characterization of varying grafting densities for AEMA-modified membranes according to some embodiments according to some embodiments of the present disclosure;

[0026] FIGs. 7A-7C are graphs portraying dynamic characterization of pure feed separation performance of dsRNA and ss-mRNA by AEMA-modified membranes according to some embodiments according to some embodiments of the present disclosure; and

[0027] FIGs. 8A-8B are graphs portraying dynamic characterization of separation performance of dsRNA and ss-mRNA by AEMA-modified membranes according to some embodiments according to some embodiments of the present disclosure with spermine.DETAILED DESCRIPTION

[0028] Referring now to FIG. 1 A, some aspects of the disclosed subject matter are directed to a method 100, e.g., 100A, of purifying a nucleic acid sample. In some embodiments, the nucleic acid sample is the product of an in vitro transcription (IVT) process, blood plasma sample, or combinations thereof. In some embodiments, the IVT components include peptides, pDNA, dsRNA, NTPs, MgCh, proteins, other cofactors, or combinations thereof. In some embodiments, the nucleic acid sample includes a target mRNA product and a concentration of impurities. In some embodiments, the mRNA product is an mRNA vaccine. In some embodiments, the concentration of impurities includes nucleic acid impurities. In some embodiments, the impurities include DNA, dsRNA, or combinations thereof. In some embodiments, the impurities include pDNA.

[0029] In some embodiments, at 102. a membrane is provided. In some embodiments, the membrane includes a separation substrate. In some embodiments, the separation substrate is composed of any suitable material that is suitable for use in separating mRNA products from impurities in a fluid sample, e.g., IVT effluent, blood plasma, etc., and further is able to be functionalized to modify a surface charge of the membrane, as will be discussed in greater detail below. In some embodiments, the separation substrate includes cellulose filter papers, cellulosic derivatives, e.g., regenerated cellulose; cellulose acetate; cellulose nitrate; aromatic and aliphatic polyamide; polyethersulfone; modified polyether sulfone; polysulfone; poly vinylidene fluoride; modified poly vinylidene fluoride; silica; polyimide; polyacyrlonitride; polyethylenimine; membranes covered with hydrophilic coatings, e.g.. cellulose, acrylate, etc.; polyvinylpyrrolidone additives, e g., to make hydrophobic polymers more polar; or combinations thereof. In some embodiments, the membrane can have any suitable geometry, e.g., flat sheet, hollow fiber, tubular, spiral wound flat and tubular, annular (Taylor vortex), etc. In some embodiments, the separation substrate has a mean pore diameter between about 0. 1-10 pm.

[0030] In some embodiments, the membrane includes a plurality of charged ligands positioned on the separation substrate. In some embodiments, the charged ligands are attached to the separation substrate to provide a desired charge modification, i.e., positive or negative, thereto. In some embodiments, the charged ligands are attached via any suitable technique, e.g., single electron transfer-living radical polymerization (SET-LRP), ultraviolet graft polymerization, etc.

[0031] In some embodiments, charged ligands are provided in a layer on a surface of the separation substrate. In some embodiments, charged ligands are provided within pores of the separation substrate, i.e., on an interior surface thereof. In some embodiments, the charged ligands have a graft density on the separation substrate of 1,000 nmol / m2to 20,000 nmol / m2In some embodiments, the charged ligands have a graft density on the separation substrate of 4,000 nmol / m2to 20,000 nmol / m2. In some embodiments, the charged ligands have a graft density on the separation substrate of 1.000 nmol / m2to 10.000 nmol / m2. In some embodiments, the charged ligands have a graft density on the separation substrate of 4,000 nmol / m2to 10,000 nmol / m2. In some embodiments, the charged ligands have a graft density on the separation substrate of about 4,000 nmol / m2. In some embodiments, the charged ligands have a graft density on the separation substrate of about 5,000 nmol / m2. In some embodiments, the charged ligands have a graft density on the separation substrate of about 6,000 nmol / m2. In some embodiments, the charged ligands have a graft density on the separation substrate of about 7,000 nmol / m2. In some embodiments, the charged ligands have a graft density on the separation substrate of about 8,000 nmol / m2. In some embodiments, the charged ligands have a graft density on the separation substrate of about 9,000 nmol / m2. In some embodiments, the charged ligands have a graft density on the separation substrate of about 10,000 nmol / m2.

[0032] In some embodiments, the charged ligands include a first set of charged ligands and at least a second set of charged ligands, where the first set of charged ligands and the second set of charged ligands are different. In some embodiments, each charged ligand provided to the separation substrate is the same or a functional equivalent, providing a substantially consistent charge profile to the membrane overall. In some embodiments, the charged ligands include primary amine ligands. In some embodiments, the charged ligands include amino acrylate; 2- aminoethyl methacrylate hydrochloride; 3-aminopropyl 2-methylprop-2-enoate; glycyl methacrylate; 2-propenoic acid; ethylene diamine methacrylate; 1 -aminoethyl 2-methylprop-2- enoate; 2-aminopropyl 2-methylprop-2-enoate; 2,3-diaminopropyl 2-methylprop-2-enoate; 2- aminobutyl 2-methylprop-2-enoate; 1 -aminopropyl 2-methylprop-2-enoate; 2-(2- aminoethoxy)ethyl 2-methylprop-2-enoate; l,3-diaminopropan-2-yl 2-methylprop-2-enoate; 3-(2-aminoethoxy)propyl 2-methylprop-2-enoate; 3-[3-(2-aminoethoxy)propoxy]propyl 2- methylprop-2-enoate; 2- [2- [2-(2-aminoethoxy)ethoxy] ethoxy] ethyl 2-methylprop-2-enoate, or combinations thereof.

[0033] Referring again to FIG. 1, in some embodiments, at 104, a medium including an mRNA product and one or more impurities is contacted with the membrane. As discussed above, in some embodiments, the medium includes the product of an IVT process, blood plasma sample, or combinations thereof. In some embodiments, the medium undergoes one or more pretreatment processes before contacting 104 the membrane. In some embodiments, contacting 104 occurs in a mobile phase having a flowrate between about 0. 1 to 20 mL / min. In some embodiments, contacting 104 occurs in a mobile phase having a flowrate between about 0.25 ml / min and about 1.5 mL / min. In some embodiments, the concentration of bound mRNA product is about 1-8 mg / m2. In some embodiments, the concentration of bound mRNA product is between about 1 mg / m2and about 2 mg / m2. In some embodiments, the concentration of bound mRNA product is about 1 .3 mg / m2.

[0034] In some embodiments, at 106, one of the impurities or the mRNA product preferentially bind to the charged ligands to form a concentration of bound product on the membrane. In some embodiments, the pH of the medium including proteins (and peptides) and mRNA product is reduced to pH values below the isoelectric point (pl) of at least some of the proteins in the medium, which causes the proteins to become net positive, while nucleic acids, e g., RNA and DNA, remain net negative. In some embodiments, the pH of the medium is reduced to pH values below the pl of all of the proteins in the medium, which causes all the proteins to become net positive, while nucleic acids remain net negative. Thus, in embodiments with a membrane having a positive charge, contacting 104 the medium with the membrane induces binding 106 between the negatively charged mRNA product, while positively charged proteins can be transported across the membrane. Additionally, in embodiments with a membrane having a negative charge, contacting 104 the medium with the membrane induces binding 106 between the positively charged proteins, while the negatively charged mRNA product can be transported across the membrane.

[0035] In some embodiments, at a pH of about 8.0 or below, negatively charged mRNA product can bind to positively charged ligands on the membrane. In some embodiments, at a pH of about 4.0 or below, binding of positively charged proteins to positively charged ligands is reduced.

[0036] In some embodiments, at 108. the pH of the medium is then raised above the pl of the bound product or the charged ligands, resulting in the elution of the bound product. In some embodiments, at a pH of about 4.0 or below, the medium is below the pl of protein impurities in the medium, resulting in those proteins adopting a net positive charge such that the proteins can diffuse across the positively charged membrane while the negatively changed mRNA product is bound 106 to the charged ligands. In some embodiments, elution 108 of mRNA product occurs above the pl of the charged ligands, i.e., where the net positive charge on the membrane becomes net negative. In some embodiments, elution 108 of mRNA product occurs above about pH 8.0. In embodiments including negatively charged ligands, at a pH of about 4.0 or below, protein impurities in the medium adopting a net positive charge bind 106 to the charged ligands, and the mRNA product can diffuse across the membrane for collection. In some embodiments, the pH can then be raised above about 4.0, resulting in elution 108 of an effluent including a concentration of protein impurities.

[0037] Referring now to FIG. 1 B, in some embodiments of method 100, e.g., 100B, prior to eluting 108 a concentration of the bound product, the medium, the membrane, or combinations thereof is treated 107A with a salt solution to adjust a corresponding ionic strength thereof. In some embodiments, increasing selectivity between proteins (and peptides) and nucleic acids is achieved by varying the ionic strength of the solution during separation and elution of the adsorbed ionic species. Changes in pH affect charge, while changes in ionic strength of the solution affect the “effective volume,” i.e., apparent volume or size, of charged species and charged surfaces, and is a method to disentangle protein-nucleic complexes from each other, as well as separate single stranded nucleic acid products from double stranded nucleic acid impurities. In some embodiments, the ionic strength is adjusted so that nucleic acid components of the medium have a double layer thickness that is tuned to the effective pore size of the charged ligand functionalized membrane.

[0038] Residual pDNA template for mRNA synthesis can be present in mRNA products, e.g., mRNA vaccines produced via IVT reactors. However, due to its immunogenicity, pDNA should be removed from those products. Adjusting the ionic strength of the medium / membrane is effective to enable elution of pDNA from the membrane, while the mRNA product remains bound to the membrane. In some embodiments, the salt solution has a concentration between about 0M and about 10M. In some embodiments, the salt solution includes a buffer, e g., 2-amino-2-hydroxymethyl-propane-l,3-diol, glycine, potassium phosphate, sodium phosphate, citric acid, etc. In some embodiments, the salt solution includes a chelating agent, e.g., ethylenediaminetetraacetic acid, triethanolamine, aminopolycarboxylicacid, etc. In some embodiments, the salt solution includes a monovalent salt, e.g., NaCL K.C1, LiCl, etc.

[0039] Still referring to FIG. IB, in some embodiments of method 100, e.g., 100B, prior to eluting 108 a concentration of the bound product, the medium, the membrane, or combinations thereof is treated 107B with a one or more cationic species. Tn some embodiments, treating 107B occurs prior to contacting 104 as well. While the immune system can tolerate low levels of dsRNA in single-dose vaccinations, dsRNA molecules longer than forty base pairs can elicit an immune response, necessitating their removal to ensure the safety and efficacy of mRNA therapeutics. Treating 107B the medium / membrane is effective to selectively reduce the negative charge of double stranded nucleic acid impurities such as ds-RNA and ds-DNA, although not completely, so that these impurities still bind to the membrane below the pl thereof. However, subsequent treatment 107A that increases the ionic strength at the surface of membrane can then flush the weakly bound double-stranded impurities, e.g., ds-RNA, from the membrane, while ss-RNA such as the mRNA product remains bound under those conditions via hydrogen bonding. In some embodiments, the cationic species has a concentration between about 0M and about 10M. In some embodiments, the cationic species includes N,N'-Bis(3- aminopropyl)-l,4-diaminobutane (spermine), N.N'-bis(3-aminopropyl)butane-l,4-diamine; N'- (3-aminopropyl)butane-I,4-diamine; N'-[3-(3-aminopropylamino)propyl]propane-l,3-diamine;N-ethyl-N'-[3-[3-(ethylamino)propylamino]propyl]propane-l,3-diamine; 1,4,8,11- tetrazacyclotetradecane; N'-(4-aminobutyl)butane- 1 ,4-diamine; N'-(3-aminopropyl)pentane- 1,5- diamine; N,N'-bis [3 -(ethylamino)propyl] butane- 1.4-diamine; N'-[3-[3-(3 aminopropylamino)propylamino]propyl]propane-l,3-diamine; 1,5,9-tnazacyclododecane; N'-[3- (4-aminobutylamino)propyl]butane-l,4-diamine; N'-(3-aminopropyl)-N-ethylbutane-l,4- diamine; N'-[3-(3-aminopropylamino)propyl]butane-l,4-diamine; N-ethyl-N'-[3- (ethylamino)propyl]butane-l,4-diamine; N'-[4-[3-(4- aminobutylamino)propylamino]butyl]butane-l,4-diamine: N'-[3-[4-(3- aminopropylamino)butylamino]propyl]butane-l,4-diamine; 4-aminobutyl-tris(3- aminopropyl)azanium; N',N'-bis(3-aminopropyl)butane-l,4-diamine; l-N-[4-(3- aminobutylamino)butyl]butane-l,3-diamine; N'-[3-[3-[3-(3- aminopropylamino)propylamino]propylamino]propyl]propane-l,3-diamine; bisbenzimide, or combinations thereof. In some embodiments, cationic species, e.g., spermine, is provided at a concentration between ImM and 20mM. In some embodiments, cationic species, e.g., spermine, is provided at a concentration between ImM and lOmM. In some embodiments, cationic species, e.g., spermine, is provided at a concentration between ImM and 4mM.

[0040] Referring to FIG. 2, some embodiments of the present disclosure are directed to a method 200 of purifying a nucleic acid sample. In some embodiments, at 202, a membrane is provided. As discussed above, in some embodiments, the membrane includes a separation substrate and a plurality of primary amine ligands positioned on the separation substrate. In some embodiments, the primary amine ligands include amino acrylate; 2-aminoethyl methacrylate hydrochloride; 3-aminopropyl 2-methylprop-2-enoate; glycyl methacrylate; 2- propenoic acid; ethylene diamine methacrylate; 1 -aminoethyl 2-methylprop-2-enoate; 2- aminopropyl 2-methylprop-2-enoate; 2,3-diaminopropyl 2-methylprop-2-enoate; 2-aminobutyl 2-methylprop-2-enoate; 1 -aminopropyl 2-methylprop-2-enoate; 2-(2-aminoethoxy)ethyl 2- methylprop-2-enoate; l,3-diaminopropan-2-yl 2-methylprop-2-enoate; 3-(2-aminoethoxy)propyl 2-methylprop-2-enoate; 3-[3-(2-aminoethoxy)propoxy]propyl 2-methylprop-2-enoate; 2-[2-[2- (2-aminoethoxy)ethoxy] ethoxy] ethyl 2-methylprop-2 -enoate, or combinations thereof. In some embodiments, the primary amine ligands have graft density the separation substrate of 4,000 nmol / m2to 10,000 nmol / m2

[0041] In some embodiments, at 204, a medium is provided. As discussed above, in some embodiments, the medium includes an mRNA product and a concentration of impurities. In some embodiments, the medium includes or is combined with a concentration of cationic species. In some embodiments, the concentration of mRNA product in the medium is between about 1 mg / mL and about 8 mg / mL. In some embodiments, the concentration of mRNA product in the medium is between about 1 mg / mL and about 2 mg / mL. In some embodiments, the concentration of bound mRNA product is about 1.3 mg / m2In some embodiment, the impurities include peptides. pDNA. dsRNA, NTPs. MgCty proteins, other cofactors, or combinations thereof.

[0042] In some embodiments, at 206, the medium is contacted with the membrane at or below a first pH. In some embodiments, the first pH is about 8.0. In some embodiments, the first pH is equivalent to the pl of one or more peptides in the medium. In some embodiments, the first pH is about 4.5. In some embodiments, the first pH is about 4.0.

[0043] In some embodiments, at 208, the mRNA product binds with the membrane to form a concentration of bound product. In some embodiments, at 210, a first impurities effluent is eluted at the first pH.

[0044] In some embodiments, at 212, the bound product is washed with one or more washes. In some embodiments, washes 212 are effective to elute a second impurities effluent. As discussed above, in some embodiments, the one or more washes includes a salt solution. Insome embodiments, the salt solution includes a buffer, e.g., 2-amino-2-hydroxymethyl-propane-1.3-diol, glycine, potassium phosphate, sodium phosphate, citric acid, etc.; a chelating agent, e.g., ethylenediaminetetraacetic acid, triethanolamine, aminopolycarboxylic acid, etc.; a monovalent salt, e.g.. NaCl, KC1, LiCl, etc.; or combinations thereof. In some embodiments, washes 212 include treatment with a cationic species prior to the salt wash. In some embodiments, the cationic species includes (spermine, N,N'-bis(3-aminopropyl)butane-l,4- diamine; N'-(3-aminopropyl)butane-l,4-diamine; N'-[3-(3-aminopropylamino)propyl]propane-1.3-diamine; N-ethyl-N'-[3-[3-(ethylamino)propylamino]propyl]propane-l,3-diamine; 1,4,8,11- tetrazacyclotetradecane; N'-(4-aminobutyl)butane-1.4-diamine; N'-(3-aminopropyl)pentane-l,5- diamine; N,N'-bis [3 -(ethylamino)propylj butane- 1,4-diamine; N'-[3-[3-(3 aminopropylamino)propylamino]propyl]propane-l,3-diamine; 1,5,9-triazacyclododecane; N'-[3- (4-aminobutylamino)propyl] butane- 1,4-diamine; N'-(3-aminopropyl)-N-ethylbutane-l,4- diamine; N'-[3-(3-aminopropylamino)propyl]butane-l,4-diamine; N-ethyl-N'-[3- (ethylamino)propyl]butane-l,4-diamine; N'-[4-[3-(4- aminobutylamino)propylamino]butyl]butane- 1,4-diamine; N'-[3-[4-(3- aminopropylamino)butylamino]propyl]butane-l,4-diamine; 4-aminobutyl-tris(3- aminopropyl)azanium; N',N'-bis(3-aminopropyl)butane- 1,4-diamine; l-N-[4-(3- aminobutylamino)butyl]butane-l,3-diamine; N'-[3-[3-[3-(3- aminopropylamino)propylamino]propylamino]propyl]propane-l,3-diamine; bisbenzimide, or combinations thereof.

[0045] Still referring to FIG. 2, in some embodiments, at 214, the bound product is contacted with a mobile phase having a second pH above a pl of the membrane, i.e., the charged ligands on the separation substrate. In some embodiments, the pl of the membrane is between about 8 and about 9. In some embodiments, the pl of the membrane is about 8.0. In some embodiments, the pl of the membrane is about 8.5. In some embodiments, the pl of the membrane is about 9.0. In some embodiments, at 216, a product effluent is eluted at the second pH. In some embodiments, the product effluent includes a concentration of the bound product.

[0046] Referring now to FIG. 3. some embodiments of the present disclosure are directed to a method 300 of purifying a nucleic acid sample. In some embodiments, at 302, a membrane is provided. As discussed above, in some embodiments, the membrane includes a separation substrate and a plurality' of primary amine ligands positioned on the separation substrate. In some embodiments, the primary amine ligands include amino acrylate; 2-aminoethyl methacrylate hydrochloride; 3-aminopropyl 2-methylprop-2-enoate; glycyl methacrylate; 2-propenoic acid; ethylene diamine methacrylate; 1 -aminoethyl 2-methylprop-2-enoate; 2- aminopropyl 2-methylprop-2-enoate; 2,3-diaminopropyl 2-methylprop-2-enoate; 2-aminobutyl 2-methylprop-2-enoate; 1 -aminopropyl 2-methylprop-2-enoate; 2-(2-aminoethoxy)ethyl 2- methylprop-2-enoate; l,3-diaminopropan-2-yl 2-methylprop-2-enoate; 3-(2-aminoethoxy)propyl 2-methylprop-2-enoate; 3-[3-(2-aminoethoxy)propoxy]propyl 2-methylprop-2-enoate; 2-[2-[2- (2-aminoethoxy)ethoxy]ethoxy]ethyl 2-methylprop-2 -enoate, or combinations thereof. In some embodiments, the primary amine ligands have graft density on the separation substrate of 4,000 nmol / m2to 10,000 nmol / m2

[0047] In some embodiments, at 304, a mobile phase having a first pH is contacted with the membrane. As discussed above, in some embodiments, the mobile phase includes a concentration of an mRNA product. In some embodiments, the mobile phase includes a concentration of one or more impurities.

[0048] In some embodiments, the mobile phase has a flowrate between about 0. 1 to 20 mL / min. In some embodiments, the mobile phase has a flowrate between about 0.25 ml / min and about 1.5 mL / min. In some embodiments, the concentration of mRNA product in the mobile phase is between about 1 mg / mL and about 2 mg / mL. In some embodiment, the impurities include peptides, pDNA, dsRNA, or combinations thereof. In some embodiments, the first pH is below about 4.0. In some embodiments, at 306, mRNA product is captured from the mobile phase. In some embodiments, the concentration of captured mRNA product is between about 1- 8 mg / m2. In some embodiments, the concentration of captured mRNA product is about 1.3 mg / m2

[0049] In some embodiments, at 308, a cationic species is added to the mobile phase. In some embodiments, adding 308 occurs prior to capturing 306. e.g., a medium including the mRNA, impurities, and the cationic species is added to the mobile phase. In some embodiments, the cationic species includes spermine, N,N'-bis(3-aminopropyl)butane-l,4-diamine; N'-(3- aminopropyl)butane-l,4-diamine; N'-[3-(3-aminopropylamino)propyl]propane-l,3-diamine; N- ethyl-N'-[3-[3-(ethylamino)propylamino]propyl]propane-l,3-diamine; 1,4,8,11- tetrazacyclotetradecane; N'-(4-aminobutyl)butane-1.4-diamine; N'-(3-aminopropyl)pentane-l,5- diamine; N,N'-bis[3-(ethylamino)propyl]butane-l ,4-diamine; N'-[3-[3-(3 aminopropylamino)propylamino]propyl]propane-l,3-diamine; 1,5,9-triazacyclododecane; N'-[3- (4-aminobutylamino)propyl] butane- 1,4-diamine; N'-(3-aminopropyl)-N-ethylbutane-l,4- diamine; N'-[3-(3-aminopropylamino)propyl]butane-l,4-diamine; N-ethyl-N'-[3- (ethylamino)propyl]butane- 1,4-diamine; N'-[4-[3-(4-aminobutylamino )propylamino]butyl]butane-l,4-diamine: N'-[3-[4-(3- aminopropylamino)butylamino]propyl]butane-l,4-diamine; 4-aminobutyl-tris(3- aminopropyl)azanium; N',N'-bis(3-aminopropyl)butane-l,4-diamine; l-N-[4-(3- aminobutylamino)butyl]butane-l,3-diamine; N'-[3-[3-[3-(3- aminopropylamino)propylamino]propylamino]propyl]propane-l,3-diamine; bisbenzimide, or combinations thereof.

[0050] In some embodiments, cationic species is added 308 to form an N / P ratio greater than about 450 in the medium / at the separation substrate. As used herein, the term “N / P ratio” is used to refer to the molar concentrations (M) of amine groups (N) provided by the cationic groups, e.g., spermine, and number of available nucleotides or phosphate backbone repeat units (P). In some embodiments, the N / P ratio of greater than 450 is specific for LMW Poly (I: C). In some embodiments, the N / P ratio at adding 308 is for another dsRNA species, yet still equivalent to greater than 450 for LWM Poly(I:C).

[0051] In some embodiments, at 310, the ionic strength of the mobile phase is increased. In some embodiments, increasing 310 results in elution of a first effluent that includes pDNA. In some embodiments, increasing 310 includes adding a concentration of salt solution to the mobile phase. In some embodiments, the salt solution includes a buffer, e g., 2-amino-2- hydroxymethyl-propane-l,3-diol, glycine, potassium phosphate, sodium phosphate, citric acid, etc.; a chelating agent, e.g., ethylenediaminetetraacetic acid, triethanolamine, aminopoly carboxylic acid, etc.; a monovalent salt, e.g., NaCl. KC1, LiCl, etc.; or combinations thereof.

[0052] In some embodiments, at 312, the pH of the mobile phase is increased above the pl of the membrane to elute a product effluent including the mRNA product. In some embodiments, the pH is increased above about 8.0. In some embodiments, the pH is increased above about 8.5. In some embodiments, the pH is increased above about 9.0.EXAMPLES

[0053] In one example consistent with embodiments of the present disclosure, a regenerated cellulose separation substrate with 0.2 |im mean pore diameter and 75 rm mean thickness was provided. To functionalize the regenerated cellulose membrane substrate via an SET-LRP process, acetonitrile (ACN), a-bromoisobutyrylbromide (BIBB), tri ethylamine (TEA), and N,N,N'.N',N'-Pentamethyldiethylenetriamine (PMDETA) were acquired. Copper discs, serving as the catalyst for the SET-LRP reaction, were obtained from the Rensselaer PolytechnicInstitute Machine Shop (10 cm O.D., Troy, NY) and McMaster-Carr (2.5 cm O.D..Elmhurst, IL). The discs were pretreated with acetic acid to remove surface oxidation. The primary' amine ligand used to functionalize the regenerated cellulose separation substrate was 2- aminoethyl methacrylate hydrochloride (AEMA). DNase and RNase free water (Neta-Scientific, Marlton, NJ) was used as the aqueous phase for the surface modification reaction.

[0054] In this embodiment, to generate free radical initiator sites on the surface of the separation substrate, regenerated cellulose was chosen for its abundance of surface hydroxyl groups, that facilitate surface modification, enhance hydrophilicity, and maintain a negative charge to reduce fouling or non-specific binding from negatively charged or hydrophobic molecules, e.g. some proteins, and nucleic acids. The mean pore size of 0.2 pm was selected due to its usage in sterile filtration and because it is much larger than 2,000 nt mRNA molecules (about 40 nm), ensuring adequate passage. The membrane pore size can be tuned to account for larger or smaller nucleic acids without impacting surface modification or operational technique.

[0055] The SET-LRP process that was used to graft the positive primary amines onto the separation substrate is a “green” graft polymerization technique offering advantages over other polymerization methods, including higher reaction speed, lower activation temperature, and the elimination of reducing agents to regenerate the reaction catalyst, resulting in lower poly dispersity'. In some embodiments, the AEMA ligands are attached via self-regulated copper catalysis to the free radical initiator (BIBB) activated surface through its acrylate group.

[0056] To generate free radical initiator sites on the surface of regenerated cellulose, the separation substrates were reacted with BIBB in the presence of acetonitrile as an organic solvent. Initially, the separation substrates were equilibrated in ACN for 1 hour. Concurrently, a reaction mixture was prepared with 0.08 mL BIBB / cm2of membrane, 0.09 mL TEA / cm2of membrane, and 0.5 mL ACN / cm2of substrate. The separation substrates were then placed in the reaction mixture and allowed to react for 3 hours with agitation on a shaker plate. After the reaction, the separation substrates were removed and washed with ACN for 1 hour to remove excess BIBB, followed by washing an equilibration with RNase free water.

[0057] Following the addition of Br free radical sites onto the separation substrate surface, primary amine ligands, e g., AEMA, were grafted via surface polymerization through acrylate groups using SET-LRP. To perform the reaction, the activated substrates (about 3 x 5 cm2) were added to various solutions containing different amounts of AEMA (5, 10, 25, 50, 75, and 100 mg) in 5 mL RNase free water, and PMDETA ligand (33.33% of the mass of AEMAadded). The reaction mixture was enclosed in a glass petri dish with a copper disc placed above the membrane to serve as a catalyst for the reaction. The reaction vessel was then placed in a vacuum chamber. To remove oxygen from the system, the chamber underwent two cycles of - 0.1 kPa vacuum depressurization and nitrogen gas pressurization at 1 kPa to ensure an inert atmosphere. The reaction proceeded for 24 hours under vacuum, after which the membranes were then thoroughly washed and then stored with RNase free water.

[0058] Referring now to FIGs. 4A-4B, the quantification of membrane AEMA surface density as a function of the amount of AEMA placed in SET-LRP reaction mixture (about 30- 600 pmol) was quantified. While substantial variation was observed, the average surface density (denoted by “X”) positively correlates with the amount of AEMA placed in reaction mixture.When a substantial amount of AEMA was added to the membrane (18,000 ± 4700 nmol / m2), the surface modification did not cause an observable variation in membrane morphology between the unmodified and modified cases.

[0059] The surface charge of the unmodified and modified membranes was estimated as a function of pH using combinations of binding (50 mM Tris-HCl, pH 8.0) and elution buffer (125 mM glycine, pH 12.0). Under these conditions, the zeta potential of AEMA membranes modified with 7,100 ± 2,900 nmol / m2and 18,000 ± 4,700 nmol / m2of AEMA is seen in FIG. 4B.

[0060] The surface charge of the membrane system was influenced by the ratio of positive - NH3+grafted species to unmodified -O' groups present on the RC membrane surface. The amount of -NHT species present can be related to the total surface density of AEMA ([NH2] Total) as a function of pH and pKa, i.e., [H+] and Ka, respectively, using the ionization fraction equation below derived from the equilibrium constant:estimating the pKa of the AEMA ligand as ~8.1. At pH values less than pH 8.1, AEMA modified membranes were found to be positive, indicating |NH? | > [O'], The pl of the modified membranes was reached at pH 8.0-9.0, where [ NHU | was about equal to [O’]. Upon closer inspection, the pH at net charge neutrality or the pl increased as the membrane surface density was increased. This behavior was expected because as primary7amine surface density7(orrelative positive charge) increases, a higher pH was used to reduce the higher concentration of protonated amine species. Above pH 8.0-9.0, the membranes were negative (| NH< | < [O’]).

[0061] For comparison, the charge of the unmodified regenerated cellulose membrane remains negative across the pH range, as expected. In the end, the tunable surface charge results highlight the sui lability of the primary amine, i.e., AEMA, for electrostatic based separations of RNA.

[0062] Referring now to FIGs. 5 A-5D, dynamic experiments were conducted to elucidate the effect of surface charge on dynamic mRNA binding and elution performance. FIGs. 5A-5D illustrate the dynamic binding and elution behavior of separation substrate stacks with a range of surface densities from the samples characterized in FIGs. 4A-4B. Analysis of binding behavior (flow-through peaks, 0-10 minutes) indicated that the lowest density7(805 ± 17 nmol / m2) (FIG. 5A) reached an appreciable degree of membrane saturation, while the higher densities (FIGs. 5B-5D) did not display flow-through behavior when challenged with 5 pg of mRNA in the feed. Specifically, in some embodiments, the membrane system elutes a high percentage of bound mRNA at the lowest pH possible (while still facilitating membrane charge inversion as described above) in order to lower the rate of mRNA hydrolytic cleavage which is greatly accelerated as the basicity7of the solution is increased. Upon examination of FIGs. 5B- 5D. anotable elution profile could be observed. For example, each density displayed the existence of a sharp elution peak at about pH 9 (for the elution buffer), while a secondary peak could also be seen above pH 11. Chromatographic analysis of the primary elution peaks showed an increase in the amount eluted from 2.7 pg by the second surface density7(4,580 ± 150 nmol / m2, FIG. 5B) to 3.4 pg by the third surface density (10,100 ± 14 nmol / m2, FIG. 5C).However, this difference could be attributed to variations of the injected mass deviating from the expected 5 pg (noted in the lower recovery of bound mass 89% to 100% for 4,580 ± 150 nmol / m2and 10,100 ± 14 nmol / m2, respectively). The amount eluted in peak one by 17,100 ± 26 nmol / m2(FIG. 5D) was reduced in comparison to the other AEMA surface densities (1.3 pg). Also, the amount eluted in peak two increased for the 17,100 ± 26 nmol / m2membranes with 3.9 pg eluted in comparison to 1.8 and 1.6 pg eluted for 4,580 ± 150 nmol / m2and 10,100 ± 14 nmol / m2, respectively. In FIG. 5D, a reduction in the primary elution peak along with a broader and more pronounced secondary peak was observed.

[0063] Referring now to FIGs. 6A-6C, a surface density range between 4,600 and 10,000 nmol / m2was chosen, and the mRNA uptake and elution performance of membranes modified with 4,800 ± 1 nmol / m2. 7,210 ± 1 nmol / m2, and 9.410 ± 1 nmol / m2of AEMA wascharacterized. These membrane systems were challenged with sufficient mRNA to enable the quantification of dynamic binding capacity at 10% breakthrough (DBCio%). A general increase in DBCio% was observed with increasing AEMA surface density, reaching a maximum value of 1.28 ± 0.04 mg / m2of mRNA adsorbed.

[0064] Referring now to FIGs. 7A-7C, the reduction of dsRNA content from mRNA- containing effluent, e.g., from IVT can reduce the immunogenicity of mRNA biologies, beneficial for therapeutic treatments. FIG. 7A displays the results of pure feed performance of an 8,700 ± 4 nmol / m2membrane challenged with about 5 pg of dsRNA compared with mRNA. The AEMA membrane was partially able to remove dsRNA in a high salt wash step (3.7 pg), while a similar portion eluted during the pH gradient (2.2 pg) where mRNA recovery' occurs.

[0065] Referring specifically to FIG. 7B, the ability to use bind / wash / elution processes and modified membranes with decreasing AEMA surface density (8,700 ± 4, 4100 ± 4, and 2,080 ± 4 nmol / m2) consistent with embodiments of the present disclosure w as investigated. Inspection of the chromatograms indicated that the amount eluted during the pH elution phase was decreased from 2.2 pg to 0.5 pg at the lowest density. Similarly, the amount washed was increased from 3.7 pg to 4.2 pg as the surface density was halved (4,100 ± 4 nmol / m2). The amount w ashed in the 2,080 ± 4 nmol / m2case w as not increased in comparison to the others, most likely due to the decreased amount bound.

[0066] While the amount bound decreased slightly (lowered to 91%), the 4,100 ± 4 nmol / m2case w as of interest as an increase in mRNA purity can be of more value than a slight loss in capacity. FIG. 7C shows the overlay of pure feed dsRNA and mRNA performance at the decreased grafting density of 4,100 ± 4 nmol / m. While the dsRNA molecule was desorbed in the wash, so too was mRNA.

[0067] Referring now FIGs. 8A-8B, performance of modified membranes consistent with embodiments of the present disclosure for dsRNA separation utilizing N / P conditions were demonstrated. An AEMA modified membrane (7,890 ± 2 nmol / m2) was tested with about 6 pg dsRNA (N / P = 0) and about 6 pg dsRNA with spermine at a sufficiently high enough N / P ratio (4 mM spermine, N / P Ratio of about 700). The addition of spermine increased the amount of dsRNA removed during the wash phase from 4.9 ± 0.2 pg to 5.8 ± 0.07 pg. Similarly, the amount present during the pH elution phase was decreased from 1.3 ± 0.04 pg to 0. 1 ± 0.06 pg. Furthermore, FIG. 8B confirmed that about 10 pg mRNA with 0.4 mM of spermine (N / P of about 50) exhibited a similar behavior seen in pure feed mRNA characterization withoutspermine. Calculations showed that 7.9 |ig of mRNA was eluted with only 0.31 pg lost during the wash phase.

[0068] While the utilization of larger quantities of dsRNA relative to mRNA enabled easier quantification and magnified the effects of spermine on the separation, it was desired to perform pure feed characterizations under more representative conditions before testing a mixed feedstock. To simulate IVT conditions while allowing for ease of quantification, about 6 wt. % dsRNA relative to mRNA was used with 4 mM spermine providing a conservative N / P ratio conditions of about 50 and about 700 for mRNA and dsRNA, respectively. A smaller 100 pL injection size was used to remain within membrane capacity limits at higher mRNA (about 100 ng / pL) and dsRNA (about 6 ng / pL) concentrations. Spermine addition rendered dsRNA undetectable where the elution of mRNA occurred.

[0069] To confirm the separation performance with a complex feedstock containing mRNA, dsRNA, and spermine, the modified AEMA membrane was tested with a 100 pL injection of about 100 ng / pL mRNA and 6 ng / pL dsRNA with about 4 mM spermine. The resulting chromatogram resembled the overlay of similar mass pure feed performance. The assay confirmed the pure feed characterization results, with dsRNA content in the mRNA elution peak below the detection limit.

[0070] Systems and methods of the present disclosure advantageously provide membranes and process conditions utilizing electrostatics to separate mRNA from protein and nucleic acid impurities, e.g., in IVT fluids and blood plasma. Some embodiments of the present disclosure include primary amine-modified microporous regenerated cellulose membranes. Manipulating the pH range of the membranes between about 4 and about 8 and further between about 8 and about 10 effectively invert the charges of the membrane and of species in the mobile phase, enabling pH-based purification of particular products, e.g., mRNA vaccines. Cationic and salt additives can also be used to enhance the separation of mRNA products from other undesired nucleic acid constructs, e.g., pDNA, dsRNA, etc. In some embodiments, addition of the multivalent cationic polyamines including spermine is used to enhance electrostatic separation, e.g., by reducing the charge of dsRNA at N / P ratios greater than 450. This charge balancing effectively improves the production of high-integrity and purity mRNA under representative in vitro transcription bioreactor effluent conditions achieving high separation capacity while reducing impurities.

[0071] Grafting density ranges of primary amine ligands, e.g., of approximately 4,000- 10,000 nmol / m2, effectively reduced secondary elution peaks, reducing the need for completedeprotonation of the membrane surface. Embodiments of the present disclosure yielded an advantageous DBCio%, e g., up to 1.28 ± 0.04 mg / m2of mRNA, with up to 100% recover}7of bound nucleic acids. The membranes not only offered a competitive binding capacity7and recovery, but also leveraged a dominant convective mechanism to rapidly purify mRNA, e.g., up to 1.5 mL / min. Additionally, the membranes of preset disclosure demonstrated reusability for up to 10 cycles without significant loss in binding capacity or recovery efficiency.

[0072] Some embodiments allow for continuous vaccine production via purification of mRNA. which can result in faster manufacturing and reduced costs of production. Embodiments of the present disclosure can replace resin-based chromatography (which is diffusive and often slow with a long residence time) with membranes (which are convective, and often faster and with shorter residence time) for purification of mRNA vaccines, reducing the residence times of the recovery process and retaining a higher percentage of folded mRNA. The footprint of the equipment used to purify mRNA products can also be reduced.

[0073] Although the invention has been described and illustrated with respect to exemplary7embodiments thereof, it should be understood by those skilled in the art that the foregoing and various other changes, omissions and additions can be made therein and thereto, without parting from the spirit and scope of the present invention.

Claims

CLAIMSWhat is claimed is:

1. A method of purifying a nucleic acid sample, comprising: providing a membrane including: a separation substrate, and a plurality of charged ligands positioned on the separation substrate, contacting a medium with the membrane at or below a pH of about 4.0, the medium including an mRNA product and one or more impurities; preferentially binding one of the impurities or the mRNA product via the charged ligands to form a concentration of bound product; and eluting a concentration of the bound product from the membrane at a pH above about 4.0.

2. The method according to claim 1, wherein the charged ligands have graft density on the separation substrate of 4,000 nmol / m2to 10,000 nmol / m23. The method according to claim 1, wherein the charged ligands include primary amine ligands, including amino acrylate; 2-aminoethyl methacrylate hydrochloride; 3- aminopropyl 2-methylprop-2-enoate; glycyl methacrylate; 2-propenoic acid; ethylene diamine methacrylate: 1 -aminoethyl 2-methylprop-2-enoate; 2-aminopropyl 2- methylprop-2-enoate; 2,3-diaminopropyl 2-methylprop-2-enoate; 2-aminobutyl 2- methylprop-2-enoate; 1-aminopropyl 2-methylprop-2-enoate; 2-(2-aminoethoxy)ethyl 2- methylprop-2-enoate; l,3-diaminopropan-2-yl 2-methylprop-2-enoate; 3-(2- aminoethoxy)propyl 2-methylprop-2-enoate; 3-[3-(2-aminoethoxy)propoxy]propyl 2- methylprop-2-enoate: 2-[2-[2-(2 -aminoethoxy )ethoxy]ethoxy]ethyl 2-methylprop-2- enoate, or combinations thereof.

4. The method according to claim 1 , wherein preferentially binding one of the impurities or the mRNA product via the charged ligands to form a concentration of bound product includes:binding the mRNA product via primary amine ligands at or below a pH of about 4.0, and eluting a first effluent including a concentration of protein impurities.

5. The method according to claim 1, further comprising: prior to eluting a concentration of the bound product, treating the medium, the membrane, or combinations thereof with a salt solution to adjust a corresponding ionic strength thereof.

6. The method according to claim 1, further comprising: prior to eluting a concentration of the bound product, treating the medium, the membrane, or combinations thereof with a solution including one or more cationic species.

7. The method according to claim 6, wherein the cationic species includes N,N'-Bis(3- aminopropyl)- 1 ,4-diaminobutane (spermine), N,N'-bis(3-aminopropyl)butane-l ,4- diamine; N'-(3-aminopropyl)butane-l,4-diamine; N'-[3-(3- aminopropylamino)propyl]propane-l,3-diamine; N-ethyl-N'-[3-[3- (ethylamino)propylamino]propyl]propane-l,3-diamine; 1,4,8,11-tetrazacyclotetradecane; N'-(4-aminobutyl)butane-l,4-diamine; N'-(3-aminopropyl)pentane- 1,5 -diamine; N,N'- bis[3-(ethylamino)propyl]butane-l,4-diamine; N'-[3-[3-(3 aminopropylamino)propylamino]propyl]propane-l,3-diamine; 1,5,9- triazacyclododecane; N'-[3-(4-aminobutylamino)propyl]butane-1.4-diamine; N'-(3- aminopropyl)-N-ethylbutane-l,4-diamine; N'-[3-(3-aminopropylamino)propyl]butane- 1,4-diamine; N-ethyl-N'-[3-(ethylamino)propyl]butane-l,4-diamine; N'-[4-[3-(4- aminobutylamino)propylamino]butyl]butane-l,4-diamine; N'-[3-[4-(3- aminopropylamino)butylamino]propyl]butane-1.4-diamine; 4-aminobutyl-tris(3- aminopropyljazanium; N',N'-bis(3-aminopropyl)butane-l,4-diamine; l-N-[4-(3- aminobutylamino)butyl]butane-l,3-diamine; N'-[3-[3-[3-(3- aminopropylamino)propylamino]propylamino]propyl]propane-l,3-di amine; bisbenzimide, or combinations thereof.

8. The method according to claim 7, wherein spermine is provided at a concentration between ImM and 20mM.

9. The method according to claim 1 , wherein contacting a medium with the membrane occurs in a mobile phase having a flowrate between about 0.1 to 20 mL / min. 0.25 ml / min and about 1.5 mL / min.

10. The method according to claim 1, wherein the separation substrate has a mean pore diameter between about 0.1-10 pm.

11. The method according to claim 1 , wherein the concentration of bound mRNA product in is about 1.3 mg / m212. A method of purifying a nucleic acid sample, comprising: providing a membrane including: a separation substrate, and a plurality of primary amine ligands positioned on the separation substrate, providing a medium including an mRNA product, a concentration of impurities, and a concentration of a cationic species; contacting the medium with the membrane at or below a first pH of about 4.0; binding the mRNA product with the membrane to form a concentration of bound product; eluting a first impurities effluent at the first pH; contacting the bound product with a salt solution to elute a second impunties effluent; contacting the bound product with a mobile phase having a second pH above a pl of the membrane; and eluting a product effluent at the second pH, the product effluent including a concentration of the bound product, wherein the impurities include peptides, pDNA, dsRNA, NTPs, MgCh, proteins, other cofactors, or combinations thereof.

13. The method according to claim 12. wherein the primary amine ligands have graft density on the separation substrate of 4,000 nmol / m2to 10,000 nmol / m214. The method according to claim 12, wherein the primary amine ligands include amino acrylate; 2-aminoethyl methacrylate hydrochloride; 3-aminopropyl 2-methylprop-2- enoate; glycyl methacrylate; 2-propenoic acid; ethylene diamine methacrylate; 1 - aminoethyl 2-methylprop-2-enoate; 2-aminopropyl 2-methylprop-2-enoate; 2,3- diaminopropyl 2-methylprop-2-enoate; 2-aminobutyl 2-methylprop-2-enoate; 1- aminopropyl 2-methylprop-2-enoate; 2-(2-aminoethoxy)ethyl 2-methylprop-2-enoate;1.3-diaminopropan-2-yl 2-methylprop-2-enoate; 3-(2-aminoethoxy)propyl 2-methylprop- 2-enoate; 3-[3-(2-aminoethoxy)propoxy]propyl 2-methylprop-2-enoate; 2-[2-[2-(2- aminoethoxy)ethoxy] ethoxy] ethyl 2-methylprop-2-enoate, or combinations thereof.

15. The method according to claim 12, wherein the cationic species includes N,N'-Bis(3- aminopropyl)-l,4-diaminobutane (spermine), N,N'-bis(3-aminopropyl)butane-l,4- diamine; N'-(3-aminopropyl)butane-l,4-diamine; N'-[3-(3- aminopropylamino)propyl]propane-l,3-diamine; N-ethyl-N'-[3-[3- (ethylamino)propylamino]propyl]propane-1.3-diamine; 1.4.8, 11-tetrazacyclotetradecane; N'-(4-aminobutyl)butane-l ,4-diamine; N'-(3-aminopropyl)pentane-l ,5-diamine; N,N'- bis[3-(ethylamino)propyl]butane-l,4-diamine; N'-[3-[3-(3 aminopropylamino)propylamino]propyl]propane-l,3-diamine; 1,5,9- triazacyclododecane; N'-[3-(4-aminobutylamino)propyl]butane-l,4-diamine; N'-(3- aminopropyl)-N-ethylbutane-l,4-diamine; N'-[3-(3-aminopropylamino)propyl]butane-1.4-diamine; N-ethyl-N'-[3-(ethylamino)propyl]butane-l,4-diamine; N'-[4-[3-(4- aminobutylamino)propylamino]butyl]butane-l,4-diamine; N'-[3-[4-(3- aminopropylamino)butylamino]propyl]butane-1.4-diamine; 4-aminobutyl-tris(3- aminopropyl)azanium; N',N'-bis(3-aminopropyl)butane-1.4-diamine; l-N-[4-(3- aminobutylamino)butyl]butane-l ,3-diamine; N'-[3-[3-[3-(3- aminopropylamino)propylamino]propylamino]propyl]propane-l,3-di amine; bisbenzimide, or combinations thereof.

16. The method according to claim 15, wherein the pl of the membrane is between about 8 and about 9.

17. The method according to claim 12. wherein contacting the medium with the membrane occurs in a mobile phase having a flowrate between about 0.25 ml / min and about 1 .5 rnL / min.

18. The method according to claim 17. wherein the concentration of bound mRNA product is between about 1 mg / m2and about 8 mg / m2.

19. A method of purifying a nucleic acid sample, comprising: providing a membrane including: a separation substrate, and a plurality of primary amine ligands positioned on the separation substrate, providing a medium including an mRNA product, a concentration of impurities, and a concentration of a cationic species to form an N / P ratio greater than about 450 or equivalent; contacting the medium with the membrane in a mobile phase having a pH below about 4.0; capturing mRNA product from the mobile phase; increasing the ionic strength of the mobile phase to elute a first effluent including nucleic acid impurities; and increasing the pH of the mobile phase above about 8.5 to elute a product effluent including mRNA product.

20. The method according to claim 19. wherein the primary amine ligands have graft density on the separation substrate of 4,000 nmol / m2to 10,000 nmol / m2, wherein the primary amine ligands include amino acrylate; 2-aminoethyl methacrylate hydrochloride; 3-aminopropyl 2-methylprop-2-enoate; glycyl methacrylate; 2-propenoic acid; ethylene diamine methacrylate; 1-aminoethyl 2- methylprop-2-enoate; 2-aminopropyl 2-methylprop-2-enoate; 2,3 -diaminopropyl 2-methylprop-2-enoate; 2-aminobutyl 2-methylprop-2-enoate; 1 -aminopropyl 2- methylprop-2-enoate; 2-(2-aminoethoxy)ethyl 2-methylprop-2-enoate; 1,3- diaminopropan-2-yl 2-methylprop-2-enoate; 3-(2-aminoethoxy)propyl 2- methylprop-2-enoate; 3-[3-(2-aminoethoxy)propoxy]propyl 2-methylprop-2-enoate; 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethyl 2-methylprop-2-enoate, or combinations thereof.

21. The method according to claim 19, wherein the cationic species includes N,N'-Bis(3- aminopropyl)-l,4-diaminobutane (spermine), N.N'-bis(3-aminopropyl)butane-l,4- diamine; N'-(3-aminopropyl)butane-l,4-diamine; N'-[3-(3- aminopropylamino)propyl]propane-l,3-diamine; N-ethyl-N'-[3-[3- (ethylamino)propylamino]propyl]propane-l,3-diamine; 1,4,8, 11-tetrazacyclotetradecane; N'-(4-aminobutyl)butane-l,4-diamine; N'-(3-aminopropyl)pentane- 1,5 -diamine; N,N'- bis[3-(ethylamino)propyl]butane-l ,4-diamine; N'-[3-[3-(3 aminopropylamino)propylamino]propyl]propane-l,3-diamine; 1,5,9- triazacyclododecane; N'-[3-(4-aminobutylamino)propyl]butane-l,4-diamine; N'-(3- aminopropyl)-N-ethylbutane-l,4-diamine; N'-[3-(3-aminopropylamino)propyl]butane- 1.4-diamine; N-ethyl-N'-[3-(ethylamino)propyl]butane-l,4-diamine; N'-[4-[3-(4- aminobutylamino)propylamino]butyl]butane-l,4-diamine; N'-[3-[4-(3- aminopropylamino)butylamino]propyl]butane-l,4-diamine; 4-aminobutyl-tris(3- aminopropyl)azanium; N',N'-bis(3-aminopropyl)butane-l,4-diamine; l-N-[4-(3- aminobutylamino)butyl]butane-l,3-diamine; N'-[3-[3-[3-(3- aminopropylamino)propylamino]propylamino]propyl]propane-l,3-di amine; bisbenzimide, or combinations thereof.

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