Method for purifying plasmid DNA

Abstract

Herein is reported a method for producing a recombinant plasmid DNA solution comprising the step of passing a crude E.coli lysate comprising recombinant plasmid DNA through a positively charged filter comprising silica, cellulose and a polymer resin to obtain a filtered recombinant plasmid DNA solution.

Description

[0001] Method For Purifying Plasmid DNA

[0002] The current invention is in the field of plasmid DNA production. In more detail, herein is reported a method for the purification of plasmid DNA recombinantly produced in bacterial cells, such as E.coli.

[0003] Background

[0004] A key step for any application in which nucleic acids are used, e.g. for introduction into an organism, is the need to produce highly purified nucleic acids. Such purified nucleic acids must meet quality standards of safety, potency and efficacy.

[0005] In addition, it is desirable to have a scalable process that can be used to produce multi-gram quantities of DNA.

[0006] Plasmids are self-replicating genetic elements that reside and multiply in host bacteria. Generally, all molecular genetic methods involving the manipulation of specific DNA fragments utilize plasmid DNA to produce large amounts of the specific DNA fragment (or protein / RNA derived from said fragment).

[0007] The choice of bacterial hosts, or sources of the plasmids, generally reflects a historical perspective. Stanley Cohen and Herb Bayer chose Escherichia coli (E. coli) strain K-12 for their groundbreaking molecular genetic experiments in the early 1970s because it was easy to grow and amenable to metabolic studies. These same properties also made E. coli K-12 the primary microorganism for bacterial geneticists to study. Molecular geneticists now use this same strain of E. coli for routine procedures because it turned out to be an extremely good host for a variety of molecular genetic applications.

[0008] Among other techniques, currently available methods for separation and purification of plasmid DNA utilize ion exchange chromatography (Duarte et al., Journal of Chromatography A, 606 (1998), 31-45) and size exclusion chromatography (Prazeres, D. M., Biotechnology Techniques Vol. 1, No. 6, June 1997, p 417-420), coupled with the use of additives such as polyethylene glycol (PEG), detergents, and other components such as hexamine cobalt, spermidine, and polyvinylpyrollidone (PVP). Additional methods of separating DNA from contaminants rely on size-exclusion chromatography, which involves separation of the nucleic acids from endotoxins and other contaminants based on the small difference in size. These methods are generally acceptable, but may be unable to provide an efficient and cost effective separation of nucleic acids (e.g., DNA, including supercoiled and / or nicked (or relaxed)) at the desired level of purity.

[0009] Also, plasmid DNA preparations, which are produced from bacterial preparations and often contain a mixture of relaxed and supercoiled plasmid DNA, frequently require endotoxin removal, as required by the FDA, as endotoxins produced by many bacterial hosts are known to cause inflammatory reactions, such as fever or sepsis, or in some cases death, in the host receiving the plasmid DNA. These endotoxins are generally lipopolysaccharides, or fragments thereof, that are components of the outer membrane of gram negative (-) bacteria, and are present in the DNA preparation of the host cells and host cell membranes or macromolecules. Hence, removal of endotoxins can be a key in the purification of plasmid DNA for therapeutic or prophylactic use. Endotoxin removal from plasmid DNA solutions primarily uses the negatively charged structure of the endotoxins. Plasmid DNA, however, also is negatively charged and thus separation is frequently achieved with anion exchange resins, which bind both these molecules and, under certain conditions, preferentially elute plasmid DNA while binding the endotoxins. Such a separation may result in only partial removal as significant amounts of endotoxins elute with the plasmid DNA and / or a very poor recovery of plasmid DNA is achieved.

[0010] Small- and large-scale isolation and purification of plasmid DNA from small or large volume microbial fermentations thus requires the development of an improved plasmid preparation process. It is also desirable for plasmid-based research and therapy, that the nucleic acids can be separated and purified keeping the same structure in a reproducible manner, and in order to avoid the adverse effect of impurities on mammalian body, the nucleic acids are required to have been separated and purified up to high purity.

[0011] Plasmid DNA used for gene therapy is typically isolated from E. coli K-12. Endotoxins, also known as lipopolysaccharides (LPS), are known to be prominent cell membrane components of gram-negative bacteria such as E. coli. In fact, some reports suggest that the lipid portion of the outer membrane of E. coli is completely composed of endotoxin molecules (Qiagen Plasmid Purification Handbook, July 1999).

[0012] WO 2001 / 46215 reported a non-chromatographic-based process for the isolation of clinical grade plasmid DNA from bacterial cells comprising post-lysis steps including, but not limited to, (1) a two part precipitation / dissolution step were plasmid DNA is precipitated with a detergent either in a single or stepwise fashion, coupled with concentration and selective dissolution of the detergent- precipitate plasmid DNA with a salt solution; (2) removal of endotoxin and other remaining impurities by adsorption onto hydrated, crystallized calcium silicate, again, either in a single or stepwise fashion; and; (3) concentration of the purified plasmid DNA by alcoholic precipitation (including but not limited to ethanol, methanol and isopropanol), or another concentrating method, including but not limited to ultrafiltration.

[0013] WO 96 / 08500 reported a filtration process for preparing nucleic acids from natural sources, wherein the nucleic acid-containing sources are disintegrated, the disintegrated product is left to rest for a certain time, the resulting disintegrated product is passed through a filtering layer made of glass, silica gel, aluminum oxide or powdery diatomaceous earth or non-woven of inter-woven or glued glass fibers and silica gel, cellulose, paper, pressed paper, non-woven of paper, particles, layers, membranes or plastics, such as non-woven fabrics based on polypropylene. The fraction that passes through the filtering layer is collected and the nucleic acids are isolated from the collected fraction and purified.

[0014] WO 96 / 21729 reported a process for reducing RNA concentration in a mixture of biological material, comprising the step of filtering a mixture of biological material having a first concentration of RNA through a diatomaceous earth material to produce a filtrate having a second concentration of RNA.

[0015] Methods in Molecular Biology (published 2003, E.coli plasmid vectors - methods and applications) reported different methods for the isolation of plasmids from E.coli, such as by alkaline lysis followed by concentration from the supernatant by ethanol precipitation, by boiling lysis, or by using silica oxides as the DNA-binding matrix. Generally, harvested cells are resuspended and then lysed with sodium hydroxide and sodium dodecyl sulfate (SDS), cellular debris is precipitated with ice-cold potassium acetate and this is removed by passing the sample through a filter plate, before DNA is concentrated by precipitation with alcohol.

[0016] WO 2004 / 60277 reported a process for purifying plasmid DNA from host cell impurities to obtain a DNA product, the process comprising: (a) lysing host cells containing the plasmid DNA to obtain a lysate; (b) clarifying the lysate to obtain a clarified lysate; (c) ultrafiltering the clarified lysate to obtain an ultrafiltered clarified lysate; (d) adding a first precipitating agent in sufficient quantity to the ultrafiltered clarified lysate to obtain a precipitate of the plasmid DNA; (e) dissolving the precipitate to obtain a first solution; (f) adding a second precipitation agent in sufficient quantity to the solution to precipitate the host cell impurities and to obtain a solute containing the plasmid DNA; (g) transferring the solute into another buffer to obtain a second solution; (h) applying the second solution to an anion exchange chromatography (AEX) material to obtain an eluate containing the plasmid DNA; and (i) applying the eluate to a hydrophobic interaction chromatography (HIC) material to obtain the DNA product.

[0017] WO 2005 / 26331 reported a process for producing and isolating highly purified plasmid DNA that includes the step of cells lysis in which there is (a) a means for turbulent flow to rapidly mix a cell suspension with a solution that lyses cells; and (b) a means for laminar flow to permit incubating a mixture formed in (a) without substantial agitation, wherein the mixture formed in (a) flows from the means for turbulent flow into the means for laminar flow. One mechanism of performing flocculate removal is through a lysate filtration step, such as through a 1 to 5 mm, and preferably a 3.5 mm grid filter, followed by a depth filtration as a polishing filtration step.

[0018] WO 2006 / 83721 reported a method of purifying supercoiled plasmid DNA from a cell lysate of a large-scale microbial fermentation which comprises: (a) lysing microbial host cells containing supercoiled plasmid DNA, forming a host cell lysate; and (b) clarifying said lysate of step (a) by flocculating host cell debris. Also reported is a method of purifying supercoiled plasmid DNA from a cell lysate of a large-scale microbial fermentation which comprises: (a) precipitating said supercoiled plasmid DNA; and, (b) concentrating said precipitated, supercoiled plasmid DNA by microfiltration under a tangential flow filtration mode.

[0019] WO 2009 / 111336 reported a method of purifying plasmid DNA from host cells for laboratory or clinical use comprises the steps of a) lysing host cells containing plasmid DNA to obtain a lysate; b) precipitating host cell impurities from the lysate of step a) by adding a first solution comprising at least one monovalent cation and at least one divalent cation; c) centrifuging or filtrating the lysate from step b) to remove the precipitation of the host cell impurities forming a clarified lysate; d) precipitating plasmid DNA from the clarified lysate of step c) by adding a second solution comprising a first plasmid DNA precipitating agent; and e) collecting the plasmid DNA precipitation by a separation step.

[0020] WO 2009 / 135048 reported a process for the purification of plasmid DNA, the process comprising: (a) pre-treating an aqueous composition containing plasmid DNA by combining the aqueous composition with an anion exchange resin; (b) treating the pre-treated aqueous composition with a polypeptide to digest colanic acid; (c) separating the plasmid DNA from the treated aqueous composition, the separation comprising combining the treated aqueous composition with an affinity chromatography resin, and thereafter combining the treated aqueous composition with a hydrophobic interaction chromatography resin; and (d) filtering the plasmid DNA.

[0021] Nunes et al. (J. Membr. Sci. 415-416 (2012) 24-35) reported the modeling and application of plasmid DNA recovery from fermentation broths by a combined tandem process of micro-and ultrafiltration. To achieve primary isolation of the plasmid from fermentation broths, immediately after cell lysis, a 0.2 pm microfiltration membrane was selected for solid / liquid separation, which was performed in a diafiltration mode, as an alternative to centrifugation. Then to attain plasmid concentration and purification, an ultrafiltration membrane with a pore radius of 4.1 nm was selected. The effect of the ionic strength on apparent genomic DNA re-solubilization was investigated, by testing the addition of two different salts to the diafiltration buffer: CH3COOK and CaC12. The results show that these salts can be used to control genomic DNA apparent resolubilization.

[0022] Praveen et al. (Int. J. Bioass. 2 (2013) 243-248) reported preparation and purification of DNA from bacterial cells and characterization of plasmid DNA using an extraction protocol, such as the chlorofom-ethanol extraction process.

[0023] Sun et al. (J. Biosci. Bioeng. 116 (2013) 281-286) reported a process consisting of alkaline lysis, tangential flow filtration, purification by anion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography for pharmaceutical-grade plasmid DNA in vaccine applications and gene therapy. The process begins with RNA precipitation of the alkaline lysate by high salt. Following alkaline lysis a dead-end filtration and a high salt solution was conducted. Low molecular weight RNA and the clarified alkaline lysate were then essentially removed using a tangential flow filtration system. The process was scaled up to yield 800 mg of pharmaceutical-grade plasmid DNA from approximately 2 kg of bacterial cell paste.

[0024] US 2013 / 0034876 reported systems and methods that increase protein yield from recombinant manufacturing processes. The systems and methods treat used depth filters with bound proteins of interest as a stationary phase exchange resin to recapture bound protein of interest from the depth filter.

[0025] Manzano et al. (Biotechnol. Biotechnol. Equip. 3 (2015) 586-591) reported plasmid DNA prepurification by tangential flow ultrafiltration in a hollow-fiber module. In more detail, a hollow fibre module operating in TFF mode was used in this work for the pre-purifi cation of plasmid pVAXl-NH36 from clarified lysates of Escherichia coli DH5a cultures. The results suggested the use of a combination of concentration-diafiltration steps for the pre-purifi cation of the plasmid lysates processed.

[0026] Padilla-Zamundio et al. (Bioprocess. Biosy st. eng. 38 (2015) 1091-1096) reported Plasmid DNA primary recovery from E. coli lysates by depth bed microfiltration, especially deep-bed microfiltration (DBM) using DBM Sartoclear P® capsule, wherein a dilute suspension is injected into a filter made of porous material, and the particles are trapped in the interstices or along the walls of the pore space by various mechanisms. For plasmid pVAXl-NH36 with final size of 4.0 kbp a concentration between 4.00 and 4.60 pg pDNA / cm3 determined by HPLC after filtration has been reported.

[0027] Li et al. (Sep. Pur. Technol. 176 (2017) 287-293) reported about the effect of ionic strength on membrane fouling during ultrafiltration of plasmid DNA using Biomax polyethersulfone ultrafiltration membranes with 300 and 1000 kDa nominal molecular weight cutoffs wherein fouling was most pronounced for the larger 16.8 kbp plasmid, consistent with the greater probability of plasmid trapping at the pore entrance.

[0028] Li et al. (Biotechnol. Bioeng. 113 (2017) 783-789) reported enhanced purification of plasmid DNA isoforms by exploiting ionic strength effects during ultrafiltration. Experiments were performed with a 3.0 kbp plasmid using composite regenerated cellulose ultrafiltration membranes. DNA transmission during ultrafiltration occurs by elongation of the DNA in the converging flow field approaching the membrane pores. It has been shown that it was possible to separate supercoiled, linear, and open-circular plasmids by exploiting differences in the elongational flexibility of these topological isoforms.

[0029] US 2018 / 0230180 reported methods of producing an aqueous formulation of an antigen-binding protein or enhancing re-oxidation of an antigen-binding protein. The methods comprise contacting an aqueous solution comprising antigen-binding protein molecules with a charged depth filter under conditions sufficient to enhance re-oxidation of the antigen-binding protein molecules and achieve a decrease in the percentage of reduced antigen-binding protein molecules. Summary Of The Invention

[0030] The current invention is based, at least in part, on the surprising result that a positively charged filter can be used to efficiently process recombinantly produced plasmid DNA.

[0031] In more detail, it has been found that a positively charged filter, e.g., composed of an inorganic filter aid, such as natural silica, cellulose and a polymer resin that is positively charged, can be used to efficiently process crude bacterial lysates comprising recombinant plasmid DNA.

[0032] It has been found that the positively charged filter, is more efficient than a neutral filter, i.e. a filter that does not have a positive charge.

[0033] With the method according to the current invention a clarified lysate comprising recombinant plasmid DNA can be produced.

[0034] Therefore, the present invention is also directed to a gram negative bacterial recombinant plasmid composition.

[0035] Furthermore, the methods described herein are scalable for purifying plasmid DNA from a few dozen microliters to hundreds of liters of cell culture, therefore fitting the needs of both laboratory applications and clinical applications.

[0036] The processes and compositions described herein have a range of uses, including diverse applications, such as, e.g., in the field of molecular biology, health and medicine.

[0037] The current invention encompasses at least the following independent and dependent embodiments:

[0038] 1. A method for producing a recombinant plasmid DNA solution comprising the following step(s): a) passing a crude E.coli lysate comprising recombinant plasmid DNA through a positively charged filter, preferably a depth filter, comprising an inorganic filter aid, cellulose and a polymer resin to obtain a filtered recombinant plasmid DNA solution, thereby producing a recombinant plasmid DNA solution. A method for producing a recombinant plasmid DNA solution comprising the following step(s): a) passing a crude E.coli lysate comprising recombinant plasmid DNA through a positively charged filter, preferably a depth filter, comprising silica, preferably natural silica, cellulose and a polymer resin to obtain a filtered recombinant plasmid DNA solution, thereby producing a recombinant plasmid DNA solution. The method according to any one of embodiments 1 to 2 further comprising before step a) the step aO) lysing E.coli cells comprising the recombinant plasmid DNA to obtain a crude E.coli lysate. The method according to any one of embodiments 1 to 3 further comprising before step a) or step aO) the step of al) cultivating an E.coli cell comprising the plasmid DNA for producing recombinant plasmid DNA. The method according to any one of embodiments 1 to 4 further comprising after step a) the step of b) purifying the filtered recombinant plasmid DNA solution with one or more chromatography steps and / or one or more filtration steps and / or combinations thereof, whereby tangential flow filtration is preferred as one of the filter steps. The method according to any one of embodiments 1 to 5, wherein the produced or obtained recombinant plasmid DNA solution is a purified recombinant plasmid DNA solution, i.e. the recombinant plasmid DNA solution is enriched in recombinant plasmid DNA content as non-plasmid DNA compounds have been removed from the recombinant plasmid DNA solution applied to the positively charged filter. The method according to any one of embodiments 3 to 6, wherein after step aO) and before step a) the buffer is not changed / wherein in step aO) and step a) the buffer is the same. 8. The method according to any one of embodiments 1 to 7, wherein the crude E.coli lysate is a solution comprising about 15 mM Tris-HCl, about 3 mM EDTA, about 60 mM NaOH, about 0.3 % SDS, about 0.9 M KAc, about 4.5 % (v / v) acetic acid, about 0.5 M CaC12 and has a pH value of about 4.6.

[0039] 9. The method according to any one of embodiments 1 to 8, wherein the crude E.coli lysate has a conductivity of about 90 mS / cm.

[0040] 10. The method according to any one of embodiments 1 to 9, wherein the recombinant plasmid DNA has a size of 5000 bps or more.

[0041] The method according to any one of embodiments 1 to 10, wherein the recombinant plasmid DNA has a size of 7000 bps or more.

[0042] 12. The method according to any one of embodiments 1 to 11, wherein the recombinant plasmid DNA has a size of 15000 bps or less.

[0043] 13. The method according to any one of embodiments 1 to 12, wherein the recombinant plasmid DNA has a size of 12000 bps or less.

[0044] 14. The method according to any one of embodiments 1 to 13, wherein the filter has a filter area of from and including about 0.23 m2 to about 3.4 m2.

[0045] 15. The method according to any one of embodiments 1 to 13, wherein the filter has a filter area of from and including about 0.0170 m2 to about 0.1020 m2.

[0046] 16. A method for producing a recombinant plasmid DNA solution comprising the following steps: i) cultivating an E.coli cell comprising plasmid DNA for producing recombinant plasmid DNA. ii) lysing E.coli cells comprising the recombinant plasmid DNA to obtain a crude E.coli lysate. iii) passing the crude E.coli lysate comprising the recombinant plasmid DNA through a positively charged filter, preferably a depth filter, comprising an inorganic filter aid, preferably silica, cellulose and a polymer resin to obtain a filtered recombinant plasmid DNA solution, thereby producing a recombinant plasmid DNA solution.

[0047] 17. The method according to embodiment 16 further comprising after step iii) the step of iv) purifying the filtered recombinant plasmid DNA solution with one or more chromatography steps and / or one or more filtration steps and / or combinations thereof, whereby tangential flow filtration is preferred as one of the filter steps.

[0048] 18. The method according to any one of embodiments 1 to 17, wherein an amount of recombinant plasmid DNA is not removed by the positively charged filter (allowing to obtain a filtered solution comprising recombinant plasmid DNA).

[0049] 18a. The method according to any one of embodiments 1 to 17, wherein a significant amount of recombinant plasmid DNA is not removed by the positively charged filter (allowing to obtain a filtered solution comprising recombinant plasmid DNA).

[0050] 19. The method according to any one of embodiments 1 to 17, wherein an amount of recombinant plasmid DNA is not bound to the positively charged filter (allowing to obtain a filtered solution comprising recombinant plasmid DNA).

[0051] 19a. The method according to any one of embodiments 1 to 17, wherein a significant amount of recombinant plasmid DNA is not bound to the positively charged filter (allowing to obtain a filtered solution comprising recombinant plasmid DNA).

[0052] 20. The method according to any one of embodiments 1 to 19a, wherein the positively charged filter contains a polymer resin containing quaternary amines, preferably quaternary amino ethyl residues or quaternary ammonium residues, resulting in a positive surface charge.

[0053] 21. The method according to any one of embodiments 1 to 20, wherein the crude E.coli lysate is subjected to separation techniques to remove at least part of the other nucleic acid, protein, and cell contaminants that are present, before applying the crude E.coli lysate comprising recombinant plasmid DNA to the positively charged filter. 22. The method according to any one of embodiments 1 to 21, wherein the crude E.coli lysate is subjected to purification steps to remove intact cells and cell debris before applying the crude E.coli lysate comprising recombinant plasmid DNA to the positively charged filter.

[0054] 23. The method according to any one of embodiments 1 to 22, wherein the crude E.coli lysate is obtained by alkaline lysis to disrupt the cell walls, release recombinant plasmid DNA molecules, and precipitate (considerable amounts of) cell debris, genomic DNA (gDNA) and proteins upon neutralization.

[0055] 24. The method according to any one of embodiments 1 to 23, wherein the crude E.coli lysate is obtained by adding an equal volume of a solution comprising 0.2 M NaOH and 0.1 % (w / v) SDS to the E.coli cell suspension.25. The method according to any one of embodiments 1 to 24, wherein the crude E.coli lysate is obtained by separating the E.coli cells from the cultivation broth, resuspending the cells in buffer, adding an volume of a solution comprising 0.2 M NaOH and 0.1 % (w / v) SDS equal to the buffer volume to the E.coli cell suspension, incubating for up to 10 minutes, precipitating RNA by adding CaC12 and removing precipitate by depth filtration.

[0056] 26. The method according to any one of embodiments 1 to 25, wherein the positively charged filter comprises cellulose fibers, a polymer resin containing quaternary amines, preferably quaternary amino ethyl residues or quaternary ammonium residues, and a silica-based filter aid.

[0057] 27. The method according to any one of embodiments 1 to 26, wherein the positively charged filter comprises two layers, wherein each layer comprises a cellulose filter matrix, and wherein the cellulose filter matrix is impregnated with a filter aid comprising silica.

[0058] 28. The method according to any one of embodiments 1 to 27, wherein the positively charged filter comprises two layers, wherein each layer comprises a cellulose filter matrix, wherein the cellulose filter matrix is impregnated with a filter aid comprising silica and wherein each layer further comprises a polymer resin containing quaternary amines, preferably quaternary amino ethyl residues or quaternary ammonium residues. 29. The method according to any one of embodiments 1 to 28, wherein the positively charged filter is a Zeta Plus depth filter with SP series media.

[0059] 30. The method according to any one of embodiments 1 to 29, wherein the positively charged filter is more efficient than a filter without a positive charge, i.e. that the filter performs better at least regarding filtered volumes and amounts of recombinant plasmid DNA.

[0060] ***

[0061] 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 and 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.

[0062] Detailed Description Of Embodiments Of The Invention

[0063] The processes for producing plasmid DNA and proteins have much in common, requiring fermentation, cell harvesting, product recovery, chromatography purification, dilution / concentration and sterilization. However, different characteristics of DNA and proteins require different processes at each step. For example, high-pressure homogenization and ultrasonication are not suitable for purification of plasmid DNA due to its sensitivity to shear forces. Alkaline lysis, first reported by Bimboim and Doly in 1979, is usually the best choice in plasmid DNA production for disruption of bacterial cells (see, e.g., Sun et al., J. Biosci. Bioeng. 116 (2013) 281-286).

[0064] Plasmid extraction kits for small laboratory scale plasmid DNA isolation are available from a variety of suppliers and typically involve alkaline lysis and removal of cellular debris, followed by binding of the DNA to a resin, which is immobilized within a filter plate or small column. The DNA is then washed to remove contaminants and eluted from the resin using a low-ionic-strength buffer. The large-scale and lab-scale protocols for the isolation of plasmid DNA in the lysate fall into three categories. First, isopropanol, polyethylene glycol (PEG), compaction agents and chaotropic salts have been used in DNA precipitation. The second category involves tangential flow filtration (TFF) for concentration and removal of contaminating RNA and proteins to purify plasmid DNA after the clarification step. In the third category, chromatography methods, such as size exclusion chromatography (SEC), ion exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), reversed-phase HPLC (RP-HPLC) and affinity chromatography (AC), are often used for refinement after primary purification of plasmid DNA. However, no single type of chromatography media can remove all residual proteins, chromosomal DNA, endotoxins and RNA. Therefore, an optimal multi-step chromatography process is required (see, e.g., Sun et al., J. Biosci. Bioeng. 116 (2013) 281-286).

[0065] Currently, there are some options forpre-purification of pDNA at laboratory scale, such as aqueous two-phase systems and solvent precipitation which have shown good results for impurities removal and high yield recovery of pDNA. However, large amounts of solvents, alcohols or salts are used to accomplish these operations, increasing the production cost and the environmental impact. In the last years, new bioprocesses to obtain purified pDNA from E. coli ferments were developed using hollow-fiber tangential flow filtration (TFF) and tandem anion-exchange membrane chromatography operating in frontal mode (see, e.g., Manzano, I., et al., Biotechnol. Biotechnol. Equip. 3 (2015) 586-591).

[0066] Alkaline lysis not only disrupts the cell wall, releasing plasmid molecules, but also removes considerable amounts of cell debris, genomic DNA (gDNA) and proteins, which precipitate upon neutralization, leaving RNA as the major contaminant, together with low quantities of gDNA, proteins and endotoxins. Alkaline lysis takes advantage of the renaturation cycle of pDNA molecules. Upon neutralization of the lysate with a high-salt solution, a large quantity of precipitates is formed that tends to flocculate. Any solid-liquid operation can be used, in principle, to remove it from the lysate. At larger scales of operation, filtration is preferred. Theodossiou et al. (Bioproc. Eng. 16 (1997) 175-183) studied several filters, with different materials and pore sizes and also the possible application of filter aids. Even the smallest pore size filter (5 pm) was unable to fully retain the solid material. With the addition of filter aids, complete separation was achieved, but at the expense of a reduced capacity and lower yield (due, in part, to plasmid adsorption on the filter aid itself). Using microfiltration no filter aids are necessary and very high yields can be expected if low plasmid adsorption on the membranes is achieved. Kendall et al. (Biotechnol. Bioeng. 79 (2002) 816-822) studied the application of a 0.45 pm nitrocellulose membrane to purify plasmids from clarified cell lysates; the clarification was carried out by gravity-driven filtration. The use of a highly hydrophobic material such as nitrocellulose enabled a considerable removal of the main hydrophobic contaminants namely RNA, gDNA and some proteins, however it caused significant membrane fouling (see, e.g., Nunes, J.C., et al., J. Membr. Sci. 415-416 (2012) 24-35).

[0067] 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.

[0068] 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).

[0069] 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). Definitions

[0070] 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.

[0071] The term “about” denotes a range of + / - 20 % of the thereafter following numerical value. In one embodiment the term about denotes a range of + / - 10 % of the thereafter following numerical value. In one embodiment the term about denotes a range of + / - 5 % of the thereafter following numerical value.

[0072] The term “comprising” also includes the term “consisting of’.

[0073] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.

[0074] In more detail, the term "isolated" refers to material, which is substantially or essentially free from components which 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.

[0075] An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g. cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0076] As used herein, "non-supercoiled plasmid DNA" refers to any DNA that is not supercoiled plasmid DNA, including any other form of plasmid DNA such as nicked, open circle, and linear, as well as host genomic DNA. As used herein, "NTU" refers to normalized turbidity units. Turbidity is defined as particle "counts" which pass by the optical field of a submerged probe. Solution turbidity can be monitored with a laser-based, light-scattering device.

[0077] As used herein, "OD600" refers to the optical density of a solution determined at 600 nm, a light scattering, spectrophotometric measurement of the number of cells / mL.

[0078] As used herein, "L.O.D." refers to the limit of detection.

[0079] As used herein, "MW" refers to molecular weight in Daltons.

[0080] The term "polynucleotide" means a polymeric form of nucleotides of at least 10 bases or base pairs in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide, and is meant to include single and double stranded forms of DNA and / or RNA. In the art, this term is often used interchangeably with "oligonucleotide".

[0081] The terms “(recombinant AAV) vector" or “rAAVv” or “transgene”, which can be used interchangeably herein, denote a nucleic acid derived from a wild-type genome of an adeno- associated virus, wherein except for the ITR (adeno-associated virus Inverted Terminal Repeat) sequences all endogenous AAV nucleic acids are replaced by one or more exogenous nucleic acid(s). For example, such an exogenous nucleic acid can be a nucleic acid transcribed into a transcript of interest or that encodes a therapeutic protein or a therapeutic nucleic acid. Typically, for a rAAVv one or both ITR sequences of the wild-type AAV genome are retained either in its original form or in modified form. Thus, a rAAVv can be distinguished from a wild-type AAV vector, since all or at least a part of the viral genome has been replaced with a non-native (i.e. exogenous) nucleic acid with respect to the virus. Incorporation of a non-native nucleic acid therefore defines the AAV vector as well as the particle comprising said AAV vector as a "recombinant" vector. It has to be pointed out that the serotype of the ITRs in the rAAVv does not need to be the same as the serotype of the adeno-associated capsid polypeptides forming the shell of the rAAVp comprising said rAAVv.

[0082] In more detail, the term “vector" or “transgene” denotes the portion of a larger nucleic acid, e.g. of a recombinant plasmid, that is ultimately packaged or encapsulated or encapsidated either directly or in form of a single strand or in form of RNA into a protein shell composed of adeno- associated virus capsid polypeptides to form a rAAVp. In cases where recombinant plasmids are used to construct or manufacture rAAVps, the viral particle does not include the portion of the "plasmid" that does not correspond to the vector part of the recombinant plasmid. For example, in case of a rAAVp the recombinant vector comprises that part of the recombinant plasmid that is interspaced between two AAV ITRs. The non-vector portion of the recombinant plasmid is referred to as the "plasmid backbone". The plasmid backbone is important for cloning and amplification of the plasmid, a process that is needed for propagation and recombinant virus production, but is not itself packaged or encapsulated or encapsidated into the recombinant AAV particle. Thus, a “vector" refers to the nucleic acid that is packaged or encapsulated or encapsidated by a protein shell composed of adeno-associated virus capsid polypeptides, i.e. in a rAAVp.

[0083] In principle, any non- AAV nucleic acid can be packaged into a shell composed of adeno-associated capsid polypeptides resulting in a rAAVp, e.g. for subsequent infection (transduction) of a cell, ex vivo, in vitro or in vivo.

[0084] As used herein, the term "serotype" is used to classify different wild-type and recombinant AAV particles based on the amino acid sequence of the polypeptides forming the protein shell (capsid) of the respective AAV particle. Originally, serologic distinctiveness was determined based on the lack of cross-reactivity between antibodies to one AAV particle as compared to another AAV particle. Such cross-reactivity differences are usually due to differences in capsid polypeptide sequences and the respective antigenic determinants (e.g., due to VP1, VP2, and / or VP3 sequence differences of AAV serotypes). Despite the possibility that AAV variants including capsid variants may not be serologically distinct from a reference or wild-type AAV or other AAV serotype, they differ by at least one amino acid residue compared to the reference or wild-type or other AAV serotype.

[0085] Under the traditional definition, a serotype means that the virus of interest has been tested against serum specific for all existing and characterized serotypes for neutralizing activity and no antibodies have been found that neutralize the virus of interest. As more naturally occurring virus isolates are discovered and / or capsid mutants generated, there may or may not be serological differences with any of the currently existing serotypes. Thus, in cases where the new AAV particle has no serological difference, this new AAV particle would be a subgroup or variant of the corresponding wild-type serotype. In many cases, serology testing for neutralizing activity has yet to be performed on mutant viruses with capsid sequence modifications to determine if they are of another serotype according to the traditional definition of serotype. DEPTH FILTRATION AND DEPTH FILTERS

[0086] In some embodiments, the method according to the invention comprises a positively charged depth filter. Thus, in some embodiments, the method according to the invention comprises a depth filtration step. A depth filtration step is a technique comprising processing samples, such as cultivation broths, via a depth filter. In some embodiments, the depth filter comprises a porous filtration medium capable of retaining portions of a sample, such as cell components and debris, wherein filtration occurs, e.g., within the depth of the filter material. In some embodiments, the depth filter comprises synthetic material, non-synthetic material, or a combination thereof, preferably an inorganic filter and / or cellulose. In some embodiments, the depth filter comprises a substrate comprising one or more of diatomaceous earth, silica, a cellulose fiber, a polymeric fiber, a cohesive resin, and an ash composition, preferably silica in combination with a cellulose fiber. In some embodiments, at least a portion of the filtration medium of the depth filter comprises a surface modification. In some embodiments, the surface modification is a quaternary amine surface modification and / or a cationic surface modification. In some embodiments, the depth filter is selected from the group consisting of an EMPHAZE™ AEX depth filter, an XOSP depth filter, a PDD1 depth filter, and a ZETA PLUS™ depth filter.

[0087] Zeta Plus™ filter:

[0088] A Zeta Plus™ filter is a depth filter. The filtration medium / layer / material is composed of a mixture of inorganic filter aids, cellulose, a crosslinking polymer binder resin and a crosslinker. The polymer binding resin contains quaternary amines resulting in a positive surface charge level. The active filtration elements are comprised primarily of natural products.

[0089] The recommended maximum flux for pre-conditioning flush is 1200 L / m2 / hour (LMH) for cartridges and 210 LMH for capsules. The maximum operating pressure is for laboratory capsule filters 2.8 bar (40 psig) maximum inlet pressure, for scale-up capsule filters 3.1 bar (45 psig) and for production capsule filters 3.4 bar @40°C (50 psig @104°F). The pressure drop across the filter should not exceed 2.4 bar (35 psid).

[0090] The filter is available in a single layer or a dual layer form. The single layer has a filter area of 0.23 cm2, multi cells have a filter area of 1.6 m2 of dual layer media or 2.5 m2 of single layer media. Generally, Zeta Plus™ capsule filters are commercially available with filter areas of 25 cm2, 170 cm2, 340 cm2, 1020 cm2, 1.6 m2, 2.5 m2, 11.2 m2, 17.5 m2 and integer multiples thereof. Cartridge filters are available with surface areas ranging from 0.26 m2to 3.9 m2per cartridge.

[0091] An exemplary filter used herein has the product / order number E0170FSA60SP02A which is a Zeta Plus filter belonging to the EXT series (capsule) with SP series media.

[0092] PP3 filter:

[0093] A PP3 filter is a depth filter. Its filter material is polypropylene fleece based. It is available in a broad variety of retention ratings from 0.45 pm all the way up to 100 pm. Sartopure® PP3 filter elements are completely made from polypropylene.

[0094] In some embodiments, the positively charged depth filter comprises cellulose fibers and a silica- based filter aid. In some embodiments, the positively charged depth filter comprises two layers, wherein each layer comprises a cellulose filter matrix, and wherein the cellulose filter matrix is impregnated with a filter aid comprising silica. In some embodiments, the positively charged depth filter comprises two layers, wherein each layer comprises a cellulose filter matrix, wherein the cellulose filter matrix is impregnated with a filter aid comprising silica and wherein each layer further comprises a polymer resin / resin binder. An exemplary positively charged depth filter is marketed as Zeta Plus™ depth filter, Thus, in a preferred embodiment, the depth filter is a Zeta Plus depth filter. In a preferred embodiment, the depth filter is a Zeta Plus depth filter with SP series media. The product / order number of the used filter was E0170FSA60SP02A.

[0095] In some embodiments, the positively charged depth filter comprises cellulose fibers, diatomaceous earth, and perlite. In some embodiments, the positively charged depth filter comprises two layers, wherein each layer comprises a cellulose filter matrix, and wherein the cellulose filter matrix is impregnated with a filter aid comprising one or more of diatomaceous earth or perlite. In some embodiments, the positively charged depth filter comprises two layers, wherein each layer comprises a cellulose filter matrix, wherein the cellulose filter matrix is impregnated with a filter aid comprising one or more of diatomaceous earth or perlite, and wherein each layer further comprises a resin binder. An exemplary positively charged depth filter is marketed as PDD1 depth filter. Thus, in some embodiments, the depth filter is a PDD1 depth filter. In some embodiments, the positively charged depth filter comprises a silica, such as a silica filter aid, and a polyacrylic fiber. In some embodiments, the positively charged depth filter comprises two layers of filter media, wherein a first layer comprises a silica, such as a silica filter aid, and a second layer comprises a polyacrylic fiber, such as a polyacrylic fiber pulp. In some embodiments, the positively charged depth filter is a positively charged depth filter comprising synthetic material and does not comprise diatomaceous earth and / or perlite. An exemplary positively charged depth filter is marketed as XOSP depth filter, Thus, in some embodiments, the depth filter is a XOSP depth filter.

[0096] In some embodiments of all aspects and embodiments, the silica filter aid is a precipitated silica filter aid. In some embodiments, the filter aid is an aspect of the filter, such as a layer, that aids with performing the filter function. In some embodiments, the silica filter aid is a silica gel filter aid. In some embodiments, the silica filter aid has about 50% of silanols ionized at pH 7. In some embodiments, the silica filter aid is a silica gel filter aid, wherein about 50% of silanols of the silica filter aid are ionized at pH 7. In some embodiments, the silica filter aid is precipitated from silicas, such as SIPERNAT® (Evonik Industries AG), or silica gels, such as Kieselgel 60 (Merck KGaA).

[0097] In some embodiments, the polyacrylic fiber is a non-woven polyacrylic fiber pulp. In some embodiments, the polyacrylic fiber is an electrospun polyacrylic nanofiber. In some embodiments, the degree of fibrillation of the polyacrylic fibers correlates with a Canadian Standard Freeness (CSF) from about 10 mL to about 800 mL.

[0098] In some embodiments of all aspects and embodiments, the positively charged depth filter has a pore size of about 0.05 pm to about 0.2 pm, such as about 0.1 pm. In some embodiments, the positively charged depth filter has a surface area of about 0.01 m2 to about 1.5 m2, such as about 0.1 m2, about 0.55 m2, or about 1.1 m2. In some embodiments, the positively charged depth filter does not comprise diatomaceous earth and / or perlite. In some embodiments, the positively charged depth filter comprises two layers of filter media, wherein a first layer comprises a silica filter aid having about 50% of silanols ionized at pH 7, and a second layer comprises a polyacrylic fiber pulp having a degree of fibrillation of the polyacrylic fibers correlating with a Canadian Standard Freeness (CSF) from about 10 mL to about 800 mL, and wherein the positively charged depth filter does not comprise diatomaceous earth. In some embodiments of all aspects and embodiment, the positively charged depth filter comprises a hydrogel Q (quaternary amine)-functionalized non-woven material, and a multizone microporous membrane. In some embodiments, the positively charged depth filter comprises four layers comprising hydrogel Q-functionalized non-woven materials, and a nine-zone microporous membrane. In some embodiments, the non-woven material comprises polypropylene. In some embodiments, the positively charged depth filter is a positively charged depth filter comprising synthetic material and does not comprise diatomaceous earth and / or perlite. Such a depth filter is marketed as EMPHAZE™ AEX depth filter, Thus, in some embodiments, the depth filter is an EMPHAZE™ AEX depth filter.

[0099] In some embodiments of all aspects and embodiments, the positively charged depth filter comprises multiple components or layers. In some embodiments, the positively charged depth filter comprises multiple layers comprising one or more layers comprising anion-exchange (AEX) functional polymers. In some embodiments, the layer comprising AEX functional polymers comprises a quaternary ammonium (Q), such as a Q functional hydrogel. In some embodiments, the layer comprising AEX functional polymers comprises a quaternary ammonium (Q) functional polymer associated with a non-woven article. In some embodiments, the layer comprising AEX functional polymers comprises a quaternary ammonium (Q) functional hydrogel covalently grafted to a fine-fiber polypropylene non-woven scaffold. In some embodiments, the depth filter comprises multiple layers comprising a layer comprising a multi-zone membrane comprising a nine-zone membrane with a pore size of about 0.05 pm to about 0.3 pm, such as about 0.22 pm. In some embodiments, the depth filter does not comprise diatomaceous earth.

[0100] As used herein the term “depth filter” denotes a filter that achieves filtration, i.e. separation of material, within the depth of the filter material. The term “depth filter” expressly encompasses positively charged depth filters. In some embodiments, the depth filter comprises a porous filtration medium capable of retaining portions of a sample, such as, e.g., cell components and debris, wherein filtration occurs, e.g., within the depth of the filter material. A common class of such filters is those that comprise a (random) matrix of bonded fibers (or otherwise fixed), to form a complex, tortuous maze of flow channels. Particle separation in these filters generally results from entrapment by or adsorption to the filter material. Frequently used depth filter media for bioprocessing of cell culture broths and other feedstocks consists of cellulose fibers (as matrix) and a inorganic filter aid such as silica or diatomaceous earth (DE). In some embodiments, the depth filter is a synthetic filter. In some embodiments, the depth filter comprises a silica filter aid, and / or cellulose fiber. In some embodiments, the depth filter comprises a silica filter aid, and / or cellulose fiber, and / or non-woven material. In some embodiments, the depth filter comprises silica and cellulose fiber as non-woven material. Depth filter media, unlike absolute filters, retain particles and other impurities throughout the porous media allowing, e.g., for retention of particles both larger and smaller than the pore size. Particle and impurity retention is thought to involve size exclusion and adsorption through hydrophobic, ionic and other interactions. Depth filters are advantageous because they remove contaminants / impurities. The depth filter may be a multi-layer depth filter comprising multiple levels of depth filter media, which are layered in series. Employing multiple depth filters ensures that more of the filtrate stream efficiently contacts the depth filter media, enabling a better adsorption profile for the impurities.

[0101] In some embodiments of all aspects and embodiments, the positively charged depth filter comprises synthetic material, non-synthetic material, or a combination thereof. In some embodiments, the positively charged depth filter comprises a substrate comprising one or more of diatomaceous earth, silica, a cellulose fiber, a polymeric fiber, a cohesive resin, and an ash composition. In some embodiments, the positively charged depth filter is selected from the group consisting of an XOSP depth filter (Millistak+® HC Pro XOSP), a PDD1 depth filter (Pall / 3M PDD1 SUPRAcap™-50 (SC050PDD1)), or a VR02 depth filter (Zeta Plus™ Biocap VR02).

[0102] In some embodiments of all aspects and embodiments, the positively charged depth filter comprises cellulose fibers, diatomaceous earth or silica, or / and perlite. In some embodiments, the positively charged depth filter comprises two layers, wherein each layer comprises a cellulose filter matrix, and wherein the cellulose filter matrix is impregnated with a filter aid comprising one or more of diatomaceous earth or perlite. In some embodiments, the positively charged depth filter comprises two layers, wherein each layer comprises a cellulose filter matrix, wherein the cellulose filter matrix is impregnated with a filter aid comprising one or more of diatomaceous earth or perlite, and wherein each layer further comprises a resin binder. In some embodiments, the positively charged depth filter is a PDD1 depth filter.

[0103] In some embodiments of all aspects and embodiments, the positively charged depth filter comprises a silica, such as a silica filter aid, and a polyacrylic or cellulose fiber. In some embodiments, the positively charged depth filter comprises two layers of filter media, wherein a first layer comprises a silica, such as a silica filter aid, and a second layer comprises a polyacrylic fiber, such as a polyacrylic fiber pulp. In some embodiments, the positively charged depth filter is a depth filter comprising synthetic material and does not comprise diatomaceous earth and / or perlite. In some embodiments, the positively charged depth filter is a XOSP depth filter.

[0104] In some embodiments of all aspects and embodiments, the positively charged depth filter comprises cellulose fibers (as matrix) and charged surface groups (ionic charge modifications). In some embodiments, the positively charged depth filter comprises cellulose fibers (as matrix) and a cationic charge modifier that is chemically bound to the matrix components. In some embodiments, the positively charged depth filter is a VR02 / Zeta Plus™ depth filter.

[0105] In some embodiments of all aspects and embodiments, the positively charged depth filter comprises a silica filter aid. In some embodiments, the silica filter aid is a precipitated silica filter aid. In some embodiments, the filter aid is an aspect of the filter, such as a layer, that aids with performing the filter function. In some embodiments, the silica filter aid is a silica gel filter aid. In some embodiments, the positively charged depth filter has a pore size of about 0.05 pm to about 0.2 pm, such as about 0.1 pm. In some embodiments, the positively charged depth filter has a surface area in the range of about 0.01 m2 to about 1.5 m2, such as at least about 0.017 m2, at least about 0.023 m2, or at least about 0.025 m2, at least about 0.1 m2 , at least about 0.55 m2 , or of at least about 1.1 m2, or greater.

[0106] PLASMID DNA COMPOSITIONS

[0107] One aspect of the present invention is a purified recombinant plasmid DNA composition.

[0108] Such a composition may generally be produced by the method according to the current invention. Therein, the method according to the current invention may or may not be combined with additional purification steps, such as one or more chromatography steps or additional filtration steps. Thus, the invention encompasses, or in addition comprises, a process of producing and isolating purified plasmid DNA compositions. The plasmid DNA composition produced with the method according to the current invention is of sufficient purity for research-use and plasmidbased gene therapy vector / particle production.

[0109] The plasmid compositions of the present invention may include any types of plasmids with any sizes. For instance, the size range of plasmid DNA that may be purified by the method according to the current invention may be from approximately 0.3 kbp (mini-circle or minimal transcription unit) to approximately 50 kbp, typically 3 kbp to 20 kbp, or larger (e.g., 5 to 100 kbp, or larger, such as phage-derived shuttle vectors, HACs, YACs, MACs, and episomes derived from EBV or other non-integrating viruses). In certain embodiments, the plasmid DNA includes a vector backbone of approximately 0.3 kbp, 0.5 kbp, 0.75 kbp, 1 kbp, 3 kbp, 5 kbp, 10 kbp, 15 kbp, or 20 kbp, a therapeutic gene, and associated regulatory sequences. This may also apply to single stranded DNA (i.e., 0.3 kb to 50 kb, etc., such as those derived from M13). Thus, for example, a plasmid backbone may be capable of carrying inserts of approximately 1-50 kbp, e.g., 3-20 kbp. The insert will generally depend on the intended application in which the plasmid composition is to be used. For gene therapy or vaccine-based applications, for example, the insert may include DNA from any organism, but will typically be of mammalian origin, and may include, in addition to a gene encoding a therapeutic protein, regulatory sequences such as promoters, polyadenylation sequences, enhancers, locus control regions, etc. The gene encoding a therapeutic protein may be of genomic origin, and therefore contain exons and introns as reflected in its genomic organization, or it may be derived from complementary DNA. Such vectors may include for example vector backbone that can replicate with high copy numbers, having a polylinker for insertion of a therapeutic gene, a gene encoding a selectable marker, e.g., the tetracycline or kanamycin resistance gene, and is physically small and stable. The vector backbone of the plasmid advantageously permits inserts of fragments of mammalian, other eukaryotic, prokaryotic or viral DNA, and the resulting plasmid may be purified in a method according to the current invention and used in vivo or ex vivo plasmid-based therapy, or other use. The plasmid compositions can also comprise other pharmaceutically acceptable components, buffers, stabilizers, or compounds for improving gene transfer and particularly plasmid DNA transfer into a cell or organism.

[0110] In general, isolated or purified recombinant plasmid DNA compositions are provided herein. In certain embodiments, the recombinant plasmid DNA composition is a gram negative bacterial recombinant plasmid DNA composition. As described herein, an efficient method has been developed that allows for the removal of contamination from a variety of bacterial materials, such as recombinant plasmid DNA.

[0111] In general, the recombinant plasmid compositions of the present invention may be utilized in a wide range of applications, including those in fields of molecular biology, health and medicine (e.g., gene therapy, diagnostics, recombinant protein expression), to name a few. The pure recombinant plasmid DNA compositions described herein may be employed in vivo or ex vivo, for example, in gene therapy and vaccine-based applications (i.e., the plasmid compositions may be administered to mammals, including humans). Additionally or alternatively, the plasmid DNA compositions may be used in conventional diagnostics techniques, for example, to improve the stability, specificity, reproducibility, and / or sensitivity of such methodologies. This may include, for example, the analysis, detection, or examination of samples from other biological or clinical samples, such as blood, saliva, sputum, semen, buccal smears, urine or fecal waste, cell and tissue biopsies and micro dissections, amniotic fluid, or tissue homogenates of plants, animals, or human patients, and the like.

[0112] The starting material for the method according to the current invention is a mass of bacterial material, or an aqueous composition comprising such biological material, such as bacterial cells or other biological matter prepared by, e.g., fermentation or cell culture, isolated from the environment, or derived from tissues or other organisms (e.g., fungi, bacteria, etc.). In one embodiment, the biological material comprises bacterial cells derived from enterobacteria, such as E. coli. In a preferred embodiment, the biological material is a gram negative bacterial recombinant plasmid DNA material or an E.coli recombinant plasmid DNA.

[0113] A variety of cell types can be used as feed for the method according to the current invention, such as bacteria (e.g., gram negative, gram positive, and Archaea), yeast, and other prokaryotic cells, including recombinant cells. Among these and other cell types, bacterial cells, and in particular gram positive and gram negative bacterial cells, such as E.coli, Salmonella, or Bacillus, are preferred, with gram negative bacterial cells being most preferred. In a particular embodiment, the bacteria is a gram negative bacteria; more preferably in this embodiment, the bacteria is E.coli. A wide selection of well-established E. coli host strains are useful according to the method according to the current invention, and are available from Stratagene (La Jolla, CA), Qiagen (Valencia, CA), New England Biolabs (Ipswich, MA), and Promega (Madison, WI), among other commercial sources.

[0114] Typically, the biological material is a bacterial lysate, or a derivative thereof. Thus, bacterial starting material (e.g., bacterial cells, etc.) must be lysed or disrupted to form the lysate. In general, the bacterial lysis step involves any conventional method for breaking open bacterial cells, thus liberating nucleic acids and other cell components therefrom. The lysis procedure may involve the use of mechanical methods, lysing agents or solutions (chemical lysis), or combinations thereof.

[0115] For biological material derived from fermentation or cell culture, the cells are disrupted by chemical or mechanical techniques as described below, forming a crude lysate. For example, where bacterial cultures are employed, the bacterial cells are lysed to form a crude bacterial lysate. In doing so, the cellular components, including DNA, RNA, proteins and polysaccharides, are released from the cells. In certain embodiments, the lysate may undergo pre-treatment steps, such as purification steps to remove cell intact cells and cell debris, thus forming a partially purified (e.g., bacterial) lysate.

[0116] Where a lysing agent is employed (chemical lysis), the lysing agent is used to break down cell membranes, thus releasing plasmid DNA, host cell DNA, RNA and host cell proteins. One preferred lysing agent comprises an alkaline solution. A variety of bases may be employed in conventional alkaline lysis procedures, including, for example, hydroxide salts, such as potassium hydroxide (KOH), lithium hydroxide (LiOH), or sodium hydroxide (NaOH). Typically, the base is sodium hydroxide. Often, detergents are employed in lysing solutions, either alone or in combination with the alkaline solution. In general, and depending on the application, the detergent may be a cationic, anionic, non-ionic, or zwitterionic detergent, or a combination thereof. One exemplary anionic detergent is sodium dodecyl sulfate (SDS). One exemplary zwitterionic or nonionic detergent is Tween 20.

[0117] In certain embodiments of all aspects and embodiments, the lysing of the host cells is by alkaline lysis, thus forming an alkaline lysate. In an embodiment, the alkaline lysate is neutralized by an acidic solution. In an embodiment, the acid is selected from weak acids such acetic acid, citric acid, formic acid, boric acid, hydrofluoric acid, and glycine. In an embodiment, the neutralization is by adding a neutralization buffer. In an embodiment, the neutralization buffer comprises an acetic solution that comprises an acetic salt. In an embodiment, the neutralizing buffer comprises acetic acid, and potassium acetate (KAc).

[0118] In an embodiment of all aspects and embodiments, the separation step after the neutralization comprises centrifugation, filtration, or a combination thereof.

[0119] Mechanical methods for lysing bacterial cells, for use either alone or in combination with lysis solutions and agents, include agitation, sonication, centrifugation, freeze / thawing, French cell press, and the like.

[0120] Alkaline lysis and mechanical techniques for lysing bacterial cells to release and extract proteins and nucleic acids are generally well known, and are described, for example, in Sambrook et al., supra.

[0121] In certain embodiments of all aspects and embodiments, the alkaline lysis of the bacterial, e.g. E.coli, host cells comprises re-suspending harvested host cells by centrifugation or filtration in a buffer solution of Sol. I (e.g., 50 mM Tris-HCl, 10 mM ethylenediamine tetraacetic acid (EDTA), pH 8.0) and then lysing the cells by adding a Sol. II (e.g., 0.2 N NaOH and 1 % SDS). The exact formulations for Sol. I and Sol. II are not limited. Sol. I generally provides host cell suspension in the solution creating an isosmotic environment and Sol. II generally provides a strong base in order to lyse the cells. Those having ordinary skill in the art will understand that any commonly known solutions for Sol. I and Sol. II may be used to carry out the lysing step.

[0122] To lyse the host cells, the volume ratio of Sol. I to Sol. II is not fixed, and can range anywhere from about 1 :2 to about 2: 1. In an embodiment, Sol. I and Sol. II with the above-described formulations are used at the ratio of about 1 : 1. The actual volume used of each Solution depends on the amount of cells to be processed. The person skilled in the art, guided by the disclosure herein, can optimize the ratio of Sol. I to Sol. II to provide optimal lysing of the host cells.

[0123] Following the lysis step, host cell impurities are precipitated from the lysate.

[0124] In an embodiment of all aspects and embodiments, a solution comprising a neutralization buffer is added to cause precipitation of host components. In an embodiment, the solution comprises acetic acid and an acetic acid salt. Various acetic acid salts may be used, such as ammonium acetate, sodium acetate and potassium acetate. The concentration of the acetic acid and acetate salt, such as potassium acetate, in the neutralization buffer can vary over a wide range. In an embodiment, the concentration of acetic acid is about 0.1 M to about 6 M. In an embodiment, the concentration of acetic acid is about 0.5 M to about 5 M. In an embodiment, the concentration of acetic acid is about 1 M to about 4 M. In an embodiment, the concentration of acetic acid is about 2 M to about 3 M. In an embodiment, the concentration of acetate salt is about 0.05 M to about 6 M. In an embodiment, the concentration of acetate salt is about 0.1 M to about 5 M. In an embodiment, the concentration of acetate salt is about 0.4 M to about 3 M. In an embodiment, the concentration of acetate salt is about 0.6 M to about 1 M.

[0125] In some embodiments of all aspects and embodiments, it may be desirable to form a cleared lysate preparation, in which the chromosomal DNA, proteins, and membrane portions of the host cells have been at least partially removed, such as by chemical treatment or centrifugation of the lysate, thereby leaving a solution containing plasmid DNA. RNAse can optionally be added at various points in the procedure to create a cleared lysate that is substantially free of RNA. As noted elsewhere herein, initial removal of many cellular and nucleic acid contaminants can improve further purification of the plasmid DNA using conventional chromatographic techniques. Methods of creating cleared lysates are well-known in the art. For example, a cleared lysate can be produced by treating the host cells with sodium hydroxide or its equivalent (0.2 N) sodium dodecyl sulfate (SDS) (1 % ), centrifuging, and discarding the supernatant. This method of creating a cleared lysate is generally described, for example, in Burnboim et al., Nucl. Acids Res., 7, 1513 (1979); and Horowicz et al., Nucl. Acids Res., 9, 2989 (1981).

[0126] For many uses, e.g., therapeutic uses such as in gene therapy or the formation of vaccines, it may be desirable to further purify the nucleic acid obtained from the bacterial or other lysate, either before or after the sample is processed with the method according to the current invention.

[0127] After forming the bacterial lysate as described above, it may be advantageous to subject the crude lysate to separation techniques to remove at least part of the other nucleic acid, protein, and cell contaminants that are present, before attempting to purify the plasmid DNA with the method according to the current invention.

[0128] Once the biological material is lysed or otherwise prepared, the material is subjected to the method according to the current invention. As noted above, the purified recombinant plasmid DNA obtained with the method according to the current invention can thereafter be subjected to one or more chromatography method steps. In one embodiment, a combination of chromatographic separations are employed. Thus, for example, the recombinant plasmid DNA purified with the method according to the current invention may be subjected to one or more chromatography steps. Suitable chromatography steps include, for example, ion exchange chromatography (such as anion exchange chromatography and cation exchange chromatography), hydrophobic interaction chromatography, and affinity chromatography, among a range of others. In one embodiment, the chromatography step is selected from the group consisting of an anion exchange chromatography step, a cation exchange chromatography step, a hydrophobic interaction chromatography step, and an affinity chromatography step.

[0129] Thus, as noted above, the method of the present invention may also use further steps of size exclusion chromatography (SEC), reversed-phase chromatography, hydroxyapatite chromatography, and / or other available chromatography techniques, methods, or systems in combination with the steps according to the current invention.

[0130] A flocculate removal step may also be employed to provide higher purity to the resulting recombinant plasmid DNA product. This step may be used to remove a large portion of precipitated material (flocculate). One mechanism of performing flocculate removal is through a lysate filtration step, such as through a 1 to 5 mm, and preferably a 3.5 mm grid filter, followed by a depth filtration according to the current invention as a polishing filtration step. Other methods of performing flocculate removal are through centrifugation or settling. Alternatively, the flocculate may be removed by ion exchange chromatography.

[0131] At various times in the recombinant plasmid DNA purification process according to the current invention, therefore, the sample may be subjected to one or more of ion exchange chromatography (e.g., anion or cation exchange chromatography), affinity chromatography, hydrophobic interaction chromatography, and filtration (e.g., filtered through a 0.2 pm and / or 0.45 pm filter), and, optionally, filtered or subjected to chromatography methods a second, third, or fourth time, or more.

[0132] Thus, for example, the sample can be subjected to a first chromatography step, subjected to the method according to the current invention, subjected to a second chromatography step, and subjected to a third chromatography step. The sample may also be filtered at various times in the sequence, such as after the first chromatographic separation, or after the third chromatographic separation. It will be understood that subj ecting the sample to a chromatographic step also involves eluting the desired portion of the sample (such as that portion containing purified recombinant plasmid DNA) from the chromatographic material, and discarding undesired portions of the sample. Depending on the nature of the chromatographic material, the desired portions may be retained within the chromatographic material and eluted in a separate step while the undesired materials flow through the material, or the undesired materials may be retained in the chromatographic material while the desired portions flow through the chromatographic material.

[0133] Analytical determination of plasmid DNA yield and purity can be performed at different steps during the method according to the current invention. Typically, such assays are performed before and after each purification step, as well as to each nucleic acid-containing fraction from, e.g., preparative ion exchange chromatography or filtration. Representative means for performing these analytical determinations include HPLC analysis of purity, spectrophotometric estimation of yield, agarose gel electrophoresis and Southern blotting for DNA analysis. In certain embodiments, the processes according to the current invention yields a purified concentrate with a recombinant plasmid DNA concentration (including, for example, predominantly supercoiled or other plasmid DNA) of around 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, and preferably 99 %, or greater.

[0134] GENERAL METHODS FOR PRODUCING rAAVp

[0135] Piras et al. employed adherent HEK293T / 17 cells cultured in Dulbecco’s Modified Eagle’s Medium with 10% fetal bovine serum supplemented with 2 mmol / 1 GlutaMAX (Life Technologies, Grand Island, NY). AAV was produced by two-plasmid transfection using PEIpro(TM).

[0136] Powers, A.D., et al. (Hum. Gene Ther. Meth. 27 (2016) 112-121) reported the development and optimization of AAV hFIX particle production by transient transfection in an iCELLis(R) fixed- bed bioreactor. On day 3 after inoculation with HEK293T / 17 cells, the vessel was transfected with plasmid scAAV-LPl-hFIXco-helpv3 and plasmid CR21+LTAAV help 2-8 using polyethylenimine.

[0137] Poulain, A., et al. (J. Biotechnol. 255 (2017) 16-27) reported rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch. Cells were transfected using linear polyethylenimine. WO 2017 / 096039 reported scalable methods for producing recombinant AAV vectors in serum- free suspension cell culture systems suitable for clinical use. Production of rAAV vectors was performed in bioreactors with HEK293F cells using triple transfection with a plasmid ratio of 1 : 1 : 1 and a PEI-based transfection reagent.

[0138] Koo, T., et al. (Nat. Commun. 9 (2018) 1855) reported that CRISPR-LbCpfl prevents choroidal neovascularization in a mouse model of age-related macular degeneration. To produce AAV vectors, they were pseudotyped in AAV9 capsids. HEK293T cells (ATCC, CRL-3216) were transfected with pAAV-ITR-LbCpfl-crRNA, pAAV2 / 9 encoding for AAV2rep and AAV9cap, and helper plasmid. Recombinant pseudotyped AAV vector stocks were generated using PEI coprecipitation and triple-transfection with plasmids at a molar ratio of 1 : 1 : 1 in HEK293T cells. After 72 h of incubation, cells were lysed and particles were purified by iodixanol step-gradient ultracentrifugati on .

[0139] The Rep proteins from AAV2 are commonly and nearly exclusively used in the production of rAAVps derived from the serotypes AAV1 to AAV13 (Daya, S., and Berns, K.I., Clin. Microbiol. Rev. 21 (2008) 583-593; Zincarelli, C., et al., Mol. Ther. 16 (2008) 1073-1080).

[0140] WO 2019 / 094253 reported means and methods for preparing viral vectors and uses thereof. Adherent HEK293 cells were cultivated in bioreactors and triple transfected (plasmid ratio 1 : 1 : 1) with PEI / DNA at a PEI-plasmid ratio of about 1 : 1 by weight.

[0141] Collaud, F. et al. (Mol. Ther. Meth. Clin. Dev. 12 (2019) 157-174) reported a fully scalable method based on triple transfection of HEK293 cells cultured in suspension. AAV vectors were recovered from both supernatant and cells by mild detergent lysis followed by AVB Sepharose affinity column purification. Purified vectors were then concentrated and tested for quality and potency.

[0142] Nyamay’antu, A., et al. (Cell Gen. Ther. Ins. 6 (2020) 655-661) reported that the efficiency of the delivery process is essential to obtain a high number of producing cells. Of the existing transfection methods, the use of PEI-based transfection reagent is predominant in gene therapy as it combines affordability and compatibility for transfection of adherent and suspension cells. In more detail, suspension HEK293T cells were transfected using the respective transfection reagent under the recommended conditions. rAAV2-GFP were harvested 72 hours post transfection. The results are almost independent of the employed cultivation medium. In a blog article entitled “Optimization of AAV production for high-yielding and scalable GMP processes with Catalent” (www.polyplus-transfection.com) different transfection reagent to DNA ratios were tested with the two serotypes AAV9 (1 :1 and 2: 1) and AAV2 (3:1.5 and 5:2.5). In a further study comparing additional AAV2 and AAV5 vectors (different from the previous AAV2 and AAV5 vectors) and using a DoE approach to optimization, experiments were conducted varying transfection reagent to DNA ratios (3:2, 3: 1.5) and plasmid DNA molar ratios (1 : 1 : 1, 2: 1 :2, 1 :2: 1) were performed.

[0143] WO 2018 / 192983 reported an adeno-associated virus (AAV) producer cell comprising nucleic acid sequences encoding rep / cap gene; helper virus genes; and the DNA genome of the AAV vector particle, wherein said nucleic acid sequences are all integrated together at a single locus within the AAV producer cell genome. Reported are also nucleic acid vectors comprising a non-mammalian origin of replication and the ability to hold at least 25 kilo bases (kb) of DNA, characterized in that said nucleic acid vector comprises nucleic acid sequences encoding: rep / cap gene, and helper virus genes as well as uses and methods using said nucleic acid vector in order to produce stable AAV packaging and producer cell lines.

[0144] WO 2020 / 132059 reported a mammalian cell for producing an adeno-associated virus (AAV), comprising (a) a nucleic acid molecule encoding a viral helper gene under control of a first derepressible promoter; (b) a nucleic acid molecule encoding an AAV gene under control of a second derepressible promoter; and (c) a nucleic acid molecule encoding a repressor element of the first and the second derepressible promoters.

[0145] EP 3 822 346 reported the use of an engineered mammalian packaging cell line for producing recombinant virus particles, wherein the cell line is engineered to lack cell surface expression of heparan sulfate. Further disclosed are a method for producing recombinant virus particles and a recombinant virus particle obtainable by the method. Further disclosed is a mammalian packaging cell line deposited under number DSM ACC3355 or DSM ACC3356.

[0146] WO 2022 / 112218 reported methods for the production of Adeno-associated vims (AAV), comprising steps of providing a stable AAV producer cell line in which at least some or all genes encoding the components necessary for the production of AAV are stably integrated into the cell genome, and culturing said cells in perfusion culture during the AAV production step (i.e., during the N step), wherein said perfusion culture encompasses continuous replacement of spent media with fresh media, and wherein said continuous replacement of spent media with fresh media continues after the induction of AAV production. In the cell at least (a) a gene encoding the AAV Rep protein Rep78 or Rep68, (b) a gene encoding the AAV Rep protein Rep52 or Rep40; (c) the genes encoding the adenoviral helper functions E4orf6 and E2A stably integrated into the host cell genome. Further at least the following genes are stably integrated into the host cell genome (a) the genes encoding the AAV Cap proteins VP1, VP2, VP3; (b) a gene encoding the AAV Rep protein Rep78 or Rep68; (c) a gene encoding the AAV Rep protein Rep52 or Rep40; (d) the genes encoding the adenoviral helper functions E4orf6 E2A; (e) the gene of interest flanked by AAV ITRs.

[0147] WO 2022 / 173944 reported methods for producing an adeno-associated virus (AAV) in an El complementary producer cell. Especially is reported a method of producing an adeno-associated virus (AAV) in an El complementary producer cell, comprising (a) transfecting the El complementary producer cell with one or more vectors comprising (1) an El A adenovirus helper gene; (2) an adenovirus helper gene selected from E2A, E4, or both; (3) a viral-associated, noncoding RNA (VA RNA); and (4) an AA V gene selected from Rep, Cap, or both; (b) culturing the transfected El complementary producer cell under conditions suitable for producing the AAV; and (c) purifying the AAV from the cultured El complementary producer cell, thereby obtaining the AAV.

[0148] WO 2022 / 192261 reported compositions and methods for producing and characterizing stable viral vector producer cell lines that enable industrial scale production of viral vectors. Novel viral vector genome constructs, in which the constructs can be precisely mapped and viral vector genome constructs precisely quantified, are also disclosed for efficient production and characterization of viral vectors in mammalian cells.

[0149] WO 2023 / 077078 reported recombinant adeno-associated virus (rAAV) packaging and / or producer cell lines which have been engineered to reduce expression and / or activity of one or more genes and / or proteins to increase rAAV titers.

[0150] WO 2023 / 102549 reported systems for increasing AAV particle production. These systems comprise producer cell lines adapted for the production of AAV particles, as well as methods of producing AAV particles using said producer cell lines. Also provided are AAV particles produced by said production systems, producer cell lines and methods. WO 2023 / 114897 reported methods for the production of recombinant adeno-associated virus (rAAV) particles. These methods are particularly useful for the large-scale production of AAV particles. Especially it is reported A method for producing recombinant AAV (rAAV) particles, comprising (a) introducing into a mammalian cell a first polynucleotide comprising an rAAV genome, to generate an AA V producer cell; (b) culturing the AA V producer cell in a first culture medium at a first temperature for a first period of time; (c) culturing the AAV producer cell in a second culture medium at a second temperature for a second period of time, wherein the second temperature is about 38°C to about 42°C, such that rAAV particles are produced by the AAV producer cell, wherein the rAAV particles comprise an rAAV genome comprising a transgene, and an AAV capsid comprising an AAV capsid protein.

[0151] The content of all documents outlined in this section are expressly incorporated by reference herein.

[0152] RECOMBINANT CELL

[0153] Generally, for efficient as well as large-scale production of a rAAVp a cell expressing and, if possible, also secreting said rAAVp is used. Such a cell is termed “recombinant producer cell” or short “producer cell”.

[0154] For the generation of a recombinant producer cell a suitable mammalian cell is transfected with the nucleic acids required for producing said rAAVp, including the required AAV helper functions.

[0155] Generally, for expression of a coding sequence, i.e. of an open reading frame, additional regulatory elements, such as a promoter and a polyadenylation signal (sequence), are necessary. Thus, for functional transcription an open reading frame has to be and is operably linked to said additional regulatory elements. This can be achieved by combining these parts into a so-called expression cassette. The minimal regulatory elements required for an expression cassette to be functional in a mammalian cell are a promoter functional in said mammalian cell, which is located upstream, i.e. 5’, to the open reading frame, and a polyadenylation signal (sequence) functional in said mammalian cell, which is located downstream, i.e. 3’, to the open reading frame. Additionally a terminator sequence may be present 3’ to the polyadenylation signal (sequence). For expression, the promoter, the open reading frame / coding region and the polyadenylation signal sequence have to be arranged in an operably linked form. Likewise, a nucleic acid that is transcribed into a non-protein coding RNA is called “RNA gene”. Also for expression of an RNA gene, additional regulatory elements, such as a promoter and a transcription termination signal or polyadenylation signal (sequence), are necessary. The nature and localization of such elements depends on the RNA polymerase that is intended to drive the expression of the RNA gene. Thus, an RNA gene is normally also integrated into an expression cassette.

[0156] In case of an rAAVp, which is composed of different (monomeric) capsid polypeptides and a therein encapsidated single stranded DNA molecule and which in addition requires other viral helper functions for production and encapsidation, a multitude of expression cassettes differing in the contained open reading frames / coding sequences are required. In this case, at least an expression cassette for each of the transgene, for the polypeptides forming the capsid of the rAAVp, for the required viral helper functions are required. Thus, individual expression cassettes at least for each of the helper functions El A, E1B, E2A, E4orf6, the rep and cap genes are required. HEK293 cells express the El A and E1B helper functions constitutively.

[0157] ADENO-ASSOCIATED VIRUS (AAV)

[0158] For a general review of AAVs and of the adenovirus or herpes helper functions see, Berns and Bohensky, Advances in Virus Research, Academic Press., 32 (1987) 243-306. The genome of AAV is described in Srivastava et al., J. Virol., 45 (1983) 555-564. In US 4,797,368 design considerations for constructing recombinant AAV vectors are described (see also WO 93 / 24641). Additional references describing AAV vectors are West et al., Virol. 160 (1987) 38-47; Kotin, Hum. Gene Ther. 5 (1994) 793-801; and Muzyczka J. Clin. Invest. 94 (1994) 1351. Construction of recombinant AAV vectors is described in US 5,173,414; Lebkowski et al., Mol. Cell. Biol. 8 (1988) 3988-3996; Tratschin et al., Mol. Cell. Biol. 5 (1985) 3251-3260; Tratschin et al., Mol. Cell. Biol., 4 (1994) 2072-2081; Hermonat and Muzyczka Proc. Natl. Acad. Sci. USA 81 (1984) 6466-6470; Samulski et al. J. Virol. 63 (1989) 3822-3828.

[0159] An AAV is a replication-deficient parvovirus. It can replicate only in cells, in which certain viral functions are provided by a co-infecting helper virus, such as adenoviruses, herpesviruses and, in some cases, poxviruses such as vaccinia. Nevertheless, an AAV can replicate in virtually any cell line of human, simian or rodent origin provided that the appropriate helper viral functions are present. Without helper viral genes being present, an AAV establishes latency in its host cell. Its genome integrates into a specific site in chromosome 19 [(Chr) 19 (ql3.4)], which is termed the adeno- associated virus integration site 1 (AAVS1). For specific serotypes, such as AAV2 other integration sites have been found, such as, e.g., on chromosome 5 [(Chr) 5 (pl3.3)], termed AAVS2, and on chromosome 3 [(Chr) 3 (p24.3)], termed AAVS3.

[0160] AAVs are categorized into different serotypes. These have been allocated based on parameters, such as hemagglutination, tumorigenicity and DNA sequence homology. Up to now, more than 12 different serotypes and more than a hundred sequences corresponding to different clades of AAV have been identified.

[0161] The capsid protein type and symmetry determines the tissue tropism of the respective AAV. For example, AAV2, AAV4 and AAV5 are specific to retina, AAV2, AAV5, AAV8, AAV9 and AAV- rh.10 are specific for brain, AAV1, AAV2, AAV6, AAV8 and AAV9 are specific for cardiac tissue, AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10 are specific for liver, AAV1, AAV2, AAV5 and AAV9 are specific for lung.

[0162] Pseudotyping denotes a process comprising the cross packaging of the AAV genome between various serotypes, i.e. the genome is packaged with differently originating capsid proteins.

[0163] The wild-type AAV genome has a size of about 4.7 kb. The AAV genome further comprises two overlapping genes named rep and cap, which comprise multiple open reading frames (see, e.g., Srivastava et al., J. Viral., 45 (1983) 555-564; Hermonat et al., J. Viral. 51 (1984) 329-339; Tratschin et al., J. Virol., 51 (1984) 611-619). The Rep protein encoding open reading frame provides for four proteins of different size, which are termed Rep78, Rep68, Rep52 and Rep40. These are involved in replication, rescue and integration of the AAV. The Cap protein encoding open reading frame provides four proteins, which are termed VP 1, VP2, VP3, and AAP. VP1, VP2 and VP3 are part of the proteinaceous capsid of the AAV particles. The combined rep and cap open reading frames are flanked at their 5'- and 3'-ends by so-called inverted terminal repeats (ITRs). For replication, an AAV requires in addition to the Rep and Cap proteins the products of the genes El A, E1B, E4orf6, E2A and VA of an adenovirus or corresponding factors of another helper virus.

[0164] In the case of an AAV of the serotype 2 (AAV2), for example, the ITRs each have a length of 145 nucleotides and flank a coding sequence region of about 4470 nucleotides. Of the ITR’s 145 nucleotides 125 nucleotides have a palindromic sequence and can form a T-shaped hairpin structure. This structure has the function of a primer during viral replication. The remaining 20, non-paired, nucleotides are denoted as D-sequence.

[0165] The wild-type AAV genome harbors three transcription promoters P5, Pl 9, and P40 (Laughlin et al., Proc. Natl. Acad. Sci. USA 76 (1979) 5567-5571) for the expression of the rep and cap genes.

[0166] The ITR sequences have to be present in cis to the coding region. The ITRs provide a functional origin of replication (ori), signals required for integration into the target cell’s genome, and efficient excision and rescue from host cell chromosomes or recombinant plasmids. The ITRs further comprise origin of replication like-elements, such as a Rep-protein binding site (RBS) and a terminal resolution site (TRS). It has been found that the ITRs themselves can have the function of a transcription promoter (Flotte et al., J. Biol. Chem. 268 (1993) 3781-3790; Flotte et al., Proc. Natl. Acad. Sci. USA 93 (1993) 10163-10167).

[0167] For replication and encapsidation, respectively, of the viral single-stranded DNA genome an in trans organization of the rep and cap gene products is required.

[0168] The rep gene locus comprises two internal promoters, termed P5 and Pl 9. It comprises open reading frames for four proteins. Promoter P5 is operably linked to a nucleic acid sequence providing for non-spliced 4.2 kb mRNA encoding the Rep protein Rep78 (chromatin nickase to arrest cell cycle), and a spliced 3.9 kb mRNA encoding the Rep protein Rep68 (site-specific endonuclease). Promoter P19 is operably linked to a nucleic acid sequence providing for a nonspliced mRNA encoding the Rep protein Rep52 and a spliced 3.3 kb mRNA encoding the Rep protein Rep40 (DNA helicases for accumulation and packaging).

[0169] The two larger Rep proteins, Rep78 and Rep68, are essential for AAV duplex DNA replication, whereas the smaller Rep proteins, Rep52 and Rep40, seem to be essential for progeny and singlestrand DNA accumulation (Chejanovsky & Carter, Virology 173 (1989) 120-128).

[0170] The larger Rep proteins, Rep68 and Rep78, can specifically bind to the hairpin conformation of the AAV ITR. They exhibit defined enzyme activities, which are required for resolving replication at the AAV termini. Expression of Rep78 or Rep68 could be sufficient for infectious particle formation (Holscher, C., et al. J. Virol. 68 (1994) 7169-7177 and 69 (1995) 6880-6885). It is deemed that all Rep proteins, primarily Rep78 and Rep68, exhibit regulatory activities, such as induction and suppression of AAV genes as well as inhibitory effects on cell growth (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894; Labow et al., Mol. Cell. Biol., 7 (1987) 1320-1325; Khleif et al., Virology, 181 (1991) 738-741).

[0171] Recombinant overexpression of Rep78 results in phenotype with reduced cell growth due to the induction of DNA damage. Thereby the host cell is arrested in the S phase, whereby latent infection by the virus is facilitated (Berthet, C., et al., Proc. Natl. Acad. Sci. USA 102 (2005) 13634-13639).

[0172] Tratschin et al. reported that the P5 promoter is negatively auto-regulated by Rep78 or Rep68 (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894). Due to the toxic effects of expression of the Rep protein, only very low expression has been reported for certain cell lines after stable integration of AAV (see, e.g., Mendelson et al., Virol. 166 (1988) 154-165).

[0173] The cap gene locus comprises one promoter, termed P40. Promoter P40 is operably linked to a nucleic acid sequence providing for 2.6 kb mRNA, which, by alternative splicing and use of alternative start codons, encodes the Cap proteins VP1 (87 kDa, non-spliced mRNA transcript), VP2 (72 kDa, from the spliced mRNA transcript), and VP3 (61 kDa, from alternative start codon). VP1 to VP3 constitute the building blocks of the viral capsid. The capsid has the function to bind to a cell surface receptor and allow for intracellular trafficking of the virus. VP3 accounts for about 90 % of total viral particle protein. Nevertheless, all three proteins are essential for effective capsid production.

[0174] It has been reported that inactivation of all three capsid proteins VP 1 to VP3 prevents accumulation of single-strand progeny AAV DNA. Mutations in the VP1 amino-terminus ("Lip-negative" or "Inf-negative") still allows for assembly of single-stranded DNA into viral particles whereby the infectious titer is greatly reduced.

[0175] The AAP open reading frame is encoding the assembly activating protein (AAP). It has a size of about 22 kDa and transports the native VP proteins into the nucleolar region for capsid assembly. This open reading frame is located upstream of the VP3 protein encoding sequence.

[0176] In individual AAV particles, only one single-stranded DNA molecule is contained. This may be either the "plus" or "minus" strand. AAV particles containing a DNA molecule are infectious. Inside the infected cell, the parental infecting single stranded DNA is converted into a double stranded DNA, which is subsequently amplified. The amplification results in a large pool of double stranded DNA molecules from which single strands are displaced and packaged into capsids.

[0177] Adeno-associated viral (AAV) vectors can transduce dividing cells as well as resting cells. It can be assumed that a transgene introduced using an AAV vector into a target cell will be expressed for a long period. One drawback of using an AAV vector is the limitation of the size of the transgene that can be introduced into cells.

[0178] Parvovirus particles, including AAV serotypes and variants thereof, provide a means for ex vivo, in vitro and in vivo delivery of nucleic acid, which encode proteins, into cells such that the infected cells express the encoded protein. AAVs are viruses useful as gene therapy vectors as they can penetrate cells and introduce nucleic acid / genetic material so that the nucleic acid / genetic material may be stably maintained in the infected cells. Because AAV are not associated with pathogenic disease in humans, AAVs are able to deliver heterologous polynucleotide sequences (e.g., therapeutic proteins and agents) to human patients without causing substantial AAV-related pathogenesis or disease.

[0179] AAV particles used as vehicles for effective gene delivery possess a number of desirable features for such applications, including tropism for dividing and non-dividing cells. Early clinical experience with these vectors also demonstrated no sustained toxicity and immune responses were minimal or undetectable. AAV are known to infect a wide variety of cell types in vivo and in vitro by receptor-mediated endocytosis or by transcytosis. These vector systems have been tested in humans targeting retinal epithelium, liver, skeletal muscle, airways, brain, joints and hematopoietic stem cells.

[0180] Recombinant AAV particles do not typically include viral genes associated with pathogenesis. Such particles typically comprise a genome, wherein one or more of the wild-type AAV genes have been deleted in whole or in part, for example, rep and / or cap genes, but retain at least one functional flanking ITR sequence, as necessary for the rescue, replication, and packaging of the recombinant vector into an rAAV. Thus, an AAV vector includes sequences required in cis for replication and packaging (i.e. functional ITR sequences).

[0181] Recombinant AAV particles, as well as methods and uses thereof, can be based on any wild-type AAV genome or serotype or combination thereof. As a non-limiting example, a rAAV can be based upon any wild-type AAV genome, i.e. comprise the respective ITR sequences, such as AAV1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, 2i8, rh.74, rh.10 or 7m8 for example. Such particles can be based on the same strain or serotype (or subgroup or variant), or be different from each other. As a non-limiting example, a rAAV based upon one wild-type genome can be identical or different to one or more of the capsid proteins that package the vector. In addition, a recombinant AAV vector can be based upon an AAV (e.g., AAV2) wild-type serotype genome distinct from one or more of the AAV capsid proteins that package the vector. For example, the AAV vector can be based upon AAV2, whereas at least one of the three capsid proteins could be an AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV-rh.74, AAV-rh.10 or AAV-7m8 or a variant thereof, for example. AAV variants include variants and chimeras of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV-rh.74, AAV-rh.10 and AAV-7m8 capsids.

[0182] In certain embodiments of all aspects and embodiments of the invention, the rAAVp is derived from a wild-type AAV particle selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV-rh.74, AAV-rh.10 and AAV-7m8, as well as variants (e.g., capsid variants, such as amino acid insertions, additions, substitutions and deletions) thereof, for example, as set forth in WO 2013 / 158879, WO 2015 / 013313 and US 2013 / 0059732 (disclosing LK01, LK02, LK03, etc.).

[0183] In certain embodiments of all aspects and embodiments of the invention, the rAAVp comprises a capsid polypeptides with an amino acid sequence having 70 % or more sequence identity to an wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV-rh.10, AAV-rh.74, or AAV-7m8 capsid sequence.

[0184] In certain embodiments of all aspects and embodiments of the invention, the rAAVp comprises one or two ITR sequence having 70 % or more sequence identity to a wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12 ITR sequence.

[0185] Recombinant AAV particles can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are useful for, among other things, administration and delivery to a subject in vivo or ex vivo. In certain embodiments, the pharmaceutical composition contains a pharmaceutically acceptable carrier or excipient. Such excipients include any pharmaceutical agent that does not itself induce an immune response harmful to the individual receiving the composition, and which may be administered without undue toxicity. Protocols for the generation of adenoviral vectors have been described in US 5,998,205; US 6,228,646; US 6,093,699; US 6,100,242; WO 94 / 17810 and WO 94 / 23744, which are incorporated herein by reference in their entirety.

[0186] RECOMBINANT ADENO- ASSOCIATED VIRAL PARTICLES (rAAVp)

[0187] Different methods are known in the art for generating recombinant AAV particles. For example, transfection with an AAV vector comprising plasmid and a plasmid comprising AAV helper sequences (rep and cap) in conjunction with co-infection with one AAV helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus) or transfection with a recombinant AAV vector comprising plasmid, an AAV helper plasmid (comprising rep and cap), and an helper function plasmid. Non-limiting methods for generating rAAV are described, for example, in US 6,001,650, US 6,004,797, WO 2017 / 096039, and WO 2018 / 226887. Following rAAV production (i.e. particle generation in cell culture systems), rAAV can be obtained from the host cells and / or cell culture supernatant and purified.

[0188] For the generation of recombinant AAV particles, expression of the Rep and Cap proteins, the helper proteins E1A, E1B, E2A and E4orf6 as well as optionally the adenoviral VA RNA in a single mammalian cell is required. The helper proteins E1A, E1B, E2A and E4orf6 can be expressed using any promoter as shown by Matsushita et al. (Gene Ther. 5 (1998) 938-945), especially the CMV IE promoter. Thus, any promoter can be operably linked to said genes for functional expression.

[0189] Generally, to produce rAAV, different, complementing plasmids are co-transfected into a host cell. One of the plasmids comprises the transgene sandwiched between the two cis acting AAV ITRs. The missing AAV elements required for replication and subsequent packaging of progeny recombinant genomes, i.e. the open reading frames for the Rep and Cap proteins, are contained in trans on a second plasmid. The overexpression of the Rep proteins results in inhibitory effects on cell growth (Li, J., et al., J. Virol. 71 (1997) 5236-5243). Additionally, a third plasmid comprising the genes of a helper virus, i.e. El, E4orf6, E2A and VA from adenovirus, is required for rAAV production.

[0190] To reduce the number of required plasmids, rep, cap and the adenovirus helper genes may be combined on a single plasmid. Alternatively, the host cell may already stably express the El gene products. Such a cell is a HEK293 cell. The human embryonic kidney clone denoted as 293 was generated back in 1977 by integrating adenoviral DNA into human embryonic kidney cells (HEK cells) (Graham, F.L., et al., J. Gen. Virol. 36 (1977) 59-74). The HEK293 cell line comprises base pair 1 to 4344 of the adenovirus serotype 5 genome. This encompasses the El A and E1B genes as well as the adenoviral packaging signals (Louis, N., et al., Virology 233 (1997) 423-429).

[0191] When using HEK293 cells the missing E2A, E4orf6 and VA genes can be introduced either by coinfection with an adenovirus or by co-transfection with an E2A-, E4orf6- and VA-expressing plasmid (see, e.g., Samulski, R.J., et al., J. Virol. 63 (1989) 3822-3828; Allen, J.M., et al., J. Virol. 71 (1997) 6816-6822; Tamayose, K., et al., Hum. Gene Ther. 7 (1996) 507-513; Flotte, T.R., et al., Gene Ther. 2 (1995) 29-37; Conway, J.E., et al., J. Virol. 71 (1997) 8780-8789; Chiorini, J.A., et al., Hum. Gene Ther. 6 (1995) 1531-1541; Ferrari, F.K., et al., J. Virol. 70 (1996) 3227-3234; Salvetti, A., et al., Hum. Gene Ther. 9 (1998) 695-706; Xiao, X., et al., J. Virol. 72 (1998) 2224- 2232; Grimm, D., et al., Hum. Gene Ther. 9 (1998) 2745-2760; Zhang, X., et al., Hum. Gene Ther. 10 (1999) 2527-2537). Alternatively, adenovirus / AAV or herpes simplex virus / AAV hybrid vectors can be used (see, e.g., Conway, J.E., et al., J. Virol. 71 (1997) 8780-8789; Johnston, K.M., et al., Hum. Gene Ther. 8 (1997) 359-370; Thrasher, A.J., et al., Gene Ther. 2 (1995) 481-485; Fisher, J.K., et al., Hum. Gene Ther. 7 (1996) 2079-2087; Johnston, K.M., et al., Hum. Gene Ther. 8 (1997) 359-370).

[0192] In order to limit the transgene activity to specific tissues, i.e. to limit the site of action, the transgene can be operably linked to an inducible or tissue specific promoter (see, e.g., Yang, Y., et al. Hum. Gene. Ther. 6 (1995) 1203-1213).

[0193] The coding sequences of E1A and E1B (open reading frames) can be derived from a human adenovirus, such as, e.g., in particular of human adenovirus serotype 2 or serotype 5. An exemplary sequence of human Ad5 (adenovirus serotype 5) is found in GenBank entries X02996, AC 000008 and that of an exemplary human Ad2 in GenBank entry AC_000007. Nucleotides 505 to 3522 comprise the nucleic acid sequences encoding E1A and E1B of human adenovirus serotype 5. Plasmid pSTK146 as reported in EP 1 230 354, as well as plasmids pGS119 and pGS122 as reported in WO 2007 / 056994, can also be used as a source for the E1A and E1B open reading frames. El A is the first viral helper gene that is expressed after adenoviral DNA enters the cell nucleus. The E1A gene encodes the 12S and 13S proteins, which are based on the same E1A mRNA by alternative splicing. Expression of the 12S and 13S proteins results in the activation of the other viral functions E1B, E2, E3 and E4. Additionally, expression of the 12S and 13S proteins force the cell into the S phase of the cell cycle. If only the El A-derived proteins are expressed, the cell will die (apoptosis).

[0194] E1B is the second viral helper gene that is expressed. It is activated by the El A-derived proteins 12S and 13S. The E1B gene derived mRNA can be spliced in two different ways resulting in a first 55 kDa transcript and a second 19 kDa transcript. The E1B 55 kDa protein is involved in the modulation of the cell cycle, the prevention of the transport of cellular mRNA in the late phase of the infection, and the prevention of ElA-induced apoptosis. The E1B 19 kDa protein is involved in the prevention of El A-induced apoptosis of cells.

[0195] The E2 gene encodes different proteins. The E2A transcript codes for the single strand-binding protein (SSBP), which is essential for AAV replication

[0196] In addition, the E4 gene encodes several proteins. The E4 gene derived 34 kDa protein (E4orf6) prevents the accumulation of cellular mRNAs in the cytoplasm together with the E1B 55 kDa protein, but also promotes the transport of viral RNAs from the cell nucleus into the cytoplasm.

[0197] The viral associated RNA (VA RNA) is a non-coding RNA of adenovirus (Ad), regulating translation. The adenoviral genome comprises two independent copies: VAI (VA RNAI) and VAII (VARNAII). Both are transcribed by RNA polymerase III (see, e.g., Machitani, M., et al., J. Contr. Rel. 154 (2011) 285-289) from a type 2 polymerases III promoter. For recombinant AAV particle production, the adenoviral VA RNA gene can be driven by any promoter.

[0198] The structure, function, and evolution of adenovirus-associated RNA using a phylogenetic approach was investigated by Ma, Y. and Mathews, M.B. (J. Virol. 70 (1996) 5083-5099). They provided alignments as well as consensus VA RNA sequences based on 47 known human adenovirus serotypes. Said disclosure is herewith incorporated by reference in its entirety into the current application.

[0199] VA RNAs, VAI and VAII, are consisting of 157-160 nucleotides (nt). Depending on the serotype, adenoviruses contain one or two VA RNA genes. VA RNAI is believed to play the dominant pro-viral role, while VA RNAII can partially compensate for the absence of VA RNAI (Vachon, V.K. and Conn, G.L., Virus Res. 212 (2016) 39-52).

[0200] The VA RNAs are not essential, but play an important role in efficient viral growth by overcoming cellular antiviral machinery. That is, although VA RNAs are not essential for viral growth, VA RNA-deleted adenovirus cannot grow during the initial step of vector generation, where only a few copies of the viral genome are present per cell, possibly because viral genes other than VA RNAs that block the cellular antiviral machinery may not be sufficiently expressed (see Maekawa, A., et al. Nature Sci. Rep. 3 (2013) 1136).

[0201] Maekawa, A., et al. (Nature Sci. Rep. 3 (2013) 1136) reported efficient production of adenovirus vector lacking genes of virus-associated RNAs that disturb cellular RNAi machinery, wherein HEK293 cells that constitutively and highly express flippase recombinase were infected to obtain VA RNA-deleted adenovirus by FLP recombinase-mediated excision of the VA RNA locus.

[0202] The human adenovirus 2 VA RNAI corresponds to nucleotides 10586-10810 of GenBank entry AC_000007 sequence. The human adenovirus 5 VA RNAI corresponds to nucleotides 10579- 10820 of GenBank entry AC_000008 sequence.

[0203] GENERAL DESCRIPTION OF RECOMBINANT AAV PARTICLE PRODUCTION

[0204] After entry into the host cell nucleus, AAV can follow either one of two distinct and interchangeable pathways of its life cycle: the lytic or the lysogenic. The former develops in cells infected with a helper virus such as Ad or herpes simplex virus (HSV) whereas the latter is established in host cells in the absence of a helper virus.

[0205] When a latently infected cell is super-infected with a helper virus, the AAV gene expression program is activated leading to the AAV Rep-mediated rescue (i.e., excision) of the provirus DNA from the host cell chromosome followed by replication and packaging of the viral genome. Finally, upon helper virus-induced cell lysis, the newly assembled virions (particles) are released. Thus, the lytic phase of the AAV life cycle is induced.

[0206] Therefore, in the presence of Ad helper functions, the rAAV vector is subjected to the wild-type AAV lytic processes by being rescued from the plasmid backbone, replicated and packaged into preformed AAV capsids as single-stranded molecules (Goncalves, M.A.F.V., Virol. J., 2 (2005) 43).

[0207] Generation of a recombinant AAV particle involves replacing a majority of the AAV's wild-type genome with a desired transgene and providing the viral genes that are essential for virus packaging in-trans on a separate plasmid. Once all components are transfected together into a packaging cell line, recombinant AAV particles are assembled using the cell’s cellular machineries. The process of viral assembly and encapsulation takes roughly two days, after which the cells are lysed to release the rAAV for further purification and concentration (https: / / old.abmgood.com / marketing / knowledge_base / Adeno_Associated_Virus_Production_and _Modifi cati on_of_A A V. php) .

[0208] AAV is not released very efficiently from the cells, although major differences have been observed between serotypes (see, e.g., Strobel, B., et al., Lamia T. Comparative Analysis of Cesium Chloride- and lodixanol -Based Purification of Recombinant Adeno- Associated Viral Vectors for Preclinical Applications. Hum. Gene Ther. Methods 26 (2015) 147-157). When harvesting the culture, a cell disruption method is usually applied to recover the vectors entrapped in the cells.

[0209] Historically, manufacturing of rAAVps was performed by double transfection of a plasmid containing the rep and the cap ORFs and a plasmid with the gene of interest flanked by ITRs. Then, a helper virus, typically Adenovirus, was co-infected (see, e.g., Aponte-Ubillus, J. J., et al., Appl. Microbiol. Biotechnol. 102 (2018) 1045-1054; Muzyczka, N., Curr. Top. Microbiol. Immunol. 158 (1992) 97-129). In this setting, the separation of the helper virus from the final product was difficult, but a critical element to avoid induction of inflammatory responses after injection into patients (see, e.g., Schnell, M.A., et al., Mol. Ther. 3 (2001) 708-722.). Therefore, production of rAAVps nowadays moved towards an adenovirus-free approach by utilizing triple transfection (see, e.g., Large, E.E., et al., Viruses 13 (2021) 1336). To this end, three components are needed: one plasmid encoding the genes for Rep and Cap without the ITRs, a second plasmid with the transgene of interest flanked by ITRs, and a helper plasmid to provide the helper genes of the helper virus (see, e.g., Aponte-Ubillus, J. J., et al., Appl. Microbiol. Biotechnol. 102 (2018) 1045- 1054; Farris, K.D. and Pintel, D.J., Hum. Gene Ther. 19 (2008) 1421-1427; Grimm, D., et al., Hum. Gene Ther. 9 (1998) 2745-2760; Ferrari, F.K., et al., Nat. Med. 3 (1997) 1295-1297). For example, the Adenovirus helper bears the minimal required adenoviral genes E2A, E4 and VA. It is important to note, that the Human Embryonic Kidney cells 293 (HEK293) constitutively express the adenoviral genes E1A / B, which are also required for production of rAAVps. Therefore, HEK293 cells are classic producer cells for rAAVps and for manufacturing. Other cell types require a supplementation of E1A / B.

[0210] Carter et al. have shown that the entire rep and cap open reading frames in the wild-type AAV genome can be deleted and replaced with a transgene (Carter, B. J., in "Handbook of Parvoviruses", ed. by P. Tijssen, CRC Press, pp. 155-168 (1990)). Further, it has been reported that the ITRs have to be maintained to retain the function of replication, rescue, packaging, and integration of the transgene into the genome of the target cell.

[0211] When cells comprising the respective viral helper genes are transduced by an AAV vector, or, vice versa, when cells comprising an integrated AAV provirus are transduced by a suitable helper virus, then the AAV provirus is activated and enters a lytic infection cycle again (Clark, K.R., et al., Hum. Gene Ther. 6 (1995) 1329-1341; Samulski, R.J., Curr. Opin. Genet. Dev. 3 (1993) 74-80).

[0212] Producer cells contain the rep and cap gene sequences, as well as the transgene cassette flanked by ITR sequences on one or more plasmids that are retained, e.g., via drug selection. Production of rAAV in these cell lines generally occurs after their infection with the required helper functions. Therefore, cells are infected either with replication-competent adenovirus (usually wild type Ad5) or a plasmid comprising the respective helper genes to supply helper virus proteins and initiate rAAV production. A packaging cell line differs from a producer cell line as it only contains the rep and cap genes.

[0213] More generally, cells transfected or transduced with DNA for the recombinant production of AAV particles can be referred to as a "recombinant cell". Such a cell can be any mammalian cell that has been used as recipient of a nucleic acid (plasmid) encoding packaging proteins, such as AAV packaging proteins, a nucleic acid (plasmid) encoding helper proteins, and a nucleic acid (plasmid) that encodes a protein or is transcribed into a transcript of interest, i.e. a transgene placed between two AAV ITRs. The term includes the progeny of the original cell, which has been transduced or transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total nucleic acid complement as the original parent, due to natural, accidental, or deliberate mutation.

[0214] Numerous cell growth media appropriate for sustaining cell viability or providing cell growth and / or proliferation are commercially available. Examples of such medium include serum free eukaryotic growth mediums, such as medium for sustaining viability or providing for the growth of mammalian (e.g., human) cells. Non-limiting examples include Ham's F12 or F12K medium (Sigma- Aldrich), FreeStyle (FS) F17 medium (Thermo-Fisher Scientific), MEM, DMEM, RPMI- 1640 (Thermo-Fisher Scientific) and mixtures thereof. Such media can be supplemented with vitamins and / or trace minerals and / or salts and / or amino acids, such as essential amino acids for mammalian (e.g., human) cells.

[0215] For producing rAAV, three plasmids are co-transfected into a mammalian cell. The transgene plasmid encodes the expression cassette, which is cloned between the AAV ITRs, whereas rep and cap genes are provided in trans by co-transfecting a second, packaging plasmid (rep / cap plasmid) to ensure AAV replication and packaging. The third plasmid, also referred to as helper plasmid, contains the minimal helper virus factors, commonly adenoviral E2A, E4orf6 and VA genes, but lacking AAV ITRs.

[0216] Diverse methods for the DNA transfer into mammalian cells have been reported in the art. These are all useful in the methods according to the current invention. In certain embodiments of all aspects and embodiments, electroporation, nucleofection, or microinjection for nucleic acid transfer / transfection is used. In certain embodiments of all aspects and embodiments, an inorganic substance (such as, e.g., calcium phosphate / DNA co-precipitation), a cationic polymer (such as, e.g., polyethylenimine, DEAE-dextran), or a cationic lipid (lipofection) is used for nucleic acid transfer / transfection is used. Calcium phosphate and polyethylenimine are the most commonly used reagents for transfection for nucleic acid transfer in larger scales (see, e.g., Baldi et al., Biotechnol. Lett. 29 (2007) 677-684), whereof polyethylenimine is preferred.

[0217] The growth in serum-free suspension culture and improvement of efficiency and reproducibility of transfection conditions using PEI as a transfection reagent permits ready scale-up the AAV production using shake-flasks, wave, or stirred-tank bioreactors.

[0218] The composition may comprise further plasmids or / and cells. Such plasmids and cells may be in contact with free PEI.

[0219] In addition to PEI, valproic acid (VP A) can be used to improve transfection efficiency. VP A, a branched short-chain fatty acid and inhibits histone deacetylase activity. Due to this reason, it is commonly added to mammalian cell culture as an enhancer of recombinant protein production. Encoded AAV packaging proteins include, in certain embodiments of all aspects and embodiments, AAV rep and / or AAV cap. Such AAV packaging proteins include, in certain embodiments of all aspects and embodiments, AAV rep and / or AAV cap proteins of any AAV serotype.

[0220] Encoded helper proteins include, in certain embodiments of all aspects and embodiments, adenovirus El A and E1B, adenovirus E2 and / or E4, VA RNA, and / or non-AAV helper proteins.

[0221] The cultivation can be performed using the generally used conditions for the cultivation of eukaryotic cells of about 37 °C, 95 % humidity and 8 vol.-% CO2. The cultivation can be performed in serum containing or serum free medium, in adherent culture or in suspension culture. The suspension cultivation can be performed in any fermentation vessel, such as, e.g., in stirred tank reactors, wave reactors, rocking bioreactors, shaker vessels or spinner vessels or so called roller bottles. Transfection can be performed in high throughput format and screening, respectively, e.g. in a 96 or 384 well format.

[0222] Methods according to the current invention can include AAV particles of any serotype, or a variant thereof. In certain embodiments of all aspects and embodiments, a recombinant AAV particle comprises any of AAV serotypes 1-12, an AAV VP1, VP2 and / or VP3 capsid protein, or a modified or variant AAV VP1, VP2 and / or VP3 capsid protein, or wild-type AAV VP1, VP2 and / or VP3 capsid protein. In certain embodiments of all aspects and embodiments, an AAV particle comprises an AAV serotype or an AAV pseudotype, where the AAV pseudotype comprises an AAV capsid serotype different from an ITR serotype.

[0223] Expression control elements include constitutive or regulatable control elements, such as a tissuespecific expression control element or promoter.

[0224] ITRs can be any of AAV2 or AAV6 or AAV8 or AAV9 serotypes, or a combination thereof. AAV particles can include any VP1, VP2 and / or VP3 capsid protein having 75 % or more sequence identity to any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV12, AAV 2i8, AAV rh.10, AAV rh.74 or AAV 7m8 VP1, VP2 and / or VP3 capsid proteins, or comprises a modified or variant VP1, VP2 and / or VP3 capsid protein selected from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV-2i8, AAV-rh.10, AAV-rh.74 and AAV-7m8 AAV serotypes. Following production of recombinant viral (e.g., AAV) particles, if desired, the viral (e.g., rAAV) particles can be purified and / or isolated from host cells using a variety of conventional methods. Such methods include column chromatography, CsCl gradients, iodixanol gradient and the like.

[0225] For example, a plurality of column purification steps such as purification over an anion exchange column, an affinity column and / or a cation exchange column can be used. (See, e.g., WO 02 / 12455 and US 2003 / 0207439). Alternatively, or in addition, an iodixanol or CsCl gradient steps can be used (see, e.g., US 2012 / 0135515; and US 2013 / 0072548). Further, if the use of infectious virus is employed to express the packaging and / or helper proteins, residual virus can be inactivated, using various methods. For example, adenovirus can be inactivated by heating to temperatures of approximately 60 °C for, e.g., 20 minutes or more. This treatment effectively inactivates the helper virus since AAV is heat stable while the helper adenovirus is heat labile.

[0226] An objective in the rAAV production and purification systems is to implement strategies to minimize / control the generation of production related impurities such as proteins, nucleic acids, and vector-related impurities, including wild-type / pseudo wild-type AAV species (wtAAV) and AAV-encapsulated residual DNA impurities.

[0227] Considering that the rAAV represents only a minor fraction of the biomass, rAAV need to be purified to a level of purity, which can be used as a clinical human gene therapy product (see, e.g., Smith P.H., et al., Mo. Therapy 7 (2003) 8348; Chadeuf G., et al, Mo. Therapy 12 (2005) 744; report from the CHMP gene therapy expert group meeting, European Medicines Agency EMEA / CHMP 2005, 183989 / 2004).

[0228] In certain embodiments of all aspects and embodiments of the method according to the current invention, as an initial step, typically the cultivated cells that produce the rAAVps are harvested, optionally in combination with harvesting cell culture supernatant (medium) in which the cells (suspension or adherent) producing recombinant AAV particles have been cultured. The harvested cells and optionally cell culture supernatant may be used as is, as appropriate, lysed or concentrated. Further, if infection is employed to express helper functions, residual helper virus can be inactivated. For example, adenovirus can be inactivated by heating to temperatures of approximately 60 °C for, e.g., 20 minutes or more, which inactivates only the helper virus since AAV is heat stable while the helper adenovirus is heat labile. The cells in the harvested cultivation broth can be lysed using methods now in the art, such as, e.g., detergent lysis or freeze-thaw cycles, to release the rAAV particles. Concurrently during cell lysis or subsequently after cell lysis, a nuclease, such as, e.g., benzonase, is added to degrade contaminating DNA. Typically, the resulting lysate is clarified to remove cell debris, e.g. by filtering or centrifuging, to render a clarified cell lysate. In a particular example, the lysate is filtered with a micron diameter pore size filter (such as a 0.1-10.0 pm pore size filter, for example, a 0.45 pm and / or pore size 0.2 pm filter), to produce a clarified lysate.

[0229] The lysate (optionally clarified) contains recombinant AAV particles (comprising full as well as empty rAAVps) and production / process related impurities, such as soluble cellular components from the host cells that can include, inter alia, cellular proteins, lipids, and / or nucleic acids, and cell culture medium components. The optionally clarified lysate is then subjected to purification steps to purify the rAAV (comprising rAAV vectors) from impurities using chromatography. The clarified lysate may be diluted or concentrated with an appropriate buffer prior to the first chromatography step.

[0230] After cell lysis, optional clarifying, and optional dilution or concentration, a plurality of subsequent and sequential chromatography steps can be used to purify the rAAV.

[0231] The first chromatography step is preferably an affinity chromatography step using an AAV affinity chromatography ligand.

[0232] If the first chromatography step is affinity chromatography the second chromatography step can be anion exchange chromatography. Thus, in certain embodiments of all aspects and embodiments, rAAV purification is via affinity chromatography, followed by purification via anion exchange chromatography or / and cation exchange chromatography or / and size exclusion chromatography, in any order or sequence or combination.

[0233] The removal of empty capsids from full ones, for example, during downstream processing is based on their different isoelectric points (pl) in anion exchange chromatography. The average calculated pl across all serotypes is 5.9 for full capsids and 6.3 for empty capsids (Venkatakrishnan, B., et al., J. Virol. 87 (2013) 4974-4984).

[0234] Cation exchange chromatography functions to separate the AAV from cellular and other components present in the clarified lysate and / or column eluate from an affinity or size exclusion chromatography. Examples of strong cation exchange resins capable of binding rAAV over a wide pH range include, without limitation, any sulfonic acid based resin as indicated by the presence of the sulfonate functional group, including aryl and alkyl substituted sulfonates, such as sulfopropyl or sulfoethyl resins. Representative matrices include but are not limited to POROS HS, POROS HS 50, POROS XS, POROS SP, and POROS S (strong cation exchangers available from Thermo Fisher Scientific, Inc., Waltham, MA, USA). Additional examples include Capto S, Capto S ImpAct, Capto S ImpRes (strong cation exchangers available from GE Healthcare, Marlborough, MA, USA), and commercial DOWEX®, AMBERLITE®, and AMBERLYST® families of resins available from Aldrich Chemical Company (Milliwaukee, WI, USA). Weak cation exchange resins include, without limitation, any carboxylic acid based resin. Exemplary cation exchange resins include carboxymethyl (CM), phospho (based on the phosphate functional group), methyl sulfonate (S) and sulfopropyl (SP) resins.

[0235] Anion exchange chromatography functions to separate rAAV from proteins, cellular and other components present in the clarified lysate and / or column eluate from an affinity or cation exchange or size exclusion chromatography. Anion exchange chromatography can also be used to reduce and thereby control the amount of empty rAAV in the eluate. For example, the anion exchange column having full and empty rAAV bound thereto can be washed with a solution comprising NaCl at a modest concentration (e.g., about 100-125 mM, such as 110-115 mM) and a portion of the empty rAAV can be eluted in the flow through without substantial elution of the full rAAV. Subsequently, full rAAV bound to the anion exchange column can be eluted using a solution comprising NaCl at a higher concentration (e.g., about 130-300 mM NaCl), thereby producing a column eluate with reduced or depleted amounts of empty rAAVps and proportionally increased amounts of full rAAV comprising an rAAV vector.

[0236] Exemplary anion exchange resins include, without limitation, those based on polyamine resins and other resins. Examples of strong anion exchange resins include those based generally on the quaternized nitrogen atom including, without limitation, quaternary ammonium salt resins such as trialkylbenzyl ammonium resins. Suitable exchange chromatography materials include, without limitation, MACRO PREP Q (strong anion-exchanger available from BioRad, Hercules, CA, USA); UNOSPHERE Q (strong anion-exchanger available from BioRad, Hercules, CA, USA); POROS 50HQ (strong anion-exchanger available from Applied Biosystems, Foster City, CA, USA); POROS XQ (strong anion-exchanger available from Applied Biosystems, Foster City, CA, USA); POROS SOD (weak anion-exchanger available from Applied Biosystems, Foster City, CA, USA); POROS 50PI (weak ani on-exchanger available from Applied Biosystems, Foster City, CA, USA); Capto Q, Capto XQ, Capto Q ImpRes, and SOURCE 30Q (strong ani on-exchanger available from GE healthcare, Marlborough, MA, USA); DEAE SEPHAROSE (weak anion- exchanger available from Amersham Biosciences, Piscataway, NJ, USA); Q SEPHAROSE (strong ani on-exchanger available from Amersham Biosciences, Piscataway, NJ, USA). Additional exemplary anion exchange resins include aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopropyl (DEPE) and quaternary amino ethyl (QAE).

[0237] A commercial manufacturing process to purify recombinant AAV particles intended as a product to treat human disease should achieve the following objectives: 1) consistent particle purity, potency and safety; 2) manufacturing process scalability; and 3) acceptable cost of manufacturing.

[0238] Exemplary processes for recombinant AAV particle purification are reported in WO 2019 / 006390.

[0239] Methods to determine infectious titer of rAAV particles containing a transgene are known in the art (see, e.g., Zhen et al., Hum. Gene Ther. 15 (2004) 709). Methods for assaying for empty rAAV and full rAAV with packaged transgenes are known (see, e.g., Grimm et al., Gene Therapy 6 (1999) 1322-1330; Sommer et al., Malec. Ther. 7 (2003) 122-128).

[0240] To determine the presence or amount of degraded / denatured capsid, purified rAAV can be subjected to SDS-polyacrylamide gel electrophoresis, consisting of any gel capable of separating the three capsid proteins, for example, a gradient gel, then running the gel until sample is separated, and blotting the gel onto nylon or nitrocellulose membranes. Anti-AAV capsid antibodies are then used as primary antibodies that bind to denatured capsid proteins (see, e.g., Wobus et al., J. Viral. 74 (2000) 9281-9293). A secondary antibody that binds to the primary antibody contains a means for detecting the primary antibody. Binding between the primary and secondary antibodies is detected semi-quantitatively to determine the amount of capsids. Another method would be analytical HPLC with a SEC column or analytical ultracentrifuge.

[0241] THE METHOD ACCORDING TO THE CURRENT INVENTION

[0242] The current invention is based, at least in part, on the unexpected finding that a positively charged filter can be used to efficiently process recombinantly produced plasmid DNA.

[0243] In more detail, it has been found that a positively charged depth filter, e.g., composed of an inorganic filter aid, such as natural silica, cellulose and a polymer resin, which is actually recommended for the reduction of negatively charged DNA, endotoxins and other host cell proteins, such as the Zeta Plus filters manufactured by 3M (comprising silica, cellulose and a polymer resin), can be used to efficiently process crude bacterial lysates comprising recombinant plasmid DNA.

[0244] Crude bacterial cell lysates typically comprise more than 50 % (by weight) host cell proteins, followed by about 25 % host cell RNA, 10 % lipids and others. Plasmid DNA accounts for the smallest part in the lysate.

[0245] The method according to the current invention is exemplified in the following using three plasmids recombinantly produced in E.coli with different sizes of about 7500 bps (plasmid A), about 11600 bps (plasmid B) and about 10000 bps (plasmid C). This data is presented in order to exemplify the working of the invention and shall not be construed as a limitation. The true scope of the invention is set forth in the appended claims.

[0246] The plasmids were produced by standard recombinant methods in E.coli. The bacterial cells were broken up using alkaline lysis.

[0247] Two different filters have been used in these experiments.

[0248] The first filter according to the current invention was a 3M Zeta Plus filter with a filter area of about 0.017 m2 (product / order number E0170FSA60SP02A9). It belongs to the Zeta Plus EXT series (capsule) with SP series media. It is composed of natural silica, cellulose, polymer resin with positive surface charges. The pore size of this filter was 0.2-5 pm. Said filter is recommended by the manufacturer for the reduction of negatively charged DNA, endotoxins and other host cell proteins and has been characterized to remove negatively charged contaminants from pharmaceutical, biological, bioprocess and cosmetic fluids, to be a pharmaceutical -grade filter exhibiting high contaminant-holding capacity and providing for economical filtration, to retain contaminants by both mechanical entrapment and electrokinetic adsorption and to allow for scalable configurations for pilot testing and scale-up using the same materials as full-size systems. A FDA Drug Master File (DMF) and USP Class VI Biological safety compliance are available.

[0249] The second comparative filter was a Sartopure PP3 filter manufactured by Sartorius. This filter is neutral and comprises a polypropylene fleece as filter aid. It is recommended for the clarification and retention of particles and colloids in water. It has a pore size of about 3 pm. The filtration experiments were performed with a constant flow of about 150 LMH (about 45 mL / min) and a pressure limit of 2 bar.

[0250] The plasmids were in a solution comprising about 15 mM Tris-HCl buffer, about 3 mM EDTA, about 60 mM NaOH, about 0.3 % (w / v)SDS, about 0.9 M KAc, about 4.5 % Acetic Acid (v / v) (corresponding to about 1.7 M), about 0.5 M CaC12 at a pH value of about 4.6 (conductivity 90 mS / cm).

[0251] The results with respect to volume and pDNA per filter area are presented in Table 1. The filtration was performed at constant flow and stopped once the pre-set pressure threshold of 2 bars was reached. It can be seen that independent of the plasmid size (7-12 kbps) the Zeta Plus filter (which has a positive charge) was better than the PP3 filter (which does not have a positive charge) by at least 4-times higher processed volumes and amounts, respectively, normalized by filter area. Thus, unexpectedly, the filter deemed suitable for the process based on the manufacturer intended use (the PP3 filter) prior to the current finding performed worse than the filter not deemed suitable for this process according to the manufacturer intended use (the Zeta Plus filter).

[0252] Table 1: Volume and pDNA per filter area with constant flow until pressure limit of 2 bar was reached.

[0253] The same effect can be seen when the process is continued once the pressure threshold has been reached with reduced flow until a lower flow limit of 10 mL / min had been reached. The respective data is shown in Table 2. Again, it can be seen that independent of the plasmid size (7-12 kbps) the Zeta Plus filter was better than the PP3 filter by at least 1.4-times higher processed volumes and amounts, respectively, normalized by filter area. Thus, unexpectedly, again the filter deemed suitable for the process based on the manufacturer intended use prior to the current finding performed worse than the filter not deemed suitable for this process according to the manufacturer intended use. Table 2: Volume and pDNA per filter area with constant flow until pressure limit of 2 bar was reached (as reference shown in italic) and continued until lower flow limit of 10 mL / min was reached.

[0254] Likewise a reduction in flow did not change the performance of the PP3 filter. The respective data is presented in Table 3. Overall reduction of flow results in increased volume until pressure limit is reached but does not substantially change volume until 10 ml / min is reached.

[0255] Table 3: Processed volumes for a lysed cell culture broth comprising plasmid C.

[0256] The Zeta Plus filter has a positive charge and as DNA has negative charge. A person skilled in the art would expect an attractive interaction between the filter aid and the recombinant plasmid DNA. Unexpectedly, this positively charged filter performs better than the neutral PP3 filter.

[0257] No substantial difference in endotoxin, host cell DNA, host cell protein, or / and RNA reduction has been seen between the different filter types.

[0258] However, the Zeta Plus filter that is actually marketed for removing DNA impurities, shows a very good performance in the filtration of bacterial cell lysates comprising recombinant plasmid DNA, i.e. pDNA is not removed by the filter despite its negative charge.

[0259] This is an unexpected effect as the Zeta Plus has a positive charge and should interact with the negatively charged plasmid DNA, but does not remove the recombinant pDNA from the processed sample. In contrast, the PP3 filter which has a neutral charge was expected to perform better. In more detail, the Zeta Plus filter achieved higher filtration area normalized filtrated volume (L / m2) and thereby also a higher filtered plasmid amount per filtration area (mg pDNA / m2). Thus, in a production environment where large volumes and amounts have to be processed these properties will save costs due to smaller filter area or less frequent filter changes required. Especially, it has to be pointed out that the effect as outlined above has not been observed with already purified plasmid preparations, i.e. it is depending on the processed plasmid solution. Therefore, the method according to the current invention is especially useful for processing crude E.coli lysate comprising a neutralized lysis buffer. That is, the method according to the current invention is especially useful in removing non-plasmid E.coli components from crude E.coli lysates containing recombinantly produced plasmid DNA. It is assumed that these non-plasmid components are depending on, i.e. are characteristic for, the microorganism used for the production of the recombinant plasmid DNA.

[0260] ***

[0261] The following Examples 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.

[0262] EXAMPLES

[0263] Alkaline Lysis

[0264] Alkaline lysis was performed for cell disruption according to literature (see, e.g., Diogo, M.M., et al., J. Chrom. A 998 (2003) 109-117; Freitas, S., et al., Biotechol. J. 4 (2009) 265-278; Bimboim, H.C. and Doly, J., Nuc. Acids Res. 7 (1979) 1513-1523).

[0265] Cells were resuspended in 25 mmol / L tri s(hydroxymethyl)aminom ethane (Tris)-HCl, and 10 mmol / L ethylenediaminetetraacetic acid (EDTA) at a pH value of pH 8.0, at a ratio of about 10 mL of buffer per 1 g of biomass (corresponding to about 50 mL of buffer per 1 OD of cell culture volume). Alkaline lysates were prepared by adding 8 mL / g biomass of a 200 mmol / L NaOH, 1 % (w / v) sodium dodecyl sulphate (SDS) solution. Lysis was halted after 5 min by adding about 10 mL / g biomass of a pre-chilled 3 mol / L potassium acetate (pH 5.5) solution prepared with 30% (v / v) acetic acid, potassium acetate and water. Cell debris, precipitated proteins and precipitated genomic DNA (gDNA) were removed by sedimentation and the resulting supernatant was used for further experiments.

[0266] Plasmid Concentration Determination

[0267] The concentration of pDNA in process samples was determined using a high performance liquid chromatography (HPLC) system (Ultimate 3000, Thermo Fischer). A CIMac™ pDNA-column (BIA separations) with a pore size of 6 pm was used. For analysis 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 % HPLC buffer B. Elution was performed using a gradient of buffer B, as shown in table 4. Table 4: HPLC method for the measurement of pDNA concentration.

[0268] Samples were diluted 1 :5 to 1 : 100 with buffer A, depending on initially expected pDNA concentration. Injected sample volumes ranged from 25 to 100 pL.

[0269] The plasmid concentration was quantified using a calibration curve constructed with standards of the plasmid prepared in the range 0-2.5 pg. HPLC purity degree was defined as the percentage of the plasmid peak area when compared with the total area of all peaks on the chromatogram.

[0270] Example 1 - Production of Lysate

[0271] Plasmid DNA was produced based on state of the art procedures. For production Top 10 E. coli cells were used in a fed-batch fermentation process. Briefly, fermentation was performed in chemically defined cultivation medium at 37°C. Automated feeding was triggered by pH and pO2 levels. Fermentation was stopped after 40-48h by turning off feeding and the cells were cooled to below 8°C.

[0272] Cells were separated from the cultivation broth using a disc stack centrifuge and stored at -20°C.

[0273] Cell lysis was adopted from a method described by Birnboim and Doly (1979). In more detail, after thawing, the cell paste was resuspended in 1 mL resuspension buffer (50 mM Tris, 10 mM EDTA, pH 8) per 50 OD*V units (Optical cell density at the end of fermentation * volume cell culture at the end of fermentation). Lysis buffer (0.2 M NaOH, 0.1% SDS) was added to the resuspended cells (same volume as the resuspension buffer). Lysis was performed for a maximum of 5 min at room temperature. Successful lysis could be visually identified by clearing of the solution and increase of viscosity. Lysis was stopped by addition of chilled neutralization buffer (3M potassium acetate, pH 5.5, same volume as the resuspension buffer). RNA impurities were removed by addition of a CaC12 solution to reach a final concentration of 0.5M CaC12. If not directly processed, the lysate was stored at 2-8°C until further processing. Example 2 - Filtration of the Lysate

[0274] To remove precipitates and cell debris, the lysate obtained according to Example 1 was clarified using a depth filter, either a 3M Zeta Plus (0.017 m2, Cat-No. E0170FSA60SP02A9) or a Sartorius Sartopure PP3 (0.018 m2, Cat-No. 5051302P4-00-B).

[0275] Filtrations were performed using an Akta Avant 150 system to apply exact flowrates of 42.5 mL / min and 45 mL / min, respectively, and record pressure profiles.

[0276] Before use, the depth filters were flushed and then equilibrated with water until the UV signal was close to 0 mAU / cm to ensure removal of any residual material. The lysate was applied to the respective filter at a flow rate of 50% of the respective flow rates as given above to avoid instant filter clogging. After 10 mL of lysate has passed the filter, the flow rate was increased. The filtrate was collected via fractionation (starting at 500 mAU / cm). Filtration was continued until a delta pressure of 2 bar was reached. In the following, the flowrate was decreased to keep the pressure of 2 bar constant. When a flow rate of 10 mL / min was reached the experiment was ended.

[0277] Filtered volume was calculated based on the chromatogram and the filtered amount of pDNA was determined using an HPLC method.

[0278] For the 3M Zeta Plus filter, the filtration was stopped after 271 L / m2for plasmid A, 118 L / m2for plasmid B after and after 59 L / m2for plasmid C after reaching the pressure limit. This yields in a filtered plasmid amount of 519 mg / m2for plasmid A, 3694 mg / m2for plasmid B and 120 mg / m2for plasmid C.

[0279] For the Sartorius Sartopure PP3, the filtration was stopped after 28 L / m2for plasmid A, 28 L / m2for plasmid B after and after 11 L / m2for plasmid C after reaching the pressure limit. This yields in a filtered plasmid amount of 53 mg / m2for plasmid A, 867 mg / m2for plasmid B and 24 mg / m2for plasmid C. Table 5: Volume and pDNA per filter area with constant flow until pressure limit of 2 bar was reached.

[0280] Example 3 - Filtration of purified plasmid

[0281] A purified plasmid (10078 bp) harboring a pUC origin of replication was applied to the depth filters 3M Zeta Plus (0.0025 m2, Cat-No. BC0025S60SP02A) and Sartorius Sartopure PP3 (0.018 m2, Cat-No. 5051302P4-00-B).

[0282] Filtrations were performed using an Akta Avant 150 system to apply exact flowrates of 6.25 mL / min and 45 mL / min, respectively, and record pressure profiles.

[0283] Before use, the depth filters were before use flushed and equilibrated with water until the UV signal was close to 0 mAU / cm to ensure removal of any residual material. The plasmid solution was applied to the respective filter at a flow rate of 50% of flow rates given above to avoid instant filter clogging and increased after 10 mL of the plasmid solution has passed the filter. The filtrate was collected via fractionation (starting at 500 mAU / cm). The filtration was continued until 300 mL of purified pDNA solution was filtered. Thereafter, the filters were washed with 50 mL water.

[0284] Filtered volume was calculated based on the chromatogram and the filtered amount of pDNA was determined using an HPLC method.

[0285] ***

[0286] The above description discloses several methods and materials of the preferred embodiments. This invention is susceptible to modifications in the methods and materials, as well as alterations in the equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it cover all modifications and alternatives coming within the true scope and spirit of the invention as embodied in the attached claims.

Claims

Patent Claims1. A method for producing a recombinant plasmid DNA solution comprising the following step(s): a) passing a crude E.coli lysate comprising recombinant plasmid DNA through a positively charged filter comprising silica, cellulose and a polymer resin to obtain a filtered recombinant plasmid DNA solution, thereby producing a recombinant plasmid DNA solution.

2. The method according to claim 1 further comprising before step a) the step aO) lysing E.coli cells comprising the recombinant plasmid DNA to obtain a crude E.coli lysate.

3. The method according to any one of claims 1 to 2 further comprising before step a) or step aO) the step of al) cultivating an E.coli cell comprising the plasmid DNA for producing recombinant plasmid DNA.

4. The method according to any one of claims 1 to 3 further comprising after step a) the step of b) purifying the filtered recombinant plasmid DNA solution with one or more chromatography steps and / or one or more filtration steps.

5. The method according to any one of claims 1 to 4, wherein the produced recombinant plasmid DNA solution is a purified recombinant plasmid DNA solution.

6. The method according to any one of claims 2 to 5, wherein after step aO) and before step a) the buffer is not changed.

7. The method according to any one of claims 1 to 6, wherein the crude E.coli lysate is a solution comprising about 15 mM Tris-HCl, about 3 mM EDTA, about 60 mM NaOH, about 0.3 % SDS, about 0.9 M KAc, about 4.5 % (v / v) acetic acid, about 0.5 M CaC12 and has a pH value of about 4.6.

8. The method according to any one of claims 1 to 7, wherein the crude E.coli lysate has a conductivity of about 90 mS / cm.

9. The method according to any one of claims 1 to 8, wherein the recombinant plasmid DNA has a size of 5000 bps or more.

10. The method according to any one of claims 1 to 9, wherein the recombinant plasmid DNA has a size of 7000 bps or more.

11. The method according to any one of claims 1 to 10, wherein the recombinant plasmid DNA has a size of 15000 bps or less.

12. The method according to any one of claims 1 to 11, wherein the recombinant plasmid DNA has a size of 12000 bps or less.

13. The method according to any one of claims 1 to 12, wherein the filter has a filter area of from and including about 0.23 m2 to about 3.4 m2.

14. The method according to any one of claims 1 to 12, wherein the filter has a filter area of from and including about 0.0170 m2 to about 0.1020 m2.

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