Methods for isolating supercoiled plasmid DNA.

Abstract

The present disclosure provides a method for isolating supercoiled plasmid DNA (pDNA) from a liquid sample, comprising: The present invention provides a method for separating pDNA from pDNA comprising the steps of: (a) applying a liquid sample comprising pDNA to a first chromatographic material comprising (i) an anion exchange chromatographic ligand for binding pDNA and (ii) a support material allowing convection through the first chromatographic material, the liquid sample being derived from a cell culture harvest and having been subjected to a step of removing RNA prior to step (a); (b) eluting the liquid sample from the first chromatographic material comprising a purified mixture of supercoiled pDNA and open circular pDNA; (c) applying the liquid sample from step (b) to a second chromatographic material comprising a ligand that binds pDNA and allows selective separation of supercoiled pDNA from open circular pDNA; and (d) eluting the purified supercoiled pDNA from the second chromatographic material, the supercoiled pDNA eluted in step (d) having a purity of at least 95% without the use of any additional chromatographic material beyond the first and second chromatographic materials. Steps (a)-(d) and any intermediate steps can be completed within 5 hours. Uses of the supercoiled pDNA obtained by the separation method are further disclosed.

Description

[Technical Field]

[0001] The present disclosure relates to the field of plasmid DNA (pDNA) isolation and is directed to methods for producing purified compositions containing pDNA and for isolating supercoiled pDNA from cell culture harvests. Uses of the supercoiled pDNA obtained by the methods are further disclosed. [Background technology]

[0002] Plasmids are small DNA molecules that are physically separate from chromosomal DNA. They are small, circular, double-stranded DNA molecules that replicate independently in bacteria.

[0003] Plasmid DNA (pDNA) is an important genetic engineering tool used in genetics and biotechnology laboratories to clone and amplify or express genes. The production of viral vectors and mRNA relies on pDNA. It can be used as a template for mRNA production, to manufacture viral vectors for producing vector-based DNA vaccines, or for cell and gene therapy applications. Good Manufacturing Practice (GMP)-grade pDNA can be used as direct DNA vaccines, in gene therapy, and in ex vivo applications, such as recombinant protein production.

[0004] Plasmid supply and quality are critical for the production of viral vectors, mRNA, and DNA vaccines. Methods for GMP production of pDNA must be designed to meet production volume and quality needs. Different plasmids and applications (e.g., different therapies) have different requirements. pDNA needs to be expressed and purified at high yields and titers to meet the yield and purity goals of each application.

[0005] Supercoiled (SC) plasmids in the 5-20 kb pair (kbp) range are the most common in bioprocessing applications. Previously known methods for purifying supercoiled plasmids have been described, for example, by Lemmens et al. (2003), Sandberg et al. (2004), and Hitchcock et al. (2010). Furthermore, Application Note 28-4094-85 AA (Cytiva, Sweden) proposes a three-step method involving 1) population separation on a size-exclusion chromatography resin, 2) capture and selective desorption of SC pDNA, and 3) polishing using a hard bead chromatography resin with a strong anion-exchange ligand. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] US20140296464A1 [Patent Document 2] US20160288089A1 [Patent Document 3] WO2018011600A1 [Patent Document 4] WO2018037244A1 [Patent Document 5] WO2013068741A1 [Patent Document 6] WO2015052465A1 [Patent Document 7] US7867784B2 [Patent Document 8] US6,602,990(Berg) [Patent Document 9] US6,428,707 [Patent Document 10] International Patent Application No. PCT / EP2022 / 077388 [Non-patent literature]

[0007] [Non-Patent Document 1] S Hjerten: Biochim Biophys Acta 79(2), pp. 393-398 (1964) [Non-patent document 2] "Styrene based polymer supports developed by suspension polymerization" (R Arshady: Chimica e L'Industria 70(9), pp. 70-75 (1988)) Summary of the Invention [Problem to be solved by the invention]

[0008] However, new purification strategies are constantly needed to increase the productivity and sustainability of large-scale methods for, for example, pDNA purification. [Means for solving the problem]

[0009] The present disclosure seeks to overcome or at least partially alleviate the shortcomings of the prior art.

[0010] It is therefore an object of the present disclosure to provide a more productive method for isolating supercoiled pDNA.

[0011] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (a) applying a liquid sample containing pDNA to a first chromatographic material comprising (i) an anion exchange chromatographic ligand for binding to the pDNA and (ii) a support material that allows for convective flow through the first chromatographic material; the liquid sample is derived from a cell culture harvest and has been subjected to a step of removing RNA prior to step (a); (b) eluting the liquid sample containing the purified mixture of supercoiled pDNA and open circular pDNA from the first chromatographic material; (c) applying the liquid sample from step (b) to a second chromatographic material containing a ligand that binds pDNA and allows for the selective separation of supercoiled pDNA from open circular pDNA; (d) eluting the purified supercoiled pDNA from the second chromatographic material. wherein the supercoiled pDNA eluted in step (d) has a purity of at least 95% without the use of any additional chromatographic materials other than the first and second chromatographic materials.

[0012] Step (a) may be preceded by a step (a1) of pretreating the liquid sample, the pretreating step comprising at least one of subjecting the cell culture harvest to cell lysis, precipitating RNA in the presence of an RNA precipitant, clarification, and filtration.

[0013] The step of subjecting the cell culture harvest to cell lysis may be performed by mixing the cell culture harvest with a lysis solution using a static mixer, contacting the cell culture harvest with the lysis solution to form a combined composition, and then flowing the combined composition through the static mixer to achieve mixing.

[0014] Step (a1) may provide for this step of RNA removal by precipitating RNA in the presence of an RNA precipitant, which may be selected from the group consisting of calcium chloride, lithium chloride, and tripotassium citrate, preferably calcium chloride.

[0015] In another aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: (I) providing a cell culture harvest comprising pDNA-producing cells in the form of a slurry; (II) contacting the cell culture harvest with a lysis solution to form a lysis composition; (III) mixing the lysis composition using a static mixer to form a cell lysate; (IV) adding a neutralizing solution to the cell lysate; (V) optionally precipitating RNA in the cell lysate by the action of an RNA precipitant. (VI) clarifying the cell lysate; (VII) filtering the cell lysate to obtain a solution containing plasmid DNA (pDNA); (VIII) subjecting the solution containing the pDNA to at least one chromatography step to provide a purified composition containing the pDNA. Including, Methods are provided for producing compositions comprising pDNA.

[0016] Steps (I) to (VIII) may be performed continuously or using disposable equipment. The method is fast and easily controllable.

[0017] The at least one chromatography step may include at least one bind-elute chromatography step for capturing pDNA, and in particular may include chromatography steps (a)-(d) as described herein.

[0018] The methods disclosed herein typically do not include a size exclusion chromatography step such as population separation. Thus, the methods of the first or second aspects may be carried out without the use of a size exclusion chromatography resin.

[0019] In contrast to size exclusion chromatography, step (a) of the present method offers high loading capacity, significantly reducing overall processing time for a given processing volume. For example, steps (a)-(d) and any intermediate steps can be completed within 5 hours. Furthermore, as demonstrated in the examples, a substantial reduction in ammonium sulfate consumption can be achieved. The method meets industry acceptance criteria for GMP-grade pDNA.

[0020] Thus, the two-step chromatography method disclosed herein provides highly pure supercoiled pDNA and allows for higher productivity. The method also reduces buffer consumption compared to previous methods, thereby providing a more sustainable method for pDNA isolation.

[0021] The method disclosed above comprises subjecting the supercoiled pDNA eluted in step (d) to the following steps: (e1) concentrating the supercoiled pDNA to a pharmaceutically relevant dose; (e2) replacing the buffer applied in step (d) of the above method with a pharmaceutically acceptable buffer, and / or (e3) Sterilizing the supercoiled pDNA to obtain a composition comprising pharmaceutical-grade supercoiled pDNA, and optionally the composition is a pharmaceutical composition or active pharmaceutical ingredient comprising pharmaceutical-grade supercoiled pDNA.

[0022] The present disclosure further provides a method for preventing or treating a disease or disorder in a subject, comprising administering to the subject a pharmaceutical composition or active pharmaceutical ingredient comprising pharmaceutical-grade supercoiled pDNA obtained by performing the isolation methods disclosed herein, optionally wherein the active pharmaceutical ingredient is administered in the form of a DNA vaccine, wherein the disease or disorder being prevented is caused by infection with a virus, such as SARS-CoV-2, Zika, influenza A, yellow fever virus, or human immunodeficiency virus (HIV).

[0023] In another aspect, the present disclosure is directed to a method for producing a viral vector comprising transfecting a cell in vitro with a composition comprising pharmaceutical-grade supercoiled pDNA obtained by performing the isolation method disclosed herein, thereby allowing for the production of a viral vector, optionally the viral vector being for use as an active pharmaceutical ingredient.

[0024] In a further aspect, the present disclosure also provides a method for producing RNA, comprising using a composition comprising pharmaceutical-grade supercoiled pDNA obtained by performing the separation methods disclosed herein as a template to produce RNA, optionally wherein the RNA is for use as an active pharmaceutical ingredient.

[0025] Preferred aspects of the present disclosure are set out below in the detailed description and in the dependent claims. It should be noted that the invention relates to all possible combinations of the features recited in the claims. [Brief explanation of the drawings]

[0026] [Figure 1] 1 is a flowchart of a method for isolating supercoiled pDNA of the present disclosure, comprising steps (a) through (d). [Figure 2] 2 is a flow chart of the method for isolating supercoiled pDNA of FIG. 1, further comprising a further step (a1) before step (a). [Figure 3] 2 is a flowchart of the method for separating supercoiled pDNA of FIG. 1, optionally including step (a1) before step (a) and further including one or more additional steps (e1), (e2) and / or (e3) after step (d). [Figure 4] 1 is a photograph of an agarose gel showing RNA depletion by CaCl 2 precipitation during cell lysis. [Figure 5] 1 is a graph showing the elution curves of three cycles of pDNA separation for the first chromatography step of the separation method described in the Examples below. [Figure 6] 1 is a graph showing the elution curves for the separation of supercoiled pDNA and open circular pDNA from the second chromatographic step of the separation method described in the Examples below. DETAILED DESCRIPTION OF THE INVENTION

[0027] The present disclosure relates to a method for manufacturing a semiconductor device, as illustrated in FIG. (a) applying a liquid sample containing pDNA to a first chromatographic material comprising (i) an anion exchange chromatographic ligand for binding to the pDNA and (ii) a support material that allows for convective flow through the first chromatographic material; the liquid sample is derived from a cell culture harvest and has been subjected to a step of removing RNA prior to step (a); (b) eluting the liquid sample containing the purified mixture of supercoiled pDNA and open circular pDNA from the first chromatographic material; (c) applying the liquid sample from step (b) to a second chromatographic material containing a ligand that binds pDNA and allows for the selective separation of supercoiled pDNA from open circular pDNA; (d) eluting the purified supercoiled pDNA from the second chromatographic material. wherein the supercoiled pDNA eluted in step (d) has a purity of at least 95% without the use of any additional chromatographic materials other than the first and second chromatographic materials.

[0028] Significant advantages of the disclosed method, which involves only two chromatography steps, include an 80% reduction in production time for downstream processing, a 50% reduction in ammonium sulfate consumption, and high purity (at least 95%) supercoiled pDNA, thereby providing increased sustainability compared to the previously known three-step method for pDNA purification described in Application Note 28-4094-85 AA (Cytiva, Sweden).

[0029] As used herein, the term "liquid sample" should be understood to encompass any type of sample that can be obtained from a cell culture or from a fluid derived from a cell culture, the fluid being at least partially purified by any means of separation and purification.

[0030] The term "supercoiled pDNA" refers to plasmid DNA in the form of a closed loop having a coiled morphology. The DNA can be positively or negatively supercoiled. For example, supercoiled plasmid DNA can form a two-start right-handed helix with loops at the ends.

[0031] The term "open circular pDNA" refers to plasmid DNA in which one strand of the DNA double helix has been broken, due to the release of at least a portion of the strain of the double helix that causes the plasmid to adopt a supercoiled configuration, thereby forming a closed loop that is not coiled but maintains a circular form.

[0032] The term "purity" with respect to supercoiled plasmid DNA refers to the content of supercoiled plasmid DNA relative to the total amount of plasmid DNA. "Plasmid DNA" generally includes supercoiled plasmid DNA, open circular plasmid DNA, and, if present, linear plasmid DNA. Thus, 95% pure supercoiled pDNA means that supercoiled pDNA constitutes 95% of the total pDNA.

[0033] The term "separation matrix" is used herein to mean a material comprising a support material to which one or more ligands containing functional groups are attached. The functional groups of the ligands bind to compounds, also referred to herein as analytes, that are to be separated from a liquid sample and / or separated from other compounds present in the liquid sample. The separation matrix may further comprise compounds that bind the ligands to the support material. The terms "linker" and "extender" may be used to describe such compounds. The term "resin" is sometimes used for separation matrices in this field. The terms "chromatographic material" and "chromatographic matrix" are used herein to mean one type of separation matrix.

[0034] In general, the separation matrix can be included in any type of separation device. The term "separation device" has its conventional meaning in the field of bioprocessing and should be understood to encompass any type of separation device capable of and suitable for the separation and purification of compounds from fluids, including by-products of the production of those compounds. A separation device can include a separation matrix as defined above. Non-limiting examples of separation devices include chromatography columns and membrane devices, as further described elsewhere herein. By way of non-limiting example, a chromatography material in the form of porous particles can be packed into a chromatography column prior to application of a liquid sample to the chromatography material contained therein. By way of another non-limiting example, a chromatography material in the form of a membrane-like structure can be included in a membrane device.

[0035] In this context, a "ligand" is a molecule with known or unknown affinity for a given analyte, including any functional group immobilized on its surface, or a capture agent, while an "analyte" includes any specific binding partner for a ligand. The term "ligand" may be used interchangeably herein with the terms "specific binding molecule," "specific binding partner," "capture molecule," and "capture agent." A molecule in a liquid sample that interacts with a ligand is referred to herein as an "analyte." For purposes of this disclosure, the analyte is supercoiled pDNA. As a result, the terms "analyte" and "supercoiled pDNA" may be used interchangeably herein.

[0036] The chromatographic material applied in the methods disclosed herein comprises a support material to which a ligand is attached.

[0037] As mentioned above, the support material of the first chromatographic material is a support material that allows convection through the first chromatographic material. The term "support material that allows convection" is intended to mean a support material that allows perfusion of the support material by application of a hydraulic pressure difference between the inlet and outlet of the support material, thereby achieving substantially convective transport of substances to or from the support material.

[0038] Non-limiting examples of support materials that allow convection are nanofibers, monoliths, membrane-like structures, and particles with attached extenders.

[0039] When the support material of the first chromatographic material comprises nanofibers, such nanofibers may comprise, for example, electrospun polymeric nanofibers. In use, such nanofibers form a stationary phase comprising a plurality of pores through which a mobile phase can permeate.

[0040] As used herein, the terms "monolith" and "monolithic structure" have their conventional meaning in the field of bioprocessing. A monolith can be described as a single-unit structure. Monoliths used in bioprocessing applications have a sponge-like structure containing pores. When the support material of the first chromatographic material comprises a monolithic structure, one skilled in the art can select a monolithic structure having a pore size appropriate for the purpose of separating pDNA from a liquid sample.

[0041] A non-limiting example of a monolithic support material for the first chromatography material is Convective Interaction Media (CIM™ Monolith (BIA Separations)), such as CIM Maltus™ DEAE (Sartorius).

[0042] The support material of the first chromatographic material may comprise a membranous structure, such as a single membrane, a stack of membranes, or a filter. The membrane may be an adsorptive membrane. When the support material of the chromatographic material comprises a membranous structure, one skilled in the art may select a membranous structure having a pore size suitable for the purpose of separating pDNA from a liquid sample. When the chromatographic material comprises a membranous structure, such a membranous structure may comprise, for example, a nonwoven web of polymeric nanofibers.

[0043] Non-limiting examples of suitable polymers may be selected from polysulfone, polyethersulfone, polyamide, nylon, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, and polyethylene oxide, and mixtures thereof.

[0044] Alternatively, the polymer may be a cellulosic polymer such as selected from the group consisting of cellulose and partial derivatives of cellulose, in particular cellulose esters, cross-linked cellulose, grafted cellulose, or ligand-coupled cellulose. Cellulose fiber chromatography (also known as Fibro chromatography; Cytiva, Sweden) is an ultra-high-speed chromatographic purification method that utilizes high flow rates and high capacities of cellulose fibers for short processing times and high productivity.

[0045] Non-limiting examples of suitable membrane structures include reinforced cellulose.

[0046] Another non-limiting example of a suitable membranous structure includes nylon.

[0047] Another non-limiting example of a suitable membrane structure includes polyethersulfone, such as Mustang™ Q (Pall).

[0048] Non-limiting examples of the first chromatographic material applied in step (a) of the separation method disclosed herein include anion exchange chromatographic ligands and support materials having a membrane-like structure comprising cellulose or a partial derivative of cellulose, as described in detail elsewhere herein.

[0049] Another non-limiting example of a membrane support for the first chromatographic material is Sartobind® Q membrane sorber (Sartorius), which has a macroporous structure with pore sizes >3 μm. Quaternary ammonium ligands are covalently bound to the entire inner surface of the membrane.

[0050] The term "membrane chromatography" has its conventional meaning in the field of bioprocessing. Membrane chromatography involves the binding of components of a fluid, such as individual molecules, associates, or particles, to the surface of a solid phase in contact with the fluid. The surface activity of the solid phase makes it accessible to molecules by convective transport. The advantage of membrane sorbers over packed chromatography columns is their suitability for higher flow rates. This is also referred to as convection-based chromatography. Convection-based chromatography matrices include any matrix in which the application of a hydraulic pressure difference between the inlet and outlet of the matrix perfuses the matrix, achieving substantially convective transport of substances into or out of the matrix at very rapid, high flow rates. Convection-based chromatography and membrane sorbers are described, for example, in US20140296464A1, US20160288089A1, WO2018011600A1, WO2018037244A1, WO2013068741A1, WO2015052465A1, US7867784B2, which are incorporated by reference in their entireties.

[0051] The support material of the first chromatographic material may be in the form of particles, such as substantially spherical particles, elongated particles, or irregularly shaped particles.

[0052] Suitable particle sizes may range from 5 to 500 μm. For substantially spherical particles, the average particle size may range from 5 to 1000 μm. Those skilled in the art will be able to select the appropriate particle size and porosity depending on the method used. For example, for large-scale methods, a more porous but rigid support may be preferred for economic reasons, particularly for the capture step, to allow for large-volume processing. In chromatography, process parameters such as column size and shape will influence the selection. In expanded-bed methods, the matrix generally comprises a high-density packing material, preferably a stainless steel packing material. For other methods, other criteria may influence the nature of the matrix.

[0053] The support material can be made from organic or inorganic materials and can be porous or non-porous. In one embodiment, the support material is prepared from natural polymers such as cross-linked carbohydrate materials, e.g., agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate, pectin, starch, etc. Natural polymer supports are easily prepared and are optionally cross-linked according to standard methods, such as inverse suspension gelation (S. Hjerten: Biochim Biophys Acta 79(2), pp. 393-398 (1964)). In a particularly advantageous embodiment, the support material is a type of relatively rigid but porous agarose, which is prepared by methods that enhance its fluidity; see, e.g., U.S. Pat. No. 6,602,990 (Berg). In an alternative embodiment, the support material is prepared from a cross-linked synthetic polymer, such as a synthetic polymer or copolymer of styrene or a styrene derivative, divinylbenzene, acrylamide, acrylic acid esters, methacrylic acid esters, vinyl esters, vinylamides, or the like. Such synthetic polymers are readily prepared and, optionally, cross-linked according to standard methods; see, for example, "Styrene-based polymer supports developed by suspension polymerization" (R. Arshady: Chimica e L'Industria 70(9), pp. 70-75 (1988)). Natural or synthetic polymer support materials are also available from commercial sources, such as Cytiva, Sweden, e.g., in the form of porous particles. In yet another alternative embodiment, the support material is prepared from an inorganic polymer, such as silica. Inorganic porous and non-porous supports are well known in the art and are readily prepared according to standard methods.

[0054] In the method for separating supercoiled pDNA disclosed herein, when the first chromatographic material comprises a support material in the form of particles, the ligand is attached to the support material via a longer linker molecule, also known as an extender. Extenders are well known in the art and are generally used to sterically increase the distance between the ligand and the support. The term "extender" can also refer to a tentacle or flexible arm. For a more detailed description of possible chemical structures, see, for example, U.S. Pat. No. 6,428,707, which is incorporated herein by reference. Briefly, the extender may be in the form of a polymer, such as a homopolymer or copolymer. The hydrophilic polymeric extender may be synthetic, i.e., have a synthetic backbone, or biological, i.e., be a biopolymer with a backbone of natural origin. Typical synthetic polymers are polyvinyl alcohol, polyacrylamide and polymethacrylamide, polyvinyl ether, etc. Typical biopolymers are polysaccharides, such as starch, cellulose, dextran, and agarose.

[0055] A non-limiting example of a particle-based support material for the first chromatographic material is Fractogel™ (Merck Millipore), which consists of synthetic methacrylic acid-based polymer beads with tentacles of linear polymer chains covalently bonded to functional ligands.

[0056] The anion exchange chromatography ligand of the first chromatographic material may be a strong anion exchange chromatography ligand, which may, for example, comprise a quaternary amine group or a quaternary aminoethyl group.

[0057] Alternatively, the anion exchange chromatography ligand of the first chromatographic material may be a weak anion exchange chromatography ligand, which may comprise, for example, a diethylaminoethyl (DEAE) group, a trimethylaminoethyl (TMAE) group, a dimethylaminoethyl (DMAE) group, or a trimethylhydroxypropyl (QA) group.

[0058] The first chromatographic material used in the examples herein is Mustang™ Q (Pall), which comprises a support in the form of a membrane-like structure that allows convection through the first chromatographic material, and a strong anion-exchange ligand bearing a quaternary amine group, which is covalently bound to the support.

[0059] The second chromatographic material comprises a support material having a ligand attached thereto, the support material comprising a nanofiber, a monolith, a membrane-like structure, or a particle.

[0060] The detailed descriptions of nanofibers, monoliths, membranous structures and particles found above with respect to the support material of the first chromatographic material are equally applicable to the nanofibers, monoliths, membranous structures and particles with respect to the support material of the second chromatographic material.

[0061] However, a difference compared to the support of the first chromatographic material is that the support of the second chromatographic material does not necessarily have to be a support that allows convection.

[0062] Therefore, when the support material of the second chromatography material used is a particle, the particle does not need to contain an extender, but it may optionally contain a linker connecting the ligand to the support. That is, the binding of the ligand to the support can be achieved by introducing a linker between the support and the ligand. The binding can be carried out according to any conventional covalent bonding method, such as by using epichlorohydrin; epibromohydrin; allyl glycidyl ether; bisepoxides such as butanediol diglycidyl ether; halogen-substituted aliphatic substances such as dichloropropanol; and divinyl sulfone. Other non-limiting examples of suitable linkers are polyethylene glycols (PEGs) having 2 to 6 carbon atoms, carbohydrates having 3 to 6 carbon atoms, and polyalcohols having 3 to 6 carbon atoms. All of these methods are well known in the art and can be easily performed by those skilled in the art. As further explained in detail above, the linker is optionally an extender.

[0063] As further mentioned above, the second chromatographic material comprises a ligand that binds to pDNA to allow selective separation of supercoiled pDNA from open circular pDNA. Selective separation can be achieved by the ligand of the second chromatographic material either by increasing its binding interaction with supercoiled pDNA compared to its binding interaction with open circular pDNA (meaning that supercoiled pDNA binds more strongly to the ligand of the second chromatographic material than open circular pDNA) or by increasing its binding interaction with open circular pDNA compared to its binding interaction with supercoiled pDNA (meaning that open circular pDNA binds more strongly to the ligand of the second chromatographic material than supercoiled pDNA).

[0064] The ligand of the second chromatographic material may enable salt-promoted affinity binding and / or sulfophilic aromatic adsorptive binding to pDNA.

[0065] The ligand of the second chromatographic material may, for example, comprise a sulfophilic group, preferably a 2-mercaptopyridine group.

[0066] The second chromatographic material used in the examples herein is Capto™ PlasmidSelect (Cytiva, Sweden), which contains a support in the form of porous agarose particles (with a cumulative volume distribution (d50V) median particle size of 36-44 μm) and a sulfophilic aromatic adsorption ligand containing 2-mercaptopyridine groups, which may enhance the binding interaction with supercoiled pDNA compared to the binding interaction with open-circular pDNA.

[0067] Another non-limiting example of a second chromatographic material is PlasmidSelect Xtra (Cytiva, Sweden), which comprises a support in the form of porous agarose particles (median particle size of cumulative volume distribution (d50V) of approximately 34 μm) and a sulfophilic aromatic adsorption ligand containing 2-mercaptopyridine groups.

[0068] The term "surface" is used herein to mean all external surfaces, and in the case of porous supports, includes outer surfaces and pore surfaces.

[0069] The term "eluent" is used in its conventional sense in the art, ie, a buffer of appropriate pH and / or ionic strength to release one or more compounds from the separation matrix.

[0070] The term "eluate" is used in its conventional sense in the art, ie, the portion of a liquid sample that elutes from a separation matrix after the liquid sample has been applied to the separation matrix.

[0071] As described above, in the method for isolating supercoiled pDNA, the liquid sample is derived from a cell culture harvest.

[0072] As used herein, the term "cell culture" refers to a culture of cells or a group of cultured cells, where the cells may be any type of cell, such as bacterial cells, viral cells, fungal cells, insect cells, or mammalian cells. Cell cultures may be unclarified, i.e., contain cells, or may be decellularized, i.e., cultures that contain no or few cells but contain biomolecules released from the cells prior to cell decellularization. Furthermore, unclarified cell cultures may contain whole cells, disrupted cells, cell homogenates, and / or cell lysates.

[0073] The term "cell culture harvest" is used herein to mean a cell culture that has been collected and removed from the container or device in which the cells were cultured.

[0074] As mentioned above, the liquid sample applied to the first chromatographic material in step (a) of the above separation method is derived from a cell culture harvest and has been subjected to a step to remove RNA prior to step (a).

[0075] Those skilled in the art will appreciate that liquid samples derived from cell culture harvests contain many different types of biological compounds, including RNA, that will need to be removed at some point in the purification process.

[0076] As used herein, the term "removing RNA" is intended to mean reducing the amount of RNA present in a liquid sample. In the context of the presently disclosed method, the step of removing RNA is carried out before step (a), since RNA competes with pDNA in the capture step, i.e., binding to the first chromatographic material. Therefore, it is advantageous to remove RNA before step (a) to improve the pDNA binding capacity of the first chromatographic material applied in step (a). Preferably, the removal of RNA before step (a) results in a significant reduction in the amount of RNA in the liquid sample.

[0077] The liquid sample applied to the first chromatographic material in step (a) may thus be a pre-treated liquid sample.

[0078] Therefore, the above-mentioned method may further include step (a1) before step (a), as shown in Figure 2, where step (a1) includes pretreating the liquid sample, and optionally the pretreating step includes subjecting the cell culture harvest to one or more of cell lysis, RNA precipitation in the presence of an RNA precipitant, clarification, and filtration. Preferably, the pretreating step includes at least cell lysis, RNA precipitation in the presence of an RNA precipitant, and clarification. The precipitated RNA may be removed by filtration.

[0079] Various techniques for performing cell lysis are well known in the art. Generally, harvested cells are mixed with an alkaline lysis solution to form a composition of lysed cells, also called a cell lysate. Lysis is then interrupted by the addition of a neutralizing solution to the cell lysate, which can then remove cellular debris, including fragmented cells, host cell proteins, and host cell genomic DNA.

[0080] For example, cell cultures can be subjected to alkaline lysis, neutralization, and / or clarification, as described in International Patent Application No. PCT / EP2022 / 077388. For example, cell cultures can be contacted with an alkaline lysis solution in a disposable container, such as a disposable bag, for alkaline lysis of the cells, where the disposable container includes an integrated mixer and the cell culture is mixed with the alkaline lysis solution. Lysis can be interrupted by the addition of a neutralizing solution to the cell culture.

[0081] Alternatively, collected cells present in a liquid sample (e.g., in the form of a slurry) can be mixed with an alkaline lysis solution by a static mixer. Such static mixers are known. A static mixer useful in this context may be an elongated tubular member including a plurality of septa arranged inside the tubular member to partially obstruct the flow path of the fluid passing through the tubular member. The static mixer thus achieves a turbid flow that provides mixing of the components of the passing fluid. The septa of the static mixer may have a rounded shape. With such a shape, the flow of the fluid containing the cell lysate and the free plasmids is gently redirected, avoiding damage to the plasmids.

[0082] To mix the harvested cell slurry with the alkaline lysis solution, the alkaline lysis solution may be added to a vessel or tube containing the cell slurry through a fluid inlet, and the combined composition may then be delivered (e.g., pumped) to and through a static mixer. For example, the cell slurry may be provided to a junction through one inlet, and the alkaline lysis solution may be provided to the junction through another inlet. The junction may be a Y-joint. The mixture may be pumped through the static mixer at a continuous, constant flow rate.

[0083] After mixing, optionally using a static mixer, the alkaline lysis solution is allowed to remain in contact with the cell slurry for a time sufficient to allow sufficient, preferably complete, cell lysis. The contact time may range from 1 minute to 10 minutes, and may be between 2 and 5 minutes, such as 2 to 3 minutes. The longer the contact time, the greater the risk of partial degradation of the plasmid DNA. Therefore, contact times shorter than 10 minutes are preferred.

[0084] The lysis reaction is terminated by mixing the resulting cell lysate with a neutralizing solution as described above.

[0085] The addition of the neutralizing solution may be to the same disposable vessel used during alkaline lysis, may occur in the disposable vessel, or may be in a separate disposable vessel, if a static mixer is used as described above. The disposable vessel in which the cell lysate contacts the neutralizing solution may include an integral mixer.

[0086] If a static mixer is used to mix the cell slurry and alkaline lysate, a neutralization solution may be added to the cell lysate through a fluid inlet located after (downstream from) the static mixer, and the neutralized cell lysate may then be transferred to a vessel where further processing, such as RNA precipitation, clarification, and / or filtration, may occur. After lysis and neutralization, the cell lysate may optionally be subjected to RNA precipitation using an RNA precipitant. The RNA precipitant may be selected from the group consisting of calcium chloride (CaCl), lithium chloride (LiCl), and tripotassium citrate (C6H5K3O7), and may preferably be calcium chloride.

[0087] After lysis and neutralization, the cell lysate may be subjected to clarification and / or filtration. Those skilled in the art are aware of various methods for clarification and filtration of liquid samples derived from cell culture harvests. For example, the cell lysate may be prepared essentially as described in PCT / EP2022 / 077388: - adding a neutralizing solution to interrupt the lysis, and then optionally transferring the cell lysate to a disposable container, such as a disposable mixing bag; - clarifying the cell lysate in the disposable container by adding a flocculate lifting agent to the disposable container, whereby at least two phases separate in the disposable container, whereby one of the phases is a neutralized and clarified alkaline solution containing the plasmid DNA; - removing the neutralized and clarified alkaline solution from the disposable container; - filtering the neutralized and clarified alkaline solution The solution can be clarified by

[0088] The disposable container in which clarification takes place may include an integrated mixer. The disposable container for the clarification step may be a disposable bag, such as a flexible plastic bag.

[0089] One or more of the process steps described herein can be performed as part of a continuous process, preferably using disposable equipment such as disposable bags, tubing, and mixers. For example, cell lysis using a static mixer as described above and subsequent neutralization can be performed in a continuous process. Advantageously, such a process can be easily controlled by adjusting the installation of one or more pumps that control the flow rates of the cell harvest slurry, lysis solution, and neutralization solution.

[0090] If not continuous, the steps are performed batchwise. For example, cell lysis and neutralization can be performed continuously, followed by batchwise RNA precipitation, clarification, and filtration. However, it is contemplated that all steps can be performed batchwise. Furthermore, whether performed continuously or batchwise, methods for purifying plasmid DNA involving cell lysis using a static mixer as described above, followed by neutralization, RNA precipitation, clarification, and filtration, can be followed by a chromatography step as described herein above, or other chromatography and / or filtration techniques.

[0091] As noted above, the supercoiled pDNA eluted in step (d) has a purity of at least 95%, such as 96%, 97%, 98%, 99%, or 99.5%, without the use of any additional chromatographic materials beyond the first and second chromatographic materials. Thus, the presently disclosed method for isolating supercoiled pDNA achieves a superior degree of purification by employing only two chromatographic steps. No additional chromatographic steps are required to obtain supercoiled pDNA having a purity of at least 95%.

[0092] The above-mentioned method may include a step (c1) between steps (b) and (c), in which step (c1) comprises diluting the liquid sample eluted in step (b) with a diluent in a volume ratio of at least 1:2, such as 1:3, 1:4, or 1:5, of liquid sample to diluent. The diluent comprises a salt. A currently preferred salt is ammonium sulfate ((NH4)2SO4). A currently preferred volume ratio is 1:4 of liquid sample to ammonium sulfate. A currently preferred concentration of ammonium sulfate is 3M. ​​It is contemplated that other salts having properties similar to ammonium sulfate according to the Hofmeister series may be suitable for use in place of ammonium sulfate. The Hofmeister series is a classification of ions in order of their ability to salt out or salt in proteins. The diluent may optionally act as a buffer.

[0093] The above-described method may include a step (d1) between steps (c) and (d), where step (d1) includes eluting the open circular pDNA from the second chromatographic material. Under some processing conditions, the open circular pDNA binds to the second chromatographic material and must be eluted from the second chromatographic material. As further described above, the second chromatographic material allows for selective separation of supercoiled pDNA from open circular pDNA. If the second chromatographic material can increase the binding interaction with supercoiled pDNA compared to the binding interaction with open circular pDNA, the supercoiled pDNA will bind more strongly to the second chromatographic material than the open circular pDNA. This allows the open circular pDNA to be eluted from the second chromatographic material more easily than the supercoiled pDNA. In this case, step (d1) is performed before step (d).

[0094] As further noted above, one advantage of the methods disclosed herein is a significant reduction in production time for downstream processing. Indeed, the methods disclosed herein for isolating supercoiled pDNA have been optimized so that steps (a) through (d) and any intermediate steps can be completed within 5 hours. This is the result of a combination of the features described above. More specifically, the combination of subjecting the liquid sample to an RNA removal step prior to step (a), a first chromatographic material comprising a support material that allows convection, and a second chromatographic material comprising a ligand that allows selective separation of supercoiled pDNA from open circular pDNA allows the above-described steps (a), (b), (c), and (d), as well as any intermediate steps, such as the above-described steps (c1) and (d1), to be completed within 5 hours.

[0095] The above-mentioned time frame of "within 5 hours" required for steps (a) to (d) and any intermediate steps does not include any steps performed before step (a), such as step (a1) described above, nor does it include any steps performed after step (d), such as steps (e1), (e2), and (e3) described below.

[0096] The method disclosed herein for isolating supercoiled pDNA, as shown in FIG. 3, comprises subjecting the supercoiled pDNA eluted in step (d) to the following steps: (e1) concentrating the supercoiled pDNA to a pharmaceutically relevant dose; (e2) replacing the buffer applied in step (d) of the above method with a pharmaceutically acceptable buffer, and / or (e3) Sterilizing the supercoiled pDNA to obtain a composition comprising pharmaceutical-grade supercoiled pDNA, and optionally the composition is a pharmaceutical composition or active pharmaceutical ingredient comprising pharmaceutical-grade supercoiled pDNA.

[0097] Those skilled in the art will understand that the pharmaceutically appropriate dose will depend on various factors, including, but not limited to, the disease or disorder being treated and the weight and condition of the subject being treated with the pharmaceutical composition or active pharmaceutical ingredient.

[0098] Pharmaceutically acceptable buffers are well known in the art and can be readily selected by one of ordinary skill in the art.

[0099] In order for the resulting composition to meet all legal regulations for pharmaceutical compositions and active pharmaceutical ingredients, all three steps e1 to e3 described above must usually be carried out.

[0100] The present disclosure further provides a method for preventing or treating a disease or disorder in a subject, comprising administering to the subject a pharmaceutical composition or active pharmaceutical ingredient comprising pharmaceutical-grade supercoiled pDNA obtained by performing the above-disclosed isolation method comprising one or more of steps e1-e3 (as described in detail above).

[0101] The active pharmaceutical ingredient may be administered in the form of a DNA vaccine, where the disease or disorder to be prevented is caused by infection with a virus such as SARS-CoV-2, Zika, influenza A, yellow fever virus, or human immunodeficiency virus (HIV).

[0102] The present disclosure also provides a method for producing a viral vector, comprising transfecting a cell in vitro with a composition comprising pharmaceutical-grade supercoiled pDNA obtained by carrying out the isolation method disclosed above, comprising one or more of steps e1 to e3, thereby allowing for the production of the viral vector.

[0103] The viral vector may be for use as an active pharmaceutical ingredient. It should be understood that the viral vector may comprise genetic material derived from therapeutically relevant, pharmaceutical-grade supercoiled pDNA. The active pharmaceutical ingredient may be in the form of a viral vector vaccine for use in methods for preventing diseases or disorders caused by infection with a virus. The active pharmaceutical ingredient may be for use in methods of cell therapy, gene therapy, or oncolytic therapy. Oncolytic therapy may include the treatment of cancers such as breast cancer, lung cancer, ovarian cancer, or leukemia. The active pharmaceutical ingredient may be for use in methods for treating or preventing diseases or disorders such as cystic fibrosis, Leber's congenital amaurosis, or spinal muscular atrophy.

[0104] The present disclosure further provides a method comprising using a composition comprising pharmaceutical-grade supercoiled pDNA obtained by performing the above-disclosed separation method comprising one or more of steps e1 to e3 as a template for producing RNA.

[0105] In some cases, the RNA produced in this manner may be for use as an active pharmaceutical ingredient. It should be understood that the RNA may include genetic material derived from therapeutically relevant, pharmaceutical-grade supercoiled pDNA. The active pharmaceutical ingredient may be in the form of an RNA vaccine for use in methods for preventing diseases or disorders caused by infection with viruses such as SARS-CoV-2, Zika, influenza A, human immunodeficiency virus (HIV), or rabies virus. The active pharmaceutical ingredient may be for use in methods of gene therapy, oncolytic therapy, or personalized medicine, or for treating or preventing diseases or disorders such as rare diseases, cystic fibrosis, Leber's congenital amaurosis, or spinal muscular atrophy. Oncolytic therapy may include the treatment of cancers such as breast cancer, lung cancer, ovarian cancer, or leukemia.

[0106] Those skilled in the art will understand that a pharmaceutical composition or active pharmaceutical ingredient must be administered to a subject in a pharmaceutically effective amount or dose to achieve the desired therapeutic effect. The pharmaceutically effective amount and dose depend on various factors, including, but not limited to, the disease or disorder being treated or prevented, and the weight and condition of the subject being treated.

[0107] A device or composition that "comprises" one or more recited components may also include other components not specifically recited. The term "comprising" includes, as a subset, "consisting essentially of," which means that the device or composition has the recited components in the absence of other features or components. Similarly, a method that "comprises" one or more recited steps may also include other steps not specifically recited.

[0108] The singular forms "a" and "an" should be construed to include the plural forms as well. [Example]

[0109] Isolation of supercoiled pDNA from cell culture harvests Methods and Materials E. coli cells (clone DH5α) containing plasmid DNA (pDNA) with a size of 7.3 kbp were harvested by centrifugation of the fermentation broth from a fed-batch process in a 15 L stirred tank reactor. Fermentation conditions included pH = 7.2 ± 0.2, dissolved oxygen (DO) = 30%, and temperature = 37°C. The feed profile was 0.25 h. -1 A specific growth rate of 1000 kJ / ml was maintained. The final targeted OD was 45-50. The process was terminated after 16.5 hours.

[0110] The resulting E. coli cell harvest was subjected to alkaline lysis to disrupt the cells, and the resulting liquid sample was then subjected to neutralization, i.e., the pH was adjusted to about 5.

[0111] RNA was then precipitated by adding CaCl2 to the liquid sample. Because RNA competes with pDNA for binding to the chromatography material in the subsequent capture chromatography step, it is advantageous to remove RNA prior to the capture step to improve pDNA binding capacity to the chromatography material. Figure 4 shows that the addition of CaCl2 as an RNA precipitant significantly reduces the amount of RNA in the liquid sample. Figure 4: Lane (1) 1 M CaCl2, Lane (2) 0.75 M CaCl2, Lane (3) 0.5 M CaCl2, and Lane (4) No CaCl2.

[0112] The liquid sample was then clarified by subjecting it to a deaggregation step followed by a filtration step, thereby removing RNA precipitates and aggregates from the liquid sample. Conditions: Depth filtration (PDP8, Pall): flow rate 100 LMH (L / m 2 / hour), input amount 220L / m 2 Ultrafiltration / Diafiltration (UF / DF): Shear rate 6000 s -1 , transmembrane pressure (TMP) 0.07 MPa (0.7 bar, 10.2 psi), input volume 70 L / m 2 . Dead end filtration (NFF) (NFF filter, Pall): Flow rate 600 L / m 2 / hour), input amount 180L / m 2 .

[0113] The resulting pretreated liquid sample was then subjected to two chromatographic steps, detailed below, which yielded supercoiled pDNA with a purity greater than 95%.

[0114] First Chromatography Step Capture of pDNA, i.e., separation of pDNA from the pretreated liquid sample, by use of a first chromatographic material comprising an anion exchange chromatographic ligand capable of binding pDNA.

[0115] First chromatographic material: Mustang™ Q membrane (Pall) with ligands containing quaternary amine groups. Mustang™ QXT5 (5 mL) membrane capsules were used.

[0116] Separation conditions: Three cycles were run on the Mustang™ Q membrane, each with a liquid sample load of 150 mL (cycle 1), 200 mL (cycle 2), and 200 mL (cycle 3). Input amount 12mg pDNA / membrane volume (MV) Binding in 500 mM NaCl, 50 mM Tris, pH 8.0 Elution in 3M NaCl, 50mM Tris, pH 8.0 Flow rate 5MV / min For increased yield, elution is stopped with UV enhancement for 30 minutes. CIP cleaning (CIP) 1M NaOH Regenerated 1M NaCl, 0.1M NaOH

[0117] Second Chromatography Step Separation of supercoiled pDNA from open circular pDNA by use of a second chromatographic material containing a ligand that binds to pDNA and allows for selective separation of supercoiled pDNA from open circular pDNA.

[0118] Second chromatographic material: Capto™ PlasmidSelect resin (Cytiva, Sweden) with a ligand containing a 2-mercaptopyridine group. The resin was packed into a HiScale™ 16 (20 mL) column, 10 cm bed height.

[0119] Separation conditions: Input amount: 2 mg pDNA / mL resin Eluate diluted 1+4 in 3M (NH4)2SO4 Bonding in 2.25M (NH4)2SO4 Flow rate: equilibration and loading 220 cm / hour, elution 120 cm / hour Elution with a linear gradient against water, 11 column volumes (CV), 55%

[0120] Results and Conclusions First Chromatography Step Figure 5 shows the reproducible, rapid cycling and consistency across different input volumes with the Mustang Q™ membrane. In the chromatograms in Figure 5, the bottom solid line represents cycle 1 (150 mL sample), the middle dashed line represents cycle 2 (200 mL sample), and the top dotted line represents cycle 3 (200 mL sample). The left peak represents elution of pDNA (a mixture of supercoiled and open-circular pDNA), the middle peak represents CIP, and the right peak represents membrane regeneration.

[0121] Second Chromatography Step Figure 6 shows the successful purification of supercoiled pDNA separated from open-circular pDNA using Capto™ PlasmidSelect resin. In the chromatogram in Figure 6, open-circular pDNA elutes first (small peak on the left), followed by supercoiled pDNA (larger peak). At the end of the gradient after the supercoiled pDNA, impurities such as RNA and endotoxin elute. The impurity peak also contains the pDNA process yield loss. The dotted line, designated "Conc B," indicates the salt gradient (decreasing salt concentration) of the elution buffer relative to pure water. After the final ultrafiltration / diafiltration, the purity of the resulting supercoiled pDNA was remarkably high (99.3%). Successful separation of supercoiled pDNA was confirmed by agarose gel electrophoresis (AGE) and capillary gel electrophoresis (CGE) (not shown).

[0122] Table 1 shows the purity of supercoiled (SC) pDNA and the content of open circular (OC) pDNA after each step of the isolation method described in this example.

[0123] [Table 1]

[0124] Table 2 shows the calculation of ammonium sulfate buffer consumption for the 200 L scale method according to the example above ("new method") and compared to the method disclosed in Application Note 28-4094-85 AA (Cytiva) ("prior art method").

[0125] [Table 2]

[0126] Compared to a previously known method for the purification of supercoiled pDNA, which involves three chromatographic steps (Application Note 28-4094-85 AA, Cytiva, Sweden), an 80% reduction in production time for downstream processing and a 50% reduction in ammonium sulfate consumption were achieved.

[0127] It should be understood that the present disclosure is not limited to its exemplary embodiments described above, and that several possible modifications of the present disclosure are possible within the scope of the appended claims.

[0128] (References) TIFF2025508416000004.tif59161

Claims

1. (a) applying a liquid sample containing pDNA to a first chromatographic material comprising (i) an anion exchange chromatographic ligand for binding the pDNA and (ii) a support material that allows for convection flow through the first chromatographic material, the liquid sample being derived from a cell culture harvest and having been subjected to a step of removing RNA prior to step (a); (b) eluting the liquid sample comprising the purified mixture of supercoiled pDNA and open circular pDNA from the first chromatographic material; (c) applying the liquid sample from step (b) to a second chromatographic material that contains a ligand that binds pDNA and allows for the selective separation of supercoiled pDNA from open circular pDNA; (d) eluting the purified supercoiled pDNA from the second chromatographic material. Including, the supercoiled pDNA eluted in step (d) has a purity of at least 95% without the use of any additional chromatographic materials other than said first and second chromatographic materials; A method for isolating supercoiled plasmid DNA (pDNA) from a liquid sample.

2. 2. The method of claim 1, wherein the anion exchange chromatography ligand is a strong anion exchange chromatography ligand, optionally wherein the strong anion exchange chromatography ligand comprises a quaternary amine group or a quaternary aminoethyl group.

3. 2. The method of claim 1, wherein the anion exchange chromatography ligand is a weak anion exchange chromatography ligand, and optionally the weak anion exchange chromatography ligand comprises a diethylaminoethyl (DEAE) group, a trimethylaminoethyl (TMAE) group, a dimethylaminoethyl (DMAE) group, or a trimethylhydroxypropyl (QA) group.

4. 4. The method of claim 1, wherein the ligand of the second chromatographic material allows salt-promoted affinity binding and / or sulfophilic aromatic adsorptive binding to pDNA.

5. 5. The method of claim 4, wherein the ligand of the second chromatographic material comprises a sulfur-philic group.

6. 6. The method of claim 5, wherein the ligand of the second chromatographic material comprises a 2-mercaptopyridine group.

7. 7. The method of any one of claims 1 to 6, wherein the support material of the first chromatography material comprises a material selected from the group consisting of nanofibers, monoliths, membranous structures, and particles having extenders attached thereto.

8. 8. The method of claim 7, wherein the support material of the first chromatographic material comprises electrospun polymeric nanofibers.

9. 8. The method of claim 7, wherein the support material of the first chromatography material comprises a membranous structure comprising a nonwoven web of polymeric nanofibers.

10. 8. The method of claim 7, wherein the support material of the first chromatographic material comprises particles having extenders attached thereto, the particles being substantially spherical particles.

11. 11. The method of any one of claims 1 to 10, wherein the second chromatographic material comprises a support material having a ligand attached thereto, the support material comprising a material selected from the group consisting of nanofibers, monoliths, membranous structures, and particles.

12. 12. The method of claim 11, wherein the support material of the second chromatographic material comprises particles, said particles being substantially spherical particles.

13. 12. The method of claim 11, wherein the support material of the second chromatographic material comprises electrospun polymeric nanofibers.

14. 12. The method of claim 11, wherein the support material of the second chromatography material comprises a membranous structure comprising a nonwoven web of polymeric nanofibers.

15. 15. The method of any one of claims 1 to 14, further comprising a step (a1) prior to step (a), wherein step (a1) comprises pretreating the liquid sample, said pretreating comprising at least one of subjecting the cell culture harvest to cell lysis, precipitating RNA in the presence of an RNA precipitation agent, clarification, and filtration.

16. Step (a1) (a1-a) subjecting the cell culture harvest to cell lysis; (a1-b) precipitating RNA in the presence of an RNA precipitant; and (a1-c) Clarification 16. The method of claim 15, comprising:

17. 17. The method of claim 15 or 16, wherein the pre-treating step comprises subjecting the cell culture harvest to cell lysis by mixing the cell culture harvest with a lysis solution using a static mixer.

18. 20. The method of claim 17, wherein a cell culture harvest is contacted with said lysing solution to form a combined composition, and said combined composition is passed through said static mixer to effect mixing thereof.

19. 19. The method of any one of claims 15 to 18, wherein the pre-treating step comprises precipitating RNA in the presence of an RNA precipitation agent, the RNA precipitation agent being selected from the group consisting of calcium chloride, lithium chloride, and tripotassium citrate, preferably calcium chloride.

20. (I) providing a cell culture harvest comprising pDNA producing cells; (II) contacting the cell culture harvest with a lysis solution to form a lysis composition; (III) mixing the lysis composition using a static mixer to form a cell lysate; (IV) adding a neutralizing solution to the cell lysate; (V) optionally precipitating RNA in the cell lysate by the action of an RNA precipitant. (VI) clarifying the cell lysate; (VII) filtering the cell lysate to obtain a solution containing plasmid DNA (pDNA); (VIII) subjecting the pDNA-containing solution to at least one chromatographic purification step to provide a purified composition comprising pDNA. A method for producing a purified composition comprising pDNA, comprising:

21. 21. The method of claim 20, wherein the at least one chromatographic purification step comprises at least one bind-elute chromatographic step to capture pDNA.

22. 22. The method of claim 20 or 21, wherein the at least one chromatographic purification step comprises applying a solution containing pDNA to a chromatographic material containing a ligand that binds pDNA and allows selective separation of supercoiled pDNA from open circular pDNA, and eluting the purified supercoiled pDNA from a second chromatographic material.

23. 23. The method of any one of claims 20 to 22, wherein the static mixer is an in-line static mixer.

24. 28. The method according to any one of claims 24 to 27, wherein steps (I) to (VIII) are carried out continuously.

25. Step (V) is carried out, and step (VIII) is carried out. (a) applying a solution containing pDNA to a first chromatographic material comprising (i) an anion exchange chromatographic ligand for binding to the pDNA and (ii) a support material that allows for convection flow through the first chromatographic material; (b) eluting the liquid sample comprising the purified mixture of supercoiled pDNA and open circular pDNA from the first chromatographic material; (c) applying the liquid sample from step (VIII-b) to a second chromatographic material that contains a ligand that binds pDNA and allows for the selective separation of supercoiled pDNA from open circular pDNA; (d) eluting the purified supercoiled pDNA from the second chromatographic material. Including, the supercoiled pDNA eluted in step (d) has a purity of at least 95% without the use of any additional chromatographic materials other than said first and second chromatographic materials; 25. The method according to any one of claims 20 to 24.

26. 26. The method of claim 25, wherein the first chromatographic material and / or the second chromatographic material is as defined in any one of claims 2 to 14.

27. 27. The method of any one of claims 1 to 19 or 25 to 26, further comprising step (c1) between step (b) and step (c), step (c1) comprising diluting the liquid sample eluted in step (b) with a diluent in a volume ratio of at least 1:2, such as 1:3, 1:4 or 1:5 liquid sample to diluent, wherein the diluent comprises a salt.

28. 28. The method of claim 27, wherein the diluent comprises ammonium sulfide.

29. 29. The method of any one of claims 1 to 19 or 25 to 28, further comprising step (d1) between step (c) and step (d), wherein step (d1) comprises eluting the open circular pDNA from the second chromatographic material.

30. The supercoiled pDNA eluted in step (d) is subjected to the following steps: (e1) concentrating the supercoiled pDNA to a pharma- ceutically relevant dose; (e2) replacing the buffer applied in step (d) of claim 1 with a pharma- ceutically acceptable buffer; and / or (e3) Sterilizing the supercoiled pDNA thereby obtaining a composition comprising pharmaceutical grade supercoiled pDNA, optionally said composition being a pharmaceutical composition or active pharmaceutical ingredient comprising pharmaceutical grade supercoiled pDNA.

30. The method of any one of claims 1 to 19 or 25 to 29.

31. 31. A method for producing a viral vector comprising the step of transfecting a cell in vitro with a composition comprising pharmaceutical grade supercoiled pDNA obtained by carrying out the method defined in claim 30, thereby allowing the production of the viral vector.

32. 31. A method for producing RNA comprising using a composition comprising pharmaceutical grade supercoiled pDNA obtained by carrying out the method as defined in claim 30 as a template for producing RNA, optionally said RNA for use as an active pharmaceutical ingredient.

33. 31. A method for preventing or treating a disease or disorder in a subject, comprising administering to the subject a pharmaceutical composition or active pharmaceutical ingredient comprising pharmaceutical grade supercoiled pDNA obtained by carrying out the method of claim 30, optionally wherein said active pharmaceutical ingredient is administered in the form of a DNA vaccine, wherein the disease or disorder to be prevented is caused by infection with a virus, such as SARS-CoV-2, Zika, Influenza A, Yellow Fever Virus, or Human Immunodeficiency Virus (HIV).

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