Method for purifying recombinant plasmid DNA

Abstract

Herein is reported a method for obtaining recombinant plasmid DNA (pDNA) from a solution comprising a mixture of different isoforms of the recombinant pDNA as well as other non-pDNA contaminants with a chromatography step with an anion- exchange (AEX) material, wherein a volume of the solution comprising the mixture is applied to the AEX material that comprises an amount of the recombinant pDNA that has been determined by applying the solution comprising the mixture to the AEX material, determining the amount of recombinant pDNA in the flow-through of the AEX material, determining the volume of the applied solution comprising the mixture that results in a fraction of about 10 % of the recombinant pDNA to be in the flow-through to be the volume of the solution comprising the mixture to be applied to the AEX material, whereby the recombinant pDNA is obtained from the AEX material by applying an elution buffer.

Description

[0001] P39516-EP (ASK)

[0002] Method for purifying recombinant plasmid DNA

[0003] The current invention is in the field of plasmid DNA purification. In more detail, herein is reported a novel method for purifying plasmid DNA using an anion exchange chromatography step at an advantageous combination of chaotropic salt concentration in the mobile phase and plasmid DNA loading amount.

[0004] Background of the Invention

[0005] Plasmid DNA (pDNA) may exist in three different conformations: supercoiled pDNA, open-circular pDNA, and linear pDNA. Given the various therapeutic applications of pDNA, including but not limited to, synthetic transient protein production, ex vivo gene therapy, RNA therapeutics, DNA vaccines, DNA antibodies, non-viral gene therapy, and viral vectors, it will be appreciated by those of skill in the art that improved methods of purification or isolation of pDNA are highly desirable.

[0006] Based on the high negative charge of pDNA, anion exchange chromatography is frequently used in methods for purifying or isolating pDNA. The anion exchange material contains positively charged groups. Thus, anion exchange chromatography separates molecules in solution according to their charges using an anion exchange material. Examples of typical anion exchange materials include various types of anion exchange membranes, beads, and resins. The compounds bound to the anion exchange material are eluted by increasing the concentrations of salt included in the mobile phase, commonly with an appropriate buffer solution. Thus, in known anion exchange chromatography methods, the loading of the sample is typically carried out with a buffer having a low salt concentration, and the salt concentration is subsequently increased during the elution step.

[0007] Following the initial binding and elution steps, further steps are required in order to obtain the pDNA in acceptable purity and yield.

[0008] Existing methods of purifying pDNA result in poor selectivity and poor recovery. The known methods are thus unable to provide efficient and cost-effective purification of the pDNA. It is also worth noting that many of these known methods suffer from the disadvantage of using PEG or other additives, which may not be desired in the preparation of plasmid DNA, as they require additional separation, disposal and quality control steps. As these additional steps add costs to the purification process and increase the time necessary for purification, there exists a need for a method for purification or isolation of pDNA that eliminates the need for many of the additional steps during purification, thereby reducing the amount of time required for purification, without compromising the purity or yield of the pDNA.

[0009] As will be appreciated by those of skill in the art, yet another drawback to known processes for the purification of biomolecules (such as pDNA) is the difficulty to apply such methods in large-scale production, particularly large-scale production of pharmaceutical -grade pDNA.

[0010] WO 2020 / 174085 reported methods for the purification or isolation of a biological molecule of interest, such as plasmid DNA (pDNA) involving an anion exchange (AEX) chromatography step.

[0011] Given these limitations, it would be desirable to provide a method of purification or isolation of pDNA that not only provides pDNA with improved purity and in high yield, but that may also be used for large-scale production.

[0012] Summary of the Invention

[0013] Herein is reported a method for isolating or purifying plasmid DNA (pDNA) of interest from a mixture comprising the pDNA of interest and other forms of pDNA, the method comprising contacting the mixture comprising the pDNA of interest with an anion exchange (AEX) material in the presence of a solution comprising a salt, preferably a chaotropic salt, most preferably sodium chloride (NaCl), at a concentration that allows selective binding of the pDNA of interest to the anion exchange material, and eluting the pDNA of interest with an eluent comprising a salt, preferably the same chaotropic salt, at a concentration that provides an eluant containing the purified pDNA of interest, whereby the amount of pDNA applied to the AEX material is based on the binding capacity of the AEX material.

[0014] In certain embodiments, the purified pDNA of interest in the eluant is collected in one or more fractions. The purified pDNA of interest is in certain embodiments subsequently isolated from any one or all of the collected fractions.

[0015] In some embodiments, the chaotropic salt is selected from ammonium chloride, potassium chloride, sodium chloride, magnesium sulfate, magnesium chloride, magnesium nitrate, guanidinium hydrochloride, or mixtures thereof. In one preferred embodiment, the chaotropic salt is sodium chloride. The pDNA to be purified by the method according to the current invention is typically a highly polar / charged biomolecule. It was found that the novel method according to the current invention is particularly suitable for a highly effective purification of pDNA, in certain embodiments of pharmaceutical-grade supercoiled pDNA (scpDNA). The purified pDNA has reduced levels of bacterial genomic DNA, RNA, protein and endotoxins and preferably is free of said compounds.

[0016] In certain embodiments, the present invention includes methods for purifying or isolating supercoiled pDNA with reduced amount of or even free from impurities, including one or more of open-circular pDNA, linear pDNA, endotoxins, RNA, lipopolysaccharides, genomic DNA, proteins, and / or combinations with an AEX chromatography with a loading amount determined according to the current invention. The method according to the current invention is particularly suitable for large-scale isolation or purification of pDNA (in particular supercoiled pDNA), in a limited number of steps, with high purity and good yield. In particular, the purification method according to the current invention can advantageously be used for large-scale production for volumes up to 10,000 liters fermentation working volume.

[0017] In certain embodiments, a sample preparation step precedes the AEX chromatography step. In the sample preparation step, solid components (such as cell debris or other materials insoluble in the buffer system) of the mixture comprising the pDNA of interest are separated by phase separation and / or filtration.

[0018] The method according to the current invention is generally applicable, i.e. it is independent of the pDNA to be processed, the AEX material and the elution conditions.

[0019] In more detail, the current invention encompasses at least the following embodiments

[0020] 1. A method for obtaining recombinant plasmid DNA (pDNA) from a solution comprising a mixture of different isoforms of the recombinant pDNA as well as other non-pDNA contaminants with a chromatography step with an anion- exchange (AEX) material, wherein a volume of the solution is applied to the AEX material that comprises less than the amount of the recombinant pDNA that results in a non-binding of about 10 % of the recombinant pDNA to the AEX material, whereby the recombinant pDNA is obtained from the AEX material by applying an elution buffer. A method for obtaining recombinant plasmid DNA (pDNA) from a solution comprising a mixture of different isoforms of the recombinant pDNA as well as other non-pDNA contaminants with a chromatography step with an anion- exchange (AEX) material, wherein a volume of the solution is applied to the AEX material that comprises less than 110 % of the amount of the recombinant pDNA that can be bound by the AEX material, whereby the recombinant pDNA is obtained from the AEX material by applying an elution buffer. A method for obtaining recombinant plasmid DNA (pDNA) from a solution comprising a mixture of different isoforms of the recombinant pDNA as well as other non-pDNA contaminants with a chromatography step with an anion- exchange (AEX) material, wherein a volume of the solution comprising the mixture is applied to the AEX material that comprises an amount of the recombinant pDNA that has been determined by applying the solution comprising the mixture to the AEX material, determining the amount of recombinant pDNA in the flow-through of the AEX material, determining the volume of the applied solution comprising the mixture that results in a fraction of about 10 % of the recombinant pDNA to be in the flow-through to be the volume of the solution comprising the mixture to be applied to the AEX material, whereby the recombinant pDNA is obtained from the AEX material by applying an elution buffer. The method according to any one of embodiments 1 to 3, wherein the recombinant pDNA is recombinant supercoiled pDNA and the different isoforms are open-circular pDNA and supercoiled pDNA. 5. The method according to any one of embodiments 1 to 4, wherein the recombinant pDNA has a size of 2,000 bps to 20,000 bps.

[0021] 6. The method according to any one of embodiments 1 to 5, wherein the recombinant pDNA has a size of 5,000 bps to 15,000 bps.

[0022] 7. The method according to any one of embodiments 1 to 6, wherein the recombinant pDNA has a size of 7,500 bps to 12,000 bps.

[0023] 8. The method according to any one of embodiments 1 to 7, wherein the recombinant pDNA comprises at least one expression cassette for the expression of a polypeptide or protein of interest.

[0024] 9. The method according to any one of embodiments 1 to 8, wherein the AEX material is a monolithic AEX material.

[0025] 10. The method according to any one of embodiments 1 to 9, wherein the volume of the solution that is applied is about 95 % of said volume.

[0026] 11. The method according to any one of embodiments 1 to 10, wherein the volume of the solution that is applied is about 90 % of said volume.

[0027] 12. The method according to any one of embodiments 1 to 11, wherein the volume of the solution that is applied is about 85 % of said volume.

[0028] 13. The method according to any one of embodiments 1 to 12, wherein the elution buffer comprises a chaotropic salt.

[0029] 14. The method according to any one of embodiments 1 to 13, wherein the chromatography step with the AEX material comprises the following steps: a) equilibrating the AEX material by applying an equilibration buffer comprising NaCl to obtain an equilibrated AEX material; b) adjusting the solution comprising the pDNA to be purified to the buffer conditions of the equilibration buffer to obtain a conditioned pDNA solution; c) applying the conditioned pDNA solution of b) to the equilibrated AEX material of a) to obtain a loaded AEX material; d) washing the loaded AEX material of c) with the equilibration buffer of a) to obtain a washed AEX material; e) in cases wherein the equilibration buffer comprises less than 0.6 M NaCl washing the washed AEX material of e) with a wash buffer comprising about 0.6 M of a chaotropic salt; f) eluting the pDNA from the washed AEX material of d) or e) by applying an elution buffer comprising at least 1 M of the chaotropic salt; and thereby obtaining the recombinant pDNA.

[0030] 15. The method according to any one of embodiments 1 to 14, wherein the obtaining is a purifying or enriching or isolating of the recombinant pDNA from a mixture comprising different isoforms of the pDNA and other contaminants.

[0031] 16. The method according to any one of embodiments 1 to 15, wherein the obtained recombinant pDNA comprises at least a purity of at least 80 % of the recombinant pDNA.

[0032] 17. The method according to any one of embodiments 1 to 16, wherein the obtained recombinant pDNA comprises at least a purity of at least 85 % of the recombinant pDNA.

[0033] 18. The method according to any one of embodiments 1 to 17, wherein the obtained recombinant pDNA comprises a purity of at least 90 % of the recombinant pDNA.

[0034] 19. The method according to any one of embodiments 14 to 18, wherein all buffers have a pH value in the range of and including about pH 7 to about pH 8 and comprise about 50 mM Tris(hydroxymethyl)aminomethane (Tris), about 10 mM EDTA and the chaotropic salt. 0. The method according to any one of embodiments 13 to 19, wherein the chaotropic salt is selected from ammonium chloride, potassium chloride, sodium chloride, magnesium sulfate, magnesium chloride, magnesium nitrate, guanidinium hydrochloride, or mixtures thereof. 21. The method according to any one of embodiments 1 to 20, wherein the elution buffer comprises NaCl.

[0035] 22. The method according to any one of embodiments 14 to 21, wherein

[0036] - the equilibration buffer has a pH value in the range of and including about pH 7 to about pH 8 and comprises about 50 mM Tris, about 10 mM EDTA and between 0.3 M und 0.6 M NaCl,

[0037] - the wash buffer has a pH value in the range of and including about pH 7 to about pH 8 and comprises about 50 mM Tris, about 10 mM EDTA and about 0.6 M NaCl, and

[0038] - the elution buffer has a pH value in the range of and including about pH 7 to about pH 8 and comprises about 50 mM Tris, about 10 mM EDTA and about 1 M NaCl.

[0039] 23. The method according to any one of embodiments 14 to 22, wherein

[0040] - the equilibration buffer has a pH value in the range of and including about pH 7 to about pH 8 and comprises about 50 mM Tris, about 10 mM EDTA and about 0.6 M NaCl;

[0041] - the elution buffer has a pH value in the range of and including about pH 7 to about pH 8 and comprises about 50 mM Tris, about 10 mM EDTA and about 1 M NaCl, and step e) is absent.

[0042] 24. The method according to any one of embodiments 1 to 23, wherein the recombinant pDNA is obtained from the AEX material by applying an elution buffer, collecting the eluant and isolating the recombinant pDNA from the eluant.

[0043] 25. The method according to embodiment 24, wherein the eluant is collected in fractions.

[0044] 26. The method according to any one of embodiments 1 to 25, wherein the mixture comprises supercoiled pDNA, open-circular pDNA, linear pDNA, bacterial genomic DNA, RNA, lipopolysaccharides and endotoxins. 27. The method according to any one of embodiments 1 to 25, wherein the mixture comprises supercoiled pDNA, open-circular pDNA, bacterial genomic DNA, RNA, and endotoxins,

[0045] 28. The method according to any one of embodiments 1 to 27, wherein the obtained recombinant pDNA has reduced levels of the unwanted isoform of the pDNA, bacterial genomic DNA, RNA or endotoxin.

[0046] 29. The method according to any one of embodiments 1 to 28, wherein the obtained recombinant pDNA has reduced levels of the unwanted isoform of the pDNA, bacterial genomic DNA, RNA and endotoxin.

[0047] 30. The method according to any one of embodiments 1 to 29, wherein the solution comprising the mixture is obtained from a bacterium.

[0048] 31. The method according to embodiments 30, wherein the bacterium is E.coli.

[0049] 32. The method according to any one of embodiments 1 to 31, wherein the method further comprises separating solid components from the solution comprising the mixture prior to contacting the solution with the AEX material.

[0050] 33. The method according to any one of embodiments 1 to 32, wherein the solution comprising the mixture has been filtered prior to the application to the AEX material.

[0051] 34. The method according to embodiment 33, wherein the solution has been filed with a depth filter and a sterile filter.

[0052] 35. The method according to any one of embodiments 14 to 34, wherein the solution comprising the mixture has been buffer exchanged to the equilibration buffer.

[0053] 36. The method according to any one of embodiments 1 to 35, wherein the method further comprises the step of precipitating the plasmid DNA from the eluant.

[0054] 37. The method according to embodiment 36, wherein the precipitation is by changing the pH of the eluant or by adding anti-solvents or other additives to the eluant. 38. The method according to any one embodiments 1 to 37, wherein the method further comprises the step of filtering the eluant by tangential -flow filtration to isolate the recombinant pDNA.

[0055] 39. The method according to embodiment 38, wherein the average pore size of the filtration membrane used in the tangential -flow filtration is < 0.45 pm or < 0.2 pm.

[0056] 40. The method according to any one of embodiments 1 to 39, wherein the method further comprises the step of lyophilizing the recombinant pDNA.

[0057] 41. The method of embodiment 40, wherein the lyophilization is in the presence of a carbohydrate.

[0058] 42. The method according to any one of embodiments 1 to 41, wherein the method further comprises one or more of the steps of diluting, concentrating, or buffer exchanging of the recombinant pDNA.

[0059] 43. The method according to any one of embodiments 1 to 42, wherein the method further comprises after the chromatography step with the AEX material a second chromatography step with a hydrophobic interaction material.

[0060] 44. The method according to any one of embodiments 1 to 43, wherein the method comprises prior to the chromatography step with the AEX material the steps of cell harvesting and washing bacterial cells used for the production of the recombinant pDNA, cell lysis, neutralization, and flocculate removal.

[0061] ***

[0062] In addition to the various embodiments depicted and claimed, the disclosed subject matter is also directed to other embodiments having other combinations of the features disclosed or claimed herein. As such, the particular features presented herein can be combined with each other in other manners within the scope of the disclosed subject matter such that the disclosed subject matter includes any suitable combination of the features disclosed herein. The foregoing description of specific embodiments of the disclosed subject matter has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed. Descrintion of the Figures

[0063] Figure 1 Exemplary chromatogram of an overloaded monolithic column of 1 ml column volume with 6 pm channel size and NaCl concentrations in the loading buffer of 0.4 M, 0.5 M and 0.6M.

[0064] Figure 2 Zoom of the peak with the deepening of Figure 1.

[0065] Figure 3 Collecting scheme of the peak of Figure 1.

[0066] Figure 4 Overlay of the analytical HPLC runs of the fractions collected according to Figure 3.

[0067] Figure 5 An exemplary chromatogram obtained with the method according to the invention with a NaCl concentration of 0.4 M and including step el).

[0068] Figure 6 An exemplary chromatogram obtained with the method according to the invention with a NaCl concentration of 0.6 M and without step el).

[0069] Detailed Descrintion of Emhodiments of the Invention

[0070] Herein are reported new and improved methods for the purification or isolation of recombinant plasmid DNA (pDNA). With the method according to the current invention the most advantageous conditions, i.e. providing for the best combination of load amount and purity, for purifying recombinant plasmid DNA on an anion exchange material can be identified. The novel method according to the current invention is particularly suitable for large-scale production of recombinant plasmid DNA and provides for purified pDNA with high quality and good yields.

[0071] The purified plasmid DNA obtained with the method according to the current invention is particularly suitable for applications in areas such as synthetic transient production, ex vivo gene therapy, RNA therapeutics, DNA vaccines, DNA antibodies, non-viral gene therapy, and viral particles. That is, the purified pDNA obtained with the method according to the current invention may be used, for example, in vaccines, viral therapy, gene and cell therapy, production of molecules such as RNA and polypeptides in vivo or in vitro, or in chimeric antigen receptor (CAR) T-cell therapy. When purifying pDNA using anion exchange chromatography, particularly on an industrial or large scale, there are several upstream steps that typically occur prior to loading the solution containing pDNA on the anion exchange material. These steps normally include fed-batch fermentation, cell harvesting and optionally washing, cell lysis, neutralization, RNA removal, flocculate removal by depth filtration, plasmid concentration and diafiltration. The pDNA purification process typically includes the use of anion exchange chromatography (AEX), which includes binding the pDNA on the anion exchange material using a salt having low conductivity, followed by elution of the pDNA with a salt having higher conductivity, and tangential flow filtration with buffer exchange. Optionally the pDNA can be subjected to a further purification step to further enrich supercoiled pDNA, e.g. by PlasmidSelect Xtra® (PSX) chromatography (available, e.g. from GE Healthcare Technology), often followed by a further tangential flow filtration step with a buffer exchange using an end fill buffer. In these known methods for purifying or isolating pDNA using anion exchange chromatography, the elution step is typically achieved by gradually increasing the salt concentration in the elution buffer.

[0072] Unless otherwise defined herein, scientific and technical terms used in connection with the current invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0073] Useful methods and techniques for carrying out the current invention are described in e.g. Ausubel, F.M. (ed.), Current Protocols in Molecular Biology, Volumes I to III (1997); Glover, N.D., and Hames, B.D., ed., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, R.I. (ed.), Animal Cell Culture - a practical approach, IRL Press Limited (1986); Watson, J.D., et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, E.L., From Genes to Clones; N.Y., VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, R.I., Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., N.Y. (1987).

[0074] The use of recombinant DNA technology enables the generation of derivatives of a nucleic acid. Such derivatives can, for example, be modified in individual or several nucleotide positions by substitution, alteration, exchange, deletion or insertion. The modification or derivatization can, for example, be carried out by means of site directed mutagenesis. Such modifications can easily be carried out by a person skilled in the art (see e.g. Sambrook, J., et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, B.D., and Higgins, S.G., Nucleic acid hybridization - a practical approach (1985) IRL Press, Oxford, England).

[0075] DEFINITIONS

[0076] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. As well, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably.

[0077] Unless otherwise defined herein the term “comprising of’ shall include the term “consisting of’.

[0078] The term “about” as used herein in connection with a specific value (e.g. temperature, concentration, time and others) shall refer to a variation of + / - 1 % of the specific value that the term “about” refers to.

[0079] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.

[0080] In more detail, the term "isolated" refers to material, which is substantially or essentially free from components that normally accompany the material as it is found in its native state. Thus, an “isolated plasmid” or “isolated plasmid DNA” does not contain materials normally associated with the plasmid or plasmid DNA in their in situ environment. For example, a nucleic acid or polynucleotide is said to be "isolated" when it is substantially separated from contaminant polynucleotides that correspond or are complementary to genes other than the target genes or that encode polypeptides other than the target gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated polynucleotide.

[0081] The terms "plasmid DNA of interest" and "pDNA of interest" are used synonymously, referring to the pDNA that is to be purified from other components or impurities present in the mixture subjected to the purification method according to the current invention. Impurities include, but are not limited to, host cell proteins, endotoxin, host cell DNA and / or RNA.

[0082] "Isolating" or "purifying" pDNA means enrichment of the pDNA of interest from other components with which the pDNA of interest is initially associated. Extents of desired and / or obtainable purification are provided herein. Preferably, the methods of the invention result in an about five-fold enrichment, preferably an about 10-fold enrichment, preferably an about 20-fold enrichment, preferably an about 50-fold enrichment, preferably an about 100-fold enrichment, preferably an about 200-fold enrichment, preferably an about 500-fold enrichment, preferably an about 1000-fold enrichment. Alternatively, the degree of purification may be expressed as a percentage of the pDNA of interest with respect to another component, or with respect to the resultant preparation. Examples of such percentages are provided herein.

[0083] The term "loading amount" as used herein denotes the absolute amount of pDNA of interest applied to a chromatography column. The term "loading density" as used herein denotes the amount of pDNA of interest that is applied per volume unit of the chromatography material. For example, a loading amount of 1 mg / mL denotes that 1 mg pDNA per 1 mL of chromatography material is applied. This does not take into account other DNA species present in the loading solution.

[0084] The term "selective binding" in the context of the present invention denotes that the pDNA of interest preferably binds to the AEX material, i.e. occupies binding sites on the AEX material, whereas other components in the mixture do bind less or not at all to the AEX material. One of skill in the art will appreciate that in biology (and chemistry) binding, elution, etc., will never be 100% exclusive, but for the purposes of the present invention, "selective binding" means that at least 70%, 80%, 85%, 90%, or even 95% of the pDNA of interest binds to the AEX material whereby less than 90%, 85%, 80%, 75%, 70%, 60% or even 50% of one or more other components ("contaminants") also present in the mixture will not bind to the AEX material upon contact. For the sake of convenience, the contacting of the mixture with the AEX material may also be referred herein as a "loading step" (as commonly used in column chromatography, although the method according to the current invention is of course not limited to column chromatography), and the chaotropic salt solution including the mixture to be purified is also referred as "loading buffer" or "loading solution". The chaotropic salt solution itself may also be referred to herein as "binding buffer". The term “between” in combination with ranges, e.g. concentration or pH ranges, as used herein denotes that the explicitly identified boundaries of the ranges are encompassed in the range, i.e. belong to the range.

[0085] THE METHOD ACCORDING TO THE INVENTION

[0086] The inventors unexpectedly found that it is possible to purify pDNA of interest, such as supercoiled plasmid DNA, in a quick, simple and but not straightforward manner. This is achieved by contacting a mixture comprising the pDNA of interest with an AEX chromatography material under conditions, especially with a loading amount, that result in selective and efficient binding of the pDNA of interest, while other components present in the mixture do not substantially bind or even do not bind at all to the AEX material.

[0087] The current inventors have found that loading a defined amount or volume of the mixture comprising the pDNA of interest on the AEX material advantageously improves the yield and purity that can be obtained. The loaded amount is based on the binding capacity of the chromatography material.

[0088] Determining the loading amount

[0089] The current inventors have found that the loading amount of the AEX chromatography has to be based on the absolute binding capacity of the employed AEX material in order to achieve the best yield and purity of the eluted pDNA of interest.

[0090] That is, the loading amount of the AEX material in the method according to the current invention is determined at the conditions intended to be used in the method according to the current invention by overloading the AEX material, determining the amount of pDNA of interest that has been applied to the column at which 10 % of the loaded amount is not bound to the AEX material, i.e. can be found in the flow- through, and setting the loading amount of the AEX chromatography step in the method according to the current invention to be 85 % of said amount at which 10 % is not bound to the AEX material.

[0091] In more detail, for the determination of the loading amount for the method according to the current invention, the AEX material is in a first step overloaded with the pDNA of interest. This is achieved by continuously applying a solution comprising the pDNA of interest to the column. Without being bound by this theory, it is assumed that thereby it is ensured that once the binding sites of the AEX material are mostly occupied, the pDNA of interest is less likely to bind and is found in the flow-through. As the UV signal determined during said overloading is normally close to or above the detection limit of the UV detector of the employed chromatography equipment, said overloading normally cannot be determined by visual inspection of the UV trace of the chromatogram. In some cases the overloading with or the break-through of the pDNA of interest can be identified by a deepening in the UV absorption recorded during the overloading of the column. This is shown in Figure 1 for a monolithic column of 1 ml column volume with 6 pm channel size and NaCl concentrations in the loading buffer of 0.4 M, 0.5 M and 0.6M, respectively.

[0092] Figure 2 shows a zoom of the peak with the deepening.

[0093] As the break-through of the pDNA of interest cannot be determined for all conditions by visual inspection of the loading peak as already outlined above, the loading peak is collected in fractions and these are analyzed by analytical HPLC for the pDNA of interest.

[0094] A respective collecting scheme is shown in Figure 3 and an overlay of the analytical HPLC runs of the collected fractions is shown in Figure 4.

[0095] The absolute loaded amount is calculated based on the concentration of the pDNA of interest in the loading solution and the applied volume.

[0096] The relative loaded amount is calculated based on the concentration of the pDNA of interest in the loading solution, the applied volume and the volume of the used AEX material.

[0097] The amount of pDNA of interest in the flow-through is expressed as a fraction of the loaded amount as a percentage.

[0098] The following Table 1 shows how this procedure is carried out. The values have been obtained with a chromatography with NaCl concentration of 0.5 M, a monolithic AEX column with 1 mL column volume and the sum of open circular pDNA and supercoiled pDNA as pDNA of interest. Table 1:

[0099] Break-through values different from zero are fitted using a linear regression y=a*x+b. The y-axis represents the break-through in percentage and the x-axis represents the load amount in mg per mL of column volume. Rearranging the equation to x and using 0.1 as y-value delivers the loading amount at 10 % breakthrough. As this value has been determined with overloading the column, the amount is reduced by multiplying with 0.85.

[0100] Applying the above outlined calculation to the values presented in Table 1 delivers a loading amount of 1.41 mg / mL for 10 % break-through and, thus, a loading amount for the method according to the current invention of 1.20 mg / mL.

[0101] Without being bound by this theory it is assumed that by using these overload conditions a displacement of contaminants (like gDNA, RNA) by more strongly binding pDNA of interest occurs.

[0102] The above outlined calculation method can likewise be applied to supercoiled pDNA as pDNA of interest. The following Table 2 shows how this procedure is carried out.

[0103] The values have been obtained with a chromatography with NaCl concentration of 0.5 M, 0.6 M and 0.7 M, a monolithic AEX column with 1 mL column volume and supercoiled pDNA as pDNA of interest.

[0104] Table 2: n.d. = not determined Applying the above outlined calculation to the values presented in Table 2 delivers a loading amount of 1.42 mg / mL, 0.80 mg / mL and 0.57 mg / mL, respectively, for 10 % break-through at a NaCl concentration of 0.5 M, 0.6 M and 0.7 M and, thus, a loading amount for the method according to the current invention of 1.21 mg / mL, 0.68 mg / mL and 0.49 mg / mL. Table 3:

[0105] It has to be pointed out that the binding capacity for supercoiled pDNA of the column is higher than for total pDNA. This is due to a different binding behavior of the two pDNA forms, i.e. supercoiled pDNA binds more strongly to the AEX material than open-circular pDNA and thereby the binding capacity for supercoiled pDNA is higher than the binding capacity for total pDNA.

[0106] These loading amounts were used to perform the pDNA purification according to the method according to the current invention.

[0107] The method according to the current invention comprises the following steps: a) determining the loading amount; b) equilibrating the AEX material by applying an equilibration buffer to the starting conditions to obtain an equilibrated AEX material; c) adjusting the solution comprising the pDNA to be purified to the buffer conditions of the equilibration buffer to obtain a conditioned pDNA solution; d) applying the conditioned pDNA solution of c) to the equilibrated AEX material of b) to obtain a loaded AEX material; e) washing the loaded AEX material of d) with the equilibration buffer of c) to obtain a washed AEX material; el) optionally further washing the washed AEX material with a solution comprising 0.6 M NaCl; the further washing is conducted only in cases wherein the equilibration buffer comprises less than 0.6 M NaCl; f) eluting the pDNA by applying an elution buffer to the washed AEX material to obtain an eluant; g) optionally obtaining the pDNA from the eluant and thereby purified pDNA of interest is obtained. An exemplary chromatogram obtained with the above method with a NaCl concentration of 0.4 M and, thus, including step el) is shown in Figure 5.

[0108] An exemplary chromatogram obtained with the above method with a NaCl concentration of 0.6 M and, thus, without step el) is shown in Figure 6.

[0109] The respective results at NaCl concentrations of 0.3 M to 0.6 M are shown in the following Table 4.

[0110] Table 4: n / a = not applicable. The purified pDNA

[0111] For the chromatography with a NaCl concentration of 0.5 M on a monolithic AEX column with 1 mL column volume some contaminants have been determined. The results are shown in Table 5. Table 5: blq = below limit of quantification; n / a = not applicable

[0112] It can be seen that the supercoiled pDNA concentration is increased, the RNA concentration is reduced by about 17 %, no E.coli host cell proteins are present, and E.coli host cell DNA is reduced by more than 60 %. In the following Table 6 an overview of the results obtained with different NaCl conditions are shown. Table 6: blq = below limit of quantification

[0113] Thus, it can be seen that the conditions of the method, according to the current invention, result in a specific binding of the pDNA of interest to the AEX material, i.e. other components in the solution are less bound to the AEX material. These loading conditions are believed to be rather unusual for AEX chromatography, wherein the sample to be purified is typically contacted with the AEX material under conditions of unspecified loading amount.

[0114] In other words, the specific loading conditions identified by the present inventors allow an effective enrichment of the pDNA of interest already during the loading phase of the AEX chromatography step, with further purification possible during elution with properly selected elution conditions.

[0115] Thus, the AEX chromatography step, when performed under the conditions according to the current invention, allows separating the pDNA of interest from unwanted contaminants in a single AEX purification step. Additionally, the conditions found by the inventors even allow omitting certain preparatory, prepurification steps commonly applied in the purification of the pDNA of interest.

[0116] The present invention generally relates to a method for isolating or purifying pDNA of interest from a mixture, wherein the method comprises contacting the mixture containing the pDNA of interest with an AEX material in the presence of a solution comprising a chaotropic salt at a defined pDNA loading amount and eluting the pDNA of interest from the AEX material with an eluent comprising a chaotropic salt at a concentration higher than in the loading step to provide an eluant containing the purified pDNA of interest. Typically, the fraction or fractions comprising the purified pDNA of interest in the eluant is / are collected, although this is not always necessary (for example in analytical purification runs where the concentration / purity of the molecule of interest can be measured directly in solution, e.g. by UV spectroscopy). Likewise, in most cases, the pDNA of interest is then isolated from any one or all of the collected fractions for further use.

[0117] Isolation does not necessarily mean precipitation and drying, but rather keeping the purified pDNA in a, typically buffered, solution that ensures stability against degradation, e.g. in long term storage. The isolation may thus also include concentration, i.e. removal of part of the solvent to increase the concentration of the pDNA of interest. As is well-known in the art, solutions comprising a pDNA can in many cases be advantageously stored in a frozen solution, although in some instances, solvent may also be removed, e.g. by lyophilization.

[0118] AEX chromatography is typically characterized by loading a solution comprising a mixture of the pDNA of interest to be purified with other components not of interest onto the AEX material in a buffer comprising a salt / buffer with low ionic strength, i.e. at rather low salt concentrations. These conditions allow highly polar and / or charged molecules to bind to the charged groups on the AEX material, while the components having lower polarity / hydrophilicity will bind less or not at all under such conditions. The molecule of interest is then typically eluted from the column by increasing the concentration of the salt / buffer so that the increased number of ions compete with the binding of the molecules still bound to the AEX material. In order to achieve the desired high degree of purity, a gradient increasing the ionic strength of the elution buffer is typically used for purifications via AEX chromatography. In many cases, however, AEX chromatography known in the art does not achieve sufficiently high purity or yield because too many contaminants (especially charged / highly polar contaminants) also bind to the AEX material during the loading phase, and are then eluted at the same time as the molecule of interest.

[0119] The inventors have surprisingly found that it is possible to carry out the loading step (i.e. the initial contact of the mixture with the AEX material) in solutions comprising chaotropic salts at relatively high concentrations (about 0.5 M). Chaotropic agents / salts (disorder-makers) disrupt the structure of water and thereby increase the solubility of non-polar solvent particles, and destabilize solute aggregates. The current inventors have found that using a chaotropic salt in the solution comprising the pDNA of interest when contacted with the AEX material at a sufficiently high concentration in combination with a loading amount determined according to the current invention leads to a selective binding of the pDNA of interest, while reducing or even preventing other components from binding to said AEX material. Thus, in some instances, the "flow-through" of the loading step of a solution comprising a chaotropic salt at a sufficiently high concentration and a loading amount of the pDNA of interest will already contain much, even most or essentially all contaminants from the mixture comprising the pDNA of interest.

[0120] It will be appreciated that the property of being chaotropic is usually ascribed to the specific ion and not a salt (which comprises a counter ion). Thus, it may well be that a salt may comprise an anion that is known to be kosmotropic, while the counter ion is rather known to be chaotropic. Such salts may (or may not) work in selectively binding the target molecule to the AEX material. However, those of skilled in the art will be able to find out whether a given salt will under the conditions of the current invention ensure the selective binding of the pDNA of interest to an AEX material by simply carrying out routine experiments.

[0121] In certain embodiments, elution of the pDNA of interest from the AEX material can then be achieved by an eluant (i.e. a solution or buffer used to detach the components bound to the AEX material) comprising a chaotropic salt in a concentration sufficiently high to elute the pDNA of interest. The elution phase is thus in many embodiments not materially different from conventional AEX chromatography, but due to the higher specificity in binding to the AEX material during the loading phase, the purity of the eluant is often advantageously increased.

[0122] In the methods of the current invention, the biological molecule to be purified is plasmid DNA. Plasmid DNA often exists in different conformations: apart from the "native" super-coiled (SC) conformation, plasmid DNA may also be present in open circular (OC), or even in linear form. Since supercoiled plasmid DNA (scpDNA) typically represents the desired (and commercially relevant) conformation for plasmid DNA, the pDNA of interest in a preferred embodiment of the current invention is scpDNA.

[0123] Since the pDNA of interest to be purified has been obtained by biological process, such as cell culture / fermentation processes, the mixture comprising the pDNA of interest to be purified in certain embodiments comprises supercoiled pDNA, open- circular pDNA, linear pDNA, genomic DNA, RNA, lipopolysaccharides, endotoxins, proteins, or any combination of the foregoing components, in one preferred embodiment supercoiled pDNA, open-circular pDNA, linear pDNA, genomic DNA, and RNA.

[0124] In certain embodiments, the pDNA of interest will comprise a coding part capable of expressing a polypeptide of interest.

[0125] The purification method according to the current invention will generally not depend on the size of the pDNA of interest, i.e. minivectors with only 350 bp as well as large plasmid vectors comprising up to 20 genes (35 kbp), and anything in-between may be effectively purified by the method according to the current invention.

[0126] The method according to the current invention allows the purification of pDNA of interest from other contaminants. Accordingly, in certain embodiments, the resulting purity of the pDNA of interest is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99%.

[0127] In certain embodiments, the method according to the current invention separates supercoiled plasmid DNA from other plasmid DNA forms, such as linear plasmid DNA, open-circular plasmid DNA, and, optionally, other nucleic acid molecules.

[0128] It will be appreciated that the concentration of the chaotropic salt allowing selective binding of the pDNA of interest to the AEX material during the loading step may depend on the size of the pDNA and the chosen AEX material. In certain, highly selective binding of the target molecule has been achieved with salt concentrations of about 0.3 M or higher, of about 0.4 M or higher, or of about 0.5 M or higher. In certain embodiments, the salt concentration of the solution allowing the selective binding of the target molecule is between about 0.3 M and about 0.7 M, or between about 0.4 M and about 0.65 M, or between about 0.5 M and about 0.6 M.

[0129] Chaotropic salts at the above concentrations are employed for the loading step of the present purification method. In any event, the best concentration of said salt for the loading step is dependent on the size of the pDNA of interest and the AEX material, but can be easily determined by one of skill in the art through routine experiments (see examples). Once a suitable concentration has been determined, it may be useful for consistent and reproducible binding to equilibrate the AEX material with a buffer comprising the chaotropic salt at the same concentration as in the loading solution comprising the pDNA of interest to be purified. Other factors besides size of the pDNA of interest and the AEX material may exert an influence on the binding behavior of the components in the mixture. One of these factors is the pH of the solution. For biomolecules, the available pH range for purifications is naturally rather limited. In order to avoid pH conditions that may negatively affect the stability of the target molecule (e.g. by promoting hydrolysis of the biomolecule), pH values during the loading and elution of the biomolecule of interest typically range between pH 2 and pH 11 , although pH values closer to neutral will generally be preferred, especially for biomolecules that are sensitive to degradation upon acidic or alkaline conditions. For example, in some embodiments, the pH of the solution comprising the chaotropic salt contacted with the AEX material (i.e. the "loading buffer") is between pH 4 and pH 9, or between pH 5 and pH 8.5, or between pH 6 and pH 8, or between pH 6.0 and pH 8.0, e.g. around pH 7.0. Those of skill in the art will appreciate that optimal pH conditions may be determined by one of skill in the art by routine experiments.

[0130] A change of the pH value of the mobile phase, for example during the elution step, may also change the binding behavior / strength of the compounds bound to the AEX material, and may therefore be used in certain embodiments for the controlled and selective release of contaminants from the AEX material. This is especially useful, as the binding of pDNA does not change within a wide pH range (as low as pH 2).

[0131] With regard to the conditions selected for elution of the pDNA of interest, the concentration of the chaotropic salt that provides an eluant containing the purified biological molecule of interest is typically between about 0.5 M and about 4.0 M, between about 0.5 M and about 3.0 M, between about 0.5 M and about 2.0 M, or between about 0.5 M and about 1.5 M, in one preferred embodiment between about 0.75 M and about 1.25 M, preferably about 1.0 M. As is well known in the art, the elution step may include a gradient elution by varying the concentration of the (preferably chaotropic) salt in the eluent, which typically involves increasing (continuously or stepwise) the concentration of the chaotropic salt in the eluent.

[0132] In some embodiments, the chaotropic salt for the loading or the elution step or the loading and the elution step is selected from the group consisting of: ammonium chloride, potassium chloride, sodium chloride, magnesium sulfate, magnesium chloride, magnesium nitrate, guanidinium hydrochloride, and mixtures thereof. A particularly preferred chaotropic salt, especially for the purification of pDNA, is sodium chloride. In these embodiments, the concentration of chaotropic salt, e.g., sodium chloride is typically at least about 0.5M, and may in some instances range from 0.5 M to about 2.0 M or from about 0.5 M to about 1.0 M.

[0133] Likewise as in the case of the chaotropic salt contained in the loading buffer, the chaotropic salt and its optimal concentration for the elution of the pDNA of interest depends on the size of the pDNA of interest and the specific AEX material chosen for the purification. Such optimal conditions can easily be determined by one of skill in the art through routine experimentation (see Examples). Since the AEX material can typically be reused for another purification run, it may be useful to add a high salt concentration elution at the end (e.g. with about 2.0 to 4.0 M NaCl) which will strip essentially any bound material from the AEX material.

[0134] Any available AEX materials may be used in the method according to the current invention. For example, the method can be applied with weak and with strong AEX materials, at high and low ligand density, with different bead chemistries and / or linkers. Suitable AEX materials are for example available as an anion exchange membrane, an anion exchange resin, a three-dimensional microporous hydrogel structure, a packed bed of superporous beads, macroporous beads, a monolith, agarose beads, cross-linked agarose, silica beads, large pore gels, methacrylate-based beads, polystyrene-based beads, cellulose-based beads, dextran-based beads, bisacrylamide-based beads, polyvinylether-based beads, ceramic-based beads, or polymer-based beads. In one preferred embodiment, the AEX material is a monolith. Examples of commercially available AEX materials include, among others, Sartobind Q®, Mustang Q®, Mustang E®, Poros XQ®, Poros HQ®, Nuvia Q®, Capto Q®, Bakerbond PolyQuat® , DEAE Sepharose®, NH2-750F®, Q Sepharose®, Giga Cap® Q-650M, Fractogel® EMD COO, NatriFlo® HD-Q, and 3M™ Emphaze™ AEX Hybrid Purifier (all registered trademark names).

[0135] Particularly for larger, industrial scale purifications, the method may be carried out using AEX membrane chromatography or resin bed chromatography.

[0136] It has to be pointed out that the method according to the current invention is independent of the kind and form of the AEX material. That is, the method according to the current invention does not require the AEX material to be in a specific form, i.e. other AEX materials than those used in the examples provided herein, i.e. in whatever form, such as the other alternatives mentioned above, may also be employed as the AEX material in the purification method according to the current invention. The method according to the current invention is carried out in the absence of any organic solvents, detergents, glycols, hexamine cobalt, spermidine, and / or polyvinylpyrrolidone, thereby no longer necessitating removal of such agents before providing the final pDNA in isolated form.

[0137] While the current invention is directed to an AEX purification step, it will be appreciated that a purification method for a pDNA of interest will typically include further method steps, including steps carried out prior to the AEX chromatography step, but also, optionally, additional steps carried out subsequent to the AEX chromatography step.

[0138] As outlined above, pDNA is obtained from cells grown in a so-called cell culture. In most cases, the pDNA of interest needs to be released from the bacterial cells used to produce the pDNA. This is normally done by destroying the cells via cell lysis, which may be accomplished by physical and / or chemical means, as is well known in the art. Cell lysis will typically yield the pDNA of interest together with a large number of host cell derived water-soluble and insoluble contaminants, such as cell membranes, cell organelles, genomic DNA, RNA and host cell proteins. Thus, a purification of a target molecule will typically require removal of these contaminants, where in particular the solid (i.e. non-soluble) contaminants are removed prior to the AEX chromatography step, typically by mechanical means.

[0139] Thus, in certain embodiments, the method according to the current invention further comprises separating solid components from the mixture comprising the pDNA of interest prior to contacting the mixture with an AEX material. The removal of solid components from the mixture to be purified is in certain embodiments done by filtration or phase separation. In certain embodiments, this step achieves removal of solid components via a two-phase separation, such as a solid-liquid phase separation, or via depth filtration.

[0140] In certain embodiments, a depth filtration is applied. The additional filtration step ensures removal of remaining solid particles in the liquid part of the mixture. Depth filtration devices are commercially available, for example under the name Clarisolve® depth filter from Merck-Millipore.

[0141] The AEX chromatography step of the method according to the current invention comprises in certain embodiments further steps after elution of the purified pDNA of interest. For example, in certain embodiments, the method further comprises the step of precipitating the plasmid DNA of interest from the eluant. Precipitation may be achieved by a variety of methods generally known to the art, such as changing the pH of the eluant fractions comprising the target molecule, or adding anti-solvents or other additives to the eluant, thereby causing the pDNA of interest to precipitate from the eluant.

[0142] After precipitation or as an alternative thereto, the method according to the current invention in certain embodiments further comprise s the step of filtering the eluant by tangential-flow filtration to isolate the purified pDNA of interest. For example, in some cases, the average pore size of the filtration membrane used in the tangential- flow filtration step will be < 0.45 pm or even < 0.2 pm.

[0143] In certain embodiments, the method according to the current invention comprises the step of lyophilizing the purified pDNA of interest, optionally in the presence of a carbohydrate that may stabilize the pDNA in lyophilized form during storage. Many mono- or disaccharides can be used for said purpose and are generally known in the art.

[0144] In certain embodiments, the method according to the current invention further includes one or more of the steps of dilution, concentration, or buffer exchange of the fraction(s) comprising the purified pDNA of interest.

[0145] As discussed above, in the AEX chromatography step a selective binding of the pDNA of interest to the AEX material is achieved. That is, a substantial part or / and fraction of unwanted components that are present in the starting mixture, i.e. of the contaminants, will not substantially bind to the AEX material under the loading conditions according to the current invention. For example, for purification of pDNA from a bacterial cell culture, the flow-through may comprise RNA, genomic DNA, proteins, cellular fractions, or combinations thereof. In certain embodiments, the flow-through will comprise a substantially part, i.e. at least 80%, at least 90%, or even at least 95%, of RNA that was present in the mixture contacted with the AEX material.

[0146] The method according to the current invention may comprise a washing step. Thus, in certain embodiments, the method according to the current invention further comprises the step of washing the AEX material with a washing buffer solution after the application of the solution comprising the pDNA of interest to be purified and prior to the elution of the pDNA of interest. In certain embodiments, washing may be with clean "loading" buffer (i.e. comprising a chaotropic salt but not the pDNA of interest). In certain embodiments, the washing buffer solution comprises the chaotropic salt at a concentration lower than the concentration required for the elution of the pDNA of interest.

[0147] The chaotropic salt for the washing buffer is preferably selected from the group consisting of: ammonium chloride, potassium chloride, sodium chloride, magnesium sulfate, magnesium chloride, magnesium nitrate, and mixtures thereof. In particular, when the chaotropic salt has no buffer capacity, the washing buffer may further comprise a suitable buffer substance to maintain the pH at the desired level. For simplification, the washing buffer will in many cases comprise the same chaotropic salt as in the loading buffer or / and the eluent.

[0148] When the purification of the pDNA of interest must yield the product at a very high level of purity, the method according to the current invention further comprises an additional purification step, which follows the step of eluting and optionally isolating the purified pDNA of interest from the AEX chromatography step. In certain embodiments, the second purification step is a hydrophobic interaction chromatography or involves the use of a thiophilic aromatic adsorption chromatography medium. An exemplary latter material is available for example from GE Healthcare Life Sciences under the trade name PlasmidSelect Xtra®, or in short "PSX". This particular resin is known to have excellent selectivity to separate supercoiled plasmid DNA from open circular and / or linear plasmid DNA, and may therefore be used for the final enrichment of the super-coiled conformation of pharmaceutical-grade DNA.

[0149] In certain embodiments, the method according to the current invention further comprises after purification and isolation of the pDNA of interest the step of using said pDNA of interest for the expression of a polypeptide encoded by said pDNA of interest. The polypeptide produced via the pDNA of interest can either be harvested and, optionally, purified or be part of a complex molecule, such as an adeno- associated virus (AAV). Finally, the method according to the current invention may include the formulation of the polypeptide or the AAV (obtained via expression of the purified plasmid DNA) into a pharmaceutical composition that may optionally comprise one or more pharmaceutically acceptable excipients.

[0150] Alternatively, when the pDNA is intended for pharmaceutical use directly, the method according to the current invention includes the formulation of the nucleic acid into a pharmaceutical composition, which may again optionally comprise one or more pharmaceutically acceptable excipients. Plasmid DNA purified by the method of the present invention may be used as DNA-based vaccine or be used as a "prodrug" wherein the plasmid will then involve the cell's transcription and translation apparatus to biosynthesize the therapeutic entity in situ.

[0151] In certain embodiments, the method according to the current invention will further comprise the use of the purified pDNA of interest for the production of RNA, including RNA-based drugs, for the production of shorter oligonucleotides such as siRNA or aptamers (short single-stranded nucleic acid segments (typically 20-60 nucleotides), or for the production of DNAzymes (ribozyme analogs with an RNA backbone replaced by DNA motifs that confer improved biological stability).

[0152] In certain embodiments, the method according to the invention comprises prior to the AEX chromatography step, the steps of cell harvesting and washing, cell lysis, neutralization, and flocculate removal prior to contacting the solution comprising the pDNA of interest with the AEX material.

[0153] The conditions identified for the AEX loading step in the method according to the current invention allows for selective binding of pDNA to the AEX material. Thus, RNA, which is typically also bound to the AEX material under standard, low salt conditions, does not or not substantially bind to the AEX material under the loading conditions according to the current invention and can, thus, be almost quantitatively removed from the pDNA of interest in a single step.

[0154] The method according to the current invention, thus, has the great advantage that RNA does not need to be removed by a calcium chloride (CaC12) precipitation step. Avoiding additional purification steps are of course beneficial in terms of yield, costs and duration of the overall purification method. Accordingly, the method according to the current invention in certain embodiments does not comprise a calcium chloride (CaC12) precipitation step.

[0155] The same is true for proteins, which also do not bind to the AEX material under the conditions identified herein. Since open-circular pDNA, linear pDNA and genomic DNA all have different interaction strength with AEX ligands compared to supercoiled plasmid DNA, their levels can be greatly reduced during the AEX chromatography step according to the current invention. Accordingly, the method according to the current invention allows for the simple, quick, highly effective and economic purification of pDNA of interest involving essentially only a single chromatographic step involving materials having AEX ligands. Finally, the method described herein allows scaling it for large production plants.

[0156] ***

[0157] All publications, patents, and patent applications cited herein are hereby incorporated by reference herein in their entirety for all purposes to the same extent as if each individual publication, patent, and patent application were specifically and individually indicated to be so incorporated by reference. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.

[0158] ***

[0159] The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0160] That is, although the disclosed teachings have been described with reference to various applications, methods, and compositions, it will be appreciated that various changes and modifications can be made without departing from the teachings herein and the claimed invention below. The examples are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings presented herein. While the present teachings have been described in terms of these exemplary embodiments, the skilled artisan will readily understand that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the current teachings.

[0161] Examnles

[0162] General Method Description

[0163] The experiments were conducted using an Akta Avant 25 system with a monolithic column (CIMultus DEAE (6 pm), column volume: 1 mL) and a 10 kbp plasmid. The system was equilibrated with a buffer adapted to the salt (NaCl) concentration of the load (50 mM Tris, 10 mM EDTA, xM NaCl, pH 7). To determine the dynamic binding capacity, 4 mg of plasmid were applied whereby the binding capacity of the column was exceeded. The flow-through was collected in 0.5 CV fractions. Subsequently, a wash step with the equilibration buffer was performed. For conditions with a salt content < 0.6 M NaCl, a second wash step was conducted (50 mM Tris, 10 mM EDTA, 0.6 M NaCl, pH 7). For elution of the bound plasmid DNA, the salt (NaCl) concentration was increased in a single step to 1 M (50 mM Tris, 10 mM EDTA, 1 M NaCl, pH 7). The eluate was collected by fractionation.

[0164] The content of supercoiled DNA isoform in the flow-through fractions was determined using an HPLC method and an analytical monolithic anion exchange column (CIMac™ pDNA-column (BIA Separations) with a pore size of 6 pm). The breakthrough of 10% supercoiled pDNA was determined by calculating its recovery in the individual fractions compared to the load material. The loading amount for the chromatographic material was set to 85% of the conditions at which 10% breakthrough is reached for each salt concentration, as well as the best loading salt conditions for plasmid isoform separation.

[0165] Load Preparation

[0166] Plasmid-producing E. coli cells were lysed using an alkaline cell lysis method adapted from Birnboim and Doly (1979). The lysate was filtered for clarification and concentrated via tangential flow filtration to a plasmid concentration of 1.2 mg / ml in NaCl-free buffer (50 mM Tris, 10 mM EDTA, pH 7). To investigate pDNA binding to the anion exchange material at different salt (NaCl) concentrations, the concentrated plasmid solution was adjusted to the target salt concentrations (0.3M, 0.4M, 0.5M, 0.55M, 0.6M, 0.65M, 0.7M, 0.75M NaCl) by first performing a buffer exchange to a low salt buffer (50 mM Tris, 10 mM EDTA, pH 7) and by second adjusting the NaCl concentration to the target value using a high salt buffer (50 mM Tris, 10 mM EDTA, 1.5 M NaCl, pH 7). The correct buffer condition adjustment was confirmed by determining the conductivity and comparing with a reference buffer. The pDNA concentration and the supercoiled percentage in each of the eight resulting loads were determined. The conditioned loads were stored at 2-8°C and were equilibrated to room temperature before use.

[0167] Chromatography Procedure

[0168] The experiments were conducted using an Akta Avant 25 system with a monolithic column (CIMultus DEAE (6 pm), Cat. no.: 311.5114-6, channel size: 6 pm, column volume: 1 mL) and a 10.6 kbp plasmid. Experiments were performed in duplicates. The system was equilibrated with 30 CV of a buffer adapted to the salt (NaCl) concentration of the load (50 mM Tris, 10 mM EDTA, xM NaCl, pH 7). A load with a total of 4 mg plasmid was applied thereafter to the column. The flow-through of the column was collected in 0.5 CV fractions. Subsequently, a wash step (20 CV) with the equilibration buffer was performed. For conditions < 0.6 M NaCl, a second wash step (20 CV, 50 mM Tris, 10 mM EDTA, 0.6 M NaCl, pH 7) was added. For elution, the salt concentration was increased in a single step to 1 M (20 CV, 50 mM Tris, 10 mM EDTA, 1 M NaCl, and pH 7). The eluate was collected in fractions starting when the UV signal exceeds 250 mAU / cm and stopping when the UV signal drops again below 250 mAU / cm.

[0169] Table 7: Chromatographic method for separating plasmid DNA isoforms. Loading Amount Determination

[0170] The loading amount or likewise dynamic binding capacity of the chromatographic material was determined by overloading the chromatographic material with pDNA to determine the break-through of pDNA. The determination of the loading amount shall ensure maximum product binding while maintaining minimum product loss. Without being bound by this theory, it is assumed that during the loading process, more binding sites will become occupied by pDNA meaning that less free binding sites remain. Therefore, pDNA binding, specifically the two isoforms open circular and supercoiled, is reduced, which can be determined by an increase in the UV260 signal (break-through curve) and pDNA detection in the flow-through fractions. To determine the loading amount according to the current invention, the flow- through of the loading step was collected in 0.5 CV fractions and pDNA content was quantified using analytical HPLC. Fractions near the visually detectable breakthrough, or when not visually detectable all fractions, were analyzed for their pDNA content and concomitantly the ratio of the two isoforms. The break-through of 10% total or supercoiled pDNA was determined by calculating its amount in the individual fractions and set in relation to the amount applied in the starting material. The total load volume up to each fraction was multiplied by the measured concentration of total pDNA or supercoiled isoform, respectively. This loading amount was then normalized by dividing by the column volume. The breakthrough was calculated using equation (1).

[0171] Analytical HPLC

[0172] The concentration of pDNA and the percentage of isoforms in samples was determined using a high-performance liquid chromatography (HPLC) system (Ultimate 3000, Thermo Fisher Scientific). A CIMac™ pDNA-column (BIA Separations) with a pore size of 6 pm was used. As mobile phase Buffer A (50 mM Tris, 10 mM EDTA, pH 8) and Buffer B (50 mM Tris, 10 mM EDTA, 1.5 M NaCl, pH 8) were used. The UV signal was continuously monitored and recorded at a wavelength of 260 nm. The volumetric flow rate was set to 1 mL / min. Column equilibration was carried out with 40% Buffer B. Elution was performed using a gradient from 40 % Buffer B to 100 % Buffer B, as shown in Table 8.

[0173] Table 8: HPLC method for the measurement of pDNA concentration. Samples were diluted from 1 :5 to 1 : 100 with Buffer A, depending on the initially expected pDNA concentration. Injected sample volumes ranged from 2.5 pL to 100 pL.

[0174] The plasmid amount in the analyzed sample was quantified using a calibration curve generated with standards of purified plasmid. The linear range covers pDNA amounts from 0 pg to 0.5 pg per sample. The HPLC method separates open circular pDNA from supercoiled pDNA. Both isoforms are eluted as distinct peaks.

[0175] If the peak area determined from the HPLC chromatogram is within the calibration curve, the chromatogram can be evaluated, and the total pDNA or supercoiled pDNA content in the sample can be calculated using the calibration curve. Taking into account the injection volume and pre-dilution, the concentration of the sample can be calculated. To specifically evaluate the two plasmid isoforms, both peaks are separately integrated. This approach was used to determine the pDNA concentration of all samples as well as their supercoiled content.

Claims

AMENDED CLAIMS received by the International Bureau on 05 January 2026 (05.01.2026)Patent Claims1. A method for obtaining recombinant plasmid DNA (pDNA) from a solution comprising a mixture of different isoforms of the recombinant pDNA as well as other non-pDNA contaminants with a chromatography step with an anion-5 exchange (AEX) material, wherein the AEX material is a monolithic AEX material, wherein a volume of the solution comprising the mixture is applied to the AEX material that comprises an amount of the recombinant pDNA that has been10 determined by- applying the solution comprising the mixture to the AEX material,- determining the amount of recombinant pDNA in the flow-through of the AEX material,- determining the volume of the applied solution comprising the mixture15 that results in a fraction of about 10 % of the recombinant pDNA to be in the flow-through to be the volume of the solution comprising the mixture to be applied to the AEX material, whereby the recombinant pDNA is obtained from the AEX material by applying an elution buffer.20 2. The method according to claim 1, wherein the recombinant pDNA is recombinant supercoiled pDNA and the different isoforms are open-circular pDNA and supercoiled pDNA.

3. The method according to any one of claims 1 to 2, wherein the recombinant pDNA has a size of 5,000 bps to 15,000 bps.

254. The method according to any one of claims 1 to 3, wherein the volume of the solution that is applied is about 85 % of said volume.

5. The method according to any one of claims 1 to 4, wherein the elution buffer comprises a chaotropic salt.AMENDED SHEET (ARTICLE 19)6. The method according to any one of claims 1 to 5, wherein the chromatography step with the AEX material comprises the following steps: a) equilibrating the AEX material by applying an equilibration buffer compnsing NaCl to obtain an equilibrated AEX matenal;5 b) adjusting the solution comprising the pDNA to be purified to the buffer conditions of the equilibration buffer to obtain a conditioned pDNA solution; c) applying the conditioned pDNA solution of b) to the equilibrated AEX material of a) to obtain a loaded AEX material;10 d) washing the loaded AEX material of c) with the equilibration buffer of a) to obtain a washed AEX material; e) in cases wherein the equilibration buffer comprises less than 0.6 M NaCl washing the washed AEX material of e) with a wash buffer comprising about 0.6 M of a chaotropic salt;15 f) eluting the pDNA from the washed AEX material of d) or e) by applying an elution buffer comprising at least 1 M of the chaotropic salt; and thereby obtaining the recombinant pDNA.

7. The method according to any one of claims 1 to 6, wherein the obtained20 recombinant pDNA comprises at least a purity of at least 80 % of the recombinant pDNA.

8. The method according to any one of claims 1 to 7, wherein the elution buffer comprises NaCl.

9. The method according to any one of claims 6 to 8, wherein25 the equilibration buffer has a pH value in the range of and including about pH 7 to about pH 8 and comprises about 50 mM Tris, about 10 mM EDTA and between 0.3 M und 0.6 M NaCl,AMENDED SHEET (ARTICLE 19)the wash buffer has a pH value in the range of an including about pH 7 to about pH 8 and comprises about 50 mM Tris, about 10 mM EDTA and about 0.6 M NaCl, and the elution buffer has a pH value in the range of and including about5 pH 7 to about pH 8 and comprises about 50 mM Tns, about 10 mM EDTA and about 1 M NaCl.

10. The method according to any one of claims 1 to 9, wherein the obtained recombinant pDNA has reduced levels of the unwanted isoform of the pDNA. bacterial genomic DNA, RNA and endotoxin.10 11. The method according to any one of claims 1 to 10, wherein the solution comprising the mixture is obtained from E.coli.

12. The method according to any one of claims 1 to 11, wherein the method comprises prior to the chromatography step with the AEX material the steps of cell harvesting and washing bacterial cells used for the production of the15 recombinant pDNA, cell lysis, neutralization, and flocculate removal.

13. The method according to any one of claims 1 to 12, wherein the method further comprises one or more of the steps of diluting, concentrating, or buffer exchanging of the obtained recombinant pDNA.

14. The method according to any one of claims 1 to 13, wherein the method further20 comprises after the chromatography step with the AEX material a second chromatography step with a hydrophobic interaction material.AMENDED SHEET (ARTICLE 19)[0001]STATEMENT UNDER ARTICLE 19(1) PCT[0002]By the present amendment, the Applicant has consolidated original claim 4 into independent claim 1.[0003]The introduction of the technical features of original claim 4, i.e. the monolithic AEX column into claim 1 serves to further define the invention and distinguish the claimed subject matter from the prior art cited in the International Search Report, specifically documents DI and D2. These features provide a specific technical effect, which the Applicant believes addresses the concerns raised in the Written Opinion.

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