Methods and compositions for purifying therapeutic-grade plasmid DNA

Abstract

Disclosed herein are methods and kits for purifying therapeutic-grade plasmid DNA using magnetic particles. In particular, the technology of the present disclosure relates to methods and kits for purifying therapeutic-grade plasmid DNA at a high degree of purity using magnetic particles with a siliceous oxide surface layer and a superparamagnetic core.

Description

METHODS AND COMPOSITIONS FOR PURIFYINGTHERAPEUTIC-GRADE PLASMID DNATECHNICAL FIELD

[0001] The present technology relates, generally, to kits and methods for purifying therapeutic- grade plasmid DNA using magnetic particles. In particular, the technology of the present disclosure relates to methods and kits for purifying large quantities of therapeutic-grade plasmid DNA at a high degree of purity using magnetic particles with a siliceous oxide surface layer and a superparamagnetic core.BACKGROUND

[0002] Genetic variability is maintained within a population through recombination, a process involving the exchange of genetic information among different DNA molecules that results in a reshuffling of genes. In the field of genetic engineering, a vector such as plasmid DNA (pDNA) is used to provide recombination. pDNA is a small genetic element that permits microorganisms to store genetic information elsewhere than in the nucleus. Plasmids are a foundational component of emerging DNA and mRNA vaccines; and gene and cell therapies. To produce recombinant proteins, genetic engineering of cells is performed by introducing plasmids that carry a gene encoding a protein, which is not expressed in the native cell. Gene therapy is a therapeutic strategy where nucleic acids are introduced into human cells to cure genetic defects. In an ex vivo approach, the patient cells are harvested and cultivated in the laboratory and then incubated with vectors, such as plasmids, to introduce the therapeutic genes. An alternative approach for delivering genes is in vivo gene therapy, wherein a viral vector is directly administered to the patient. An increased use of such applications results in a requirement for large quantities of therapeutic-grade pDNA. An efficient, large-scale purification process needs to meet specifications in purity and yield for therapeutic applications.

[0003] The production of pDNA involves fermentation, cell harvest, cell lysis, clarification, concentration, chromatography (capture and polishing steps), followed by concentration and final filtration. The fermentation step produces the pDNA in bacteria, such as Escherichia co / i. and will also involve a step for release of pDNA from the bacterial cells. Cell lysis can be achieved by chemical addition of alkali. Alkaline lysis pDNA extraction from bacteria is acommonly used method, initially described by Bimboim and Doly (1979). It is based on the principle that alkaline solution selectively denatures high molecular weight chromosomal DNA, leaving covalently bonded, circular pDNA intact (Ali et al., 2017). Upon neutralization, chromosomal DNA renatures and becomes insoluble precipitates, whereas pDNA remains in the supernatant. In a typical bacterial lysate, pDNA comprises only about 3% of the cleared lysate, while proteins account for 55%, RNA for 21%, cDNA for 3%, lipopolysaccharides for 3% and other host components for 15% (Stadler et al., 2004). While many pDNA purification methods are used, the final pDNA preparations are typically contaminated with varied degrees of RNA and are not suitable for therapeutic applications.

[0004] Chromatography is a commonly used technique for pDNA separation and purification. It is based on the principle that molecules applied onto a porous support medium (i.e., stationary phase) will separate from each other according to different chemical affinities between the molecules and the support. Thus, in a fluid medium (the mobile phase) the solutes move at different speeds through the tube / column that contains the support. The interactions exploit differences in the physicochemical properties of the components in the sample. In an ion exchange chromatography process, there is a competitive binding of ions of one kind, such as proteins or nucleic acids, for ions of another kind, such as salt ions, to an oppositely charged resin. The interaction between the target molecule and the resin depends on several factors, such as net charge and surface charge distribution of the target molecule, the ionic strength, and the nature of the particular ions in the solvent, pH, etc. Anion exchange chromatography (AEX) is a key mode for the purification of pDNA. An ethanol wash may be used to remove endotoxins (i.e., lipopolysaccharides) and other impurities. By increasing the amount of organic solvent in the wash step, the method shifts from an ionic mode to that of a "mixed" mode. Suitable AEX resins have a particle diameter of 2 - 500 pm and an average pore diameter of 1500 - 4000A (U.S. Pat. No. 8,093,373).

[0005] US Patent No. 8,093,373 describes a chromatography -based purification method relying on anion exchange technology, wherein a positively charged resin is used to generate a retentate with negatively charged compounds and a permeate of all other compounds, including positively charged compounds. The methods described therein include use of a separation matrix comprised of one or more porous carriers, which carrier(s) present anion exchangegroups on external surfaces as well as on pore surfaces and a pore size distribution that does not allow plasmids to pass through the pores due to steric hinderance. The matrix is then contacted with a liquid containing a plasmid of interest to adsorb the plasmid to ligands present on the external surfaces of the separation matrix. Once other components of the liquid have passed through the pores the separation matrix is contacted with an eluent to release the plasmid and recover it from a fraction of said eluent.

[0006] pDNA purification using hydrophobic interaction chromatography (HIC), may be combined with an AEX step. In the HIC step, protein and RNA are adsorbed at a salt concentration at which plasmids are not adsorbed to produce an eluate comprising pDNA. The liquid applied to the AEX column should not contain any RNA. A disadvantage of using HIC is the requirement of high salt concentrations at the beginning of a chromatography column run, such that crystallization and precipitation may be difficult to manage.

[0007] Other sorbents for nucleic acid purification have been described but not yet implemented in large scale pDNA processing for cell and gene therapy applications. For example, mixed-bed solid phases may be used for isolation of target nucleic acids, which are comprised of at least two different solid phases (U.S. Pat. No. 6,270,970). Both phases bind the target nucleic acids, but under different solution conditions, and they release the nucleic acid under similar elution conditions. The solid phase of the different beds comprises magnetic silica particles. At the benchtop scale, magnetic bead immobilization can be used to select or exclude nucleic acids of particular sizes by varying the binding conditions (Oberacker et al.. 2019); and the technology can allow for selective removal even from viscous sample suspensions (Berensmeier, 2006).

[0008] US patent No. 6,027,945 (“the ’945 patent”) describes methods for isolating biological target molecules, in particular nucleic acids, using magnetic silica particles. The ’945 patent describes methods of isolating a biological target material from other materials in a medium by: providing a medium including the biological target material; providing silica magnetic particles; forming a complex of the silica magnetic particles and the biological target material by combining the silica magnetic particles and the medium; removing the complex from the medium by application of an external magnetic field; and separating the biological target material from the complex by eluting the biological target material whereby the isolated biological target material is obtained. The ’945 patent is primarily concerned with nucleic acidpurification for molecular biological techniques, such as reverse transcription, cloning, restriction analysis, and sequencing, and not for therapeutic uses. Indeed, the exemplary plasmid DNA purification described in the ’945 patent includes the use of RNase enzymes to reduce RNA contamination. While RNase contamination may not present challenges for restriction analysis, for example, it does preclude the product from being considered a therapeutic-grade plasmid. Additionally, the ’945 patent fails to disclose the form of the plasmid, whereas therapeutic-grade plasmid DNA must be primarily supercoiled (e.g., at least 80% supercoiled) for efficacy in therapeutic applications. See Folarin O, et al.. Application of Plasmid Engineering to Enhance Yield and Quality of Plasmid for Vaccine and Gene Therapy. Bioengineering (Basel). 2019 Jun 19;6(2):54 tySupercoiling is essential for the application of DNA as vaccines and for gene therapy applications.”). Additionally, it is well known in the art that plasmids with sufficient purity for therapeutic use must have an A260 / A280 ratio of about 1.8 to about 2.0 and an A260 / A230 ratio greater than about 1.5, however the ’945 patent fails to disclose the purity of the pDNA in terms of the A260 / A280 ratio or the A260 / A230. Accordingly, the ’945 patent is properly understood as disclosing methods of nucleic acid purification for research purposes, and it does not disclose methods for producing a therapeutic-grade plasmid product as are described herein.

[0009] The intended use of a purified plasmid is an important factor when choosing a purification method. Plasmids purified for research or industrial purposes, as described in the ’945 patent, can have higher levels of contamination, so long as the contamination does not interfere with the intended use. For example, plasmids used to transform bacteria can be contaminated with some amount of endotoxin, RNA, genomic DNA. or RNase without impeding the transformation process. This is in sharp contrast to therapeutic-grade pDNA, which must be substantially or entirely free of contaminants like endotoxin, RNA, genomic DNA, or RNase, as is well known in the art. Such contamination in a therapeutic context raises substantial health risks for subjects receiving treatment.

[0010] The inventors have previously described improved siliceous oxide-coated magnetic particles having a high resistance to leaching of the magnetic material on exposure to aqueous acidic environments while also possessing an excellent magnetic response (U.S. Pat. No. 6,296,937). The magnetic particles have been used only in small scale (microliter to lowmilliliter) nucleic acid capture, whether in manual or automated operation for diagnostics applications, and have not been previously shown to be effective at for producing large yields of therapeutic-grade plasmid DNA.

[0011] Accordingly, there is a need for novel and efficient, large-scale plasmid DNA purification processes that meet specifications in purity and yield in order for the pDNA to qualify as therapeutic-grade.SUMMARY

[0012] In some embodiments, the present disclosure provides methods for purifying therapeutic- grade plasmid DNA (pDNA) comprising the use of a magnetic particle, wherein the magnetic particle comprises a siliceous oxide surface layer and a superparamagnetic core material.

[0013] In some embodiments, the particles have a nitrogen BET surface area of about 6 m2 / g to about 62 m2 / g. In some embodiments, the superparamagnetic core comprises about 15 wt.% to about 60 wt.% of the particles. In some embodiments, the particle is about 1 pm to about 15 pm in diameter. In some embodiments, the particle exhibits less than 10 parts per million of magnetic core leaching. In some embodiments, the particle comprises about 40% to about 85% siliceous oxide.

[0014] In some embodiments, the method does not comprise use of an RNase enzyme. In some embodiments, the method further comprises a step (i) of concentrating a volume of bacteria, wherein the bacteria comprise the pDNA. In some embodiments, the method further comprises a step (ii) of resuspending the concentrated bacteria in a resuspension buffer. In some embodiments, the method further comprises a step (iii) of adding a lysis buffer to the bacteria resuspended in resuspension buffer to generate a lysate. In some embodiments, the method further comprises a step (iv) of adding a neutralization buffer to the lysate.

[0015] In some embodiments, the method further comprises a step (v) of performing one or more centrifugation and / or filtration steps on the lysate to generate a cleared lysate, wherein the cleared lysate comprises a portion of the pDNA. In some embodiments, the method further comprises a step (vi) of adding the magnetic particles to the cleared lysate and incubating the mixture, wherein the portion of the pDNA binds to the magnetic particles. In someembodiments, the method further comprises a step (vii) of performing one or more wash steps. In some embodiments, the method further comprises a step (viii) of performing an elution step, wherein the elution step releases the bound pDNA from the magnetic particles.

[0016] In some embodiments, the method further comprises a magnetic separation step of the solution comprising the magnetic particles and the cleared lysate, wherein the magnetic separation step comprises applying a magnetic field to the solution and wherein the magnetic separation step results in about 10% to about 80% transmittance through the solution after 5 seconds of magnetic field exposure, about 45% to about 100 % transmittance through the solution after 40 seconds of magnetic field exposure, and about 80% to about 100 % transmittance through the solution after 600 seconds of magnetic field exposure.

[0017] In some embodiments, the volume of bacteria comprises about 4ml bacteria to about 3L bacteria. In some embodiments, the resuspension buffer comprises about 50 mM glucose, about 25 mM Tris-HCl pH 8.0, and about 10 mM EDTA pH 8.0. In some embodiments, the lysis buffer comprises about 0.2 M NaOH and about 0.5% to about 2% SDS. In some embodiments, the neutralization buffer comprises about 2 M to about 4 M potassium acetate, about 1 M to about 3 M acetic acid, and about 0.5M to about 2 M CaCh, and wherein the neutralization buffer is at about pH 4.5 to about 6.5.

[0018] In some embodiments, step (v) comprises one or more of centrifugation, use of micromolar filters, use of depth filters, use of ultrafiltration centrifuge tubes, or use of tangential flow filtration capsules. In some embodiments, step (v) comprises use of ultrafiltration centrifuge tubes in an ultrafiltration centrifuge step, and wherein the ultrafiltration centrifugation step comprises a buffer exchange step, wherein a new buffer replaces the buffer of the lysate and wherein the new buffer comprises 120 mM potassium acetate at pH 5.5 and 25 mM EDTA. In some embodiments, step (v) comprises use of tangential flow filtration capsules in a tangential flow filtration step, and wherein the tangential flow filtration step comprises a buffer exchange step wherein a new buffer replaces the buffer of the lysate and wherein the new buffer comprises about 100 mM to about 10 mM potassium acetate at about pH 4.5 to about pH 6.5 and about 20 mM to about 30 mM EDTA.

[0019] In some embodiments, step (vi) further comprises adding guanidine hydrochloride to the cleared lysate to achieve a final concentration of about 1 M to about 5 M prior to the addition of the magnetic particles. In some embodiments, a roller mixer is used to mix solutions comprising the pDNA during one or more of steps (iii), (iv), (vi), (vii), and (viii). In some embodiments, the method does not comprise a chromatography step or use of a chromatography column.

[0020] In some embodiments, the purified therapeutic-grade pDNA is at a concentration of about 300 pg / g wet cell mass to about 1200 pg / g wet cell mass. In some embodiments, the purified therapeutic-grade pDNA is at a concentration of about 400 pg / g wet cell mass to about 1100 pg / g wet cell mass. In some embodiments, the purified therapeutic-grade pDNA is at a concentration of about 300 pg / g wet cell mass to about 500 pg / g wet cell mass.

[0021] In some embodiments, the purified therapeutic-grade pDNA has an A260 / A280 ratio of about 1 .80 to about 1.97. In some embodiments, the purified therapeutic-grade pDNA has an A260 / A230 ratio of about 1.5 to about 2.4. In some embodiments, the purified therapeutic-grade pDNA is about 80% to about 99% supercoiled. In some embodiments, the purified therapeutic- grade pDNA comprises about 1 to about 200 EU endotoxin per mg pDNA.

[0022] In some embodiments, the present disclosure provides an isolated therapeutic-grade plasmid DNA (pDNA) produced by a process comprising the use of a magnetic particle, wherein the magnetic particle comprises a siliceous oxide surface layer and a superparamagnetic core material.

[0023] In some embodiments, the particles have a nitrogen BET surface area of about 6 m2 / g to about 62 m2 / g. In some embodiments, the superparamagnetic core comprises about 15 wt.% to about 60 wt.% of the particles. In some embodiments, the particle is about 1 pm to about 15 pm in diameter. In some embodiments, the particle exhibits less than 10 parts per million of magnetic core leaching. In some embodiments, the particle comprises about 40% to about 85% siliceous oxide.

[0024] In some embodiments, the method does not comprise use of an RNase enzy me. In some embodiments, the method further comprises a step (i) of concentrating a volume of bacteria, wherein the bacteria comprise the pDNA. In some embodiments, the method further comprisesa step (ii) of resuspending the concentrated bacteria in a resuspension buffer. In some embodiments, the method further comprises a step (iii) of adding a lysis buffer to the bacteria resuspended in resuspension buffer to generate a lysate. In some embodiments, the method further comprises a step (iv) of adding a neutralization buffer to the lysate.

[0025] In some embodiments, the method further comprises a step (v) of performing one or more centrifugation and / or filtration steps on the lysate to generate a cleared lysate, wherein the cleared lysate comprises a portion of the pDNA. In some embodiments, the method further comprises a step (vi) of adding the magnetic particles to the cleared lysate and incubating the mixture, wherein the portion of the pDNA binds to the magnetic particles. In some embodiments, the method further comprises a step (vii) of performing one or more wash steps. In some embodiments, the method further comprises a step (viii) of performing an elution step, wherein the elution step releases the bound pDNA from the magnetic particles.

[0026] In some embodiments, the method further comprises a magnetic separation step, wherein the magnetic separation step of the solution comprising the magnetic particles and the cleared lysate, wherein the magnetic separation step comprises applying a magnetic field to the solution and wherein the magnetic separation step results in about 10% to about 80% transmittance through the solution after 5 seconds of magnetic field exposure, about 45% to about 100 % transmittance through the solution after 40 seconds of magnetic field exposure, and about 80% to about 100 % transmittance through the solution after 600 seconds of magnetic field exposure.

[0027] In some embodiments, the volume of bacteria comprises about 4ml bacteria to about 3L bacteria. In some embodiments, the resuspension buffer comprises about 50 mM glucose, about 25 mM Tris-HCl pH 8.0, and about 10 mM EDTA pH 8.0. In some embodiments, the lysis buffer comprises about 0.2 M NaOH and about 0.5% to about 2% SDS. In some embodiments, the neutralization buffer comprises about 2 M to about 4 M potassium acetate, about 1 M to about 3 M acetic acid, and about 0.5M to about 2 M CaCh, and wherein the neutralization buffer is at about pH 4.5 to about 6.5.

[0028] In some embodiments, step (v) comprises one or more of centrifugation, use of micromolar filters, use of depth filters, use of ultrafiltration centrifuge tubes, or use of tangential flow filtration capsules. In some embodiments, step (v) comprises use of ultrafiltration centrifugetubes in an ultrafiltration centrifuge step, and wherein the ultrafiltration centrifugation step comprises a buffer exchange step, wherein a new buffer replaces the buffer of the lysate and wherein the new buffer comprises 120 mM potassium acetate at pH 5.5 and 25 mM EDTA. In some embodiments, step (v) comprises use of tangential flow filtration capsules in a tangential flow filtration step, and wherein the tangential flow filtration step comprises a buffer exchange step wherein a new buffer replaces the buffer of the lysate and wherein the new buffer comprises about 100 mM to about 10 mM potassium acetate at about pH 4.5 to about pH 6.5 and about 20 mM to about 30 mM EDTA.

[0029] In some embodiments, step (vi) further comprises adding guanidine hydrochloride to the cleared lysate to achieve a final concentration of about 1 M to about 5 M prior to the addition of the magnetic particles. In some embodiments, a roller mixer is used to mix solutions comprising the pDNA during one or more of steps (iii), (iv), (vi), (vii), and (viii).

[0030] In some embodiments, the method does not comprise a chromatography step or use of a chromatography column. In some embodiments, the purified therapeutic-grade pDNA is at a concentration of about 300 pg / g wet cell mass to about 1200 pg / g wet cell mass. In some embodiments, the purified therapeutic-grade pDNA is at a concentration of about 400 pg / g wet cell mass to about 1100 pg / g wet cell mass. In some embodiments, the purified therapeutic- grade pDNA is at a concentration of about 300 pg / g wet cell mass to about 500 pg / g wet cell mass. In some embodiments, the purified therapeutic-grade pDNA has an A260 / A280 ratio of about 1.80 to about 1.97. In some embodiments, the purified therapeutic-grade pDNA has an A260 / A230 ratio of about 1.5 to about 2.4. In some embodiments, the purified therapeutic-grade pDNA is about 80% to about 99% supercoiled. In some embodiments, the purified therapeutic- grade pDNA comprises about 1 to about 200 EU endotoxin per mg pDNA.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIGs. 1A-1B are a gel and densitometry analysis for the 3-L grow th experiments. FIG. 1A shows an agarose gel electrophoresis of pDNA from the lysis of 3-liter E. coll grow th for Trial 6 bead binding at 2% dosing. Detection of pDNA on a 1% agarose gel, with Gel Green fluorescent dye. MW ladder (lane 1): 0.5, 1.0. 1.5, 2.0, 3.0, 4.0, 5.0 6.0, 8.0, and 10.0 kilobases. Up to -200 ng nucleic acid w as loaded per well for Reference 1 (non-magnetic, spin columnkit) purified samples (lanes 3, 4, and 5) and duplicate Trial 6 purified samples (lanes 7 and 8). The microfiltered lysate (lane 10), prior to the TFF processing step (lane 9), has a high potassium acetate concentration that may quench the fluorescence. FIG. IB is a densitometry analysis for determination of % supercoiled (SC) after image inversion.

[0032] FIG. 2 shows a 1% agarose gel for the detection of pDNA with GelGreen fluorescent dye, at magnetic bead dosing levels of 2%. pDNA binding / release was observed on Trial 1-5 beads of different SiCh composition / particle size. Primarily supercoiled isoform was observed for Trial 1-5 beads, while Comparative Trial 1 and 2 beads did not produce similar levels of supercoiled DNA (FIG. 3). Comparative Trial 5 and 6 beads produced UV concentrations that reflect high levels of impurities; pDNA fluorescence was weak on agarose gels. T = trial; CT = Comparative Trial.

[0033] FIG. 3 is a table showing a summary of magnetic bead physicochemical properties and associated nucleic acid yield, purity, and % supercoiled isoform for all 1-ml and 150-ml binding studies discussed in Examples 1-3.

[0034] FIG. 4 is a table showing a summary of magnetic bead physicochemical properties and associated nucleic acid yield, purity, and % supercoiled isoform for binding studies discussed in Example 5.

[0035] FIG. 5 is a table showing a summary of magnetic bead physicochemical properties and associated nucleic acid yield, purity, and % supercoiled isoform for binding studies discussed in Example 6.DETAILED DESCRIPTION

[0036] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present disclosure are described below in various levels of detail in order to provide a substantial understanding of the present technology. The definitions of certain terms as used in this specification are provided below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary' skill in the art to which this present technology belongs.I. Definitions

[0037] All technical terms employed in this specification are commonly used in biochemistry, molecular biology and agriculture; hence, they are understood by those skilled in the field to which the present technology belongs. Those technical terms can be found, for example in: Molecular Cloning: A Laboratory Manual 3rded., vol. 1-3, ed. Sambrook and Russel (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001); Current Protocols In Molecular Biology, ed. Ausubel et al. (Greene Publishing Associates and Wiley-Interscience, New York, 1988) (including periodic updates); Short Protocols In Molecular Biology: A Compendium Of Methods From Current Protocols In Molecular Biology 5thed., vol. 1-2, ed. Ausubel et al. (John Wiley & Sons, Inc., 2002); Genome Analysis: A Laboratory Manual, vol. 1-2, ed. Green et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1997).

[0038] As used herein, the term "about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term.

[0039] The terms “encoding” and “coding” refer to the process by which a gene, through the mechanisms of transcription and translation, provides information to a cell from which a series of amino acids can be assembled into a specific amino acid sequence to produce an active enzyme. Because of the degeneracy of the genetic code, certain base changes in DNA sequence do not change the amino acid sequence of a protein.

[0040] As used herein, “expression” denotes the production of an RNA product through transcription of a gene or the production of the polypeptide product encoded by a nucleotide sequence. “Overexpression” or “up-regulation” is used to indicate that expression of a particular gene sequence or variant thereof, in a cell, has been increased by genetic engineering, relative to a control cell.

[0041] By “isolated nucleic acid molecule” is intended a nucleic acid molecule, DNA, or RNA, which has been removed from its native environment. For example, recombinant DNA molecules contained in a DNA construct are considered isolated for the purposes of the present technology. Further examples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or DNA molecules that are purified, partially or substantially, in solution. Isolated RNA molecules include in vitro RNA transcripts of the DNA molecules of the present technology. Isolated nucleic acid molecules, according to the present technology, further include such molecules produced synthetically.

[0042] ‘ ‘Promoter” connotes a region of DNA upstream from the start of transcription that is involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “constitutive promoter” is one that is active throughout the life of the cell and under most conditions. Tissue-specific, tissue-preferred, cell type-specific, and inducible promoters constitute the class of “non-constitutive promoters.” “Operably linked” or “operatively linked” refers to a functional linkage between a promoter and a second sequence, where the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. In general, “operably linked” or “operatively linked” means that the nucleic acid sequences being linked are contiguous. For example, an operatively linked promoter, enhancer elements, open reading frame, 5' and 3' UTR, and terminator sequences result in the accurate production of an RNA molecule. In some aspects, operatively linked nucleic acid elements result in the transcription of an open reading frame and ultimately the production of a polypeptide (i.e.. expression of the open reading frame).

[0043] “Sequence identity” or “identity” in the context of two polynucleotide (nucleic acid) or polypeptide sequences includes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified region. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties, such as charge and hydrophobicity, and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well-known to those of skill in the art. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1 . The scoring of conservative substitutions is calculated, for example, according to the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4: 11-17 (1988), as implemented in the program PC / GENE (Intelligenetics, Mountain View. California, USA).

[0044] Use in this description of a percentage of sequence identity denotes a value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (z.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

[0045] As used herein, “therapeutic-grade plasmid DNA” refers to plasmid DNA that can be safely and effectively used in therapeutic applications. Therapeutic-grade plasmid DNA: (i) is free of RNase contamination and has never been exposed to bovine sourced RNases, (ii) is at least 80% supercoiled, (iii) has an A260 / A280 ratio of about 1.80 to about 1.97 (iv) has an A260 / A230 ratio greater than about 1.5 and (v) has less than 100 EU endotoxin per mg pDNA.

[0046] As used herein, “superparamagnetic” refers to substances that are temporarily magnetic, such as ferromagnetic or ferrimagnetic materials with low Curie temperatures, provided that such temporarily magnetic materials are paramagnetic in the temperature range at which silica magnetic particles containing such materials are used according to the present methods to isolate biological materials.

[0047] The term “siliceous-oxide coated magnetic particle’' or “SOCM particle” as used herein refers to particles comprised of siliceous oxide coating a core of at least one particle of superparamagnetic or paramagnetic material. The SOCM particle used in the present methods has an adsorptive surface of hydrous siliceous oxide, a surface characterized by having silanol groups thereon. Target nucleic acid material, such as pDNA, adheres to the adsorptive surfaceof the particle while other material, particularly deleterious contaminants such as exonucleases, do not adhere to or co-elute from the particle with the target materials.

[0048] As used herein, “transformation"’ refers to the introduction of exogenous nucleic acid into cells, so as to produce transgenic cells stably transformed with the exogenous nucleic acid.

[0049] A “variant” is a nucleotide or amino acid sequence that deviates from the standard, or given, nucleotide or amino acid sequence of a particular gene or polypeptide. The terms “isoform,"’ ■'isotype.” and “analog” also refer to “variant” forms of a nucleotide or an amino acid sequence. An amino acid sequence that is altered by the addition, removal, or substitution of one or more amino acids, or a change in nucleotide sequence, may be considered a variant sequence. A polypeptide variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. A polypeptide variant may have “nonconservative” changes, e.g. replacement of a glycine with a tryptophan. Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted may be found using computer programs well known in the art such as Vector NTI Suite (InforMax. MD) software. Variant may also refer to a “shuffled gene” such as those described in Maxygen-assigned patents (see, e.g., U. S. Patent No. 6,602,986).

[0050] As used herein, the terms “vector,"’ “vehicle,” “construct,"’ and “plasmid” are used in reference to any recombinant polynucleotide molecule that can be propagated and used to transfer nucleic acid segment(s) from one organism to another. Vectors generally comprise parts which mediate vector propagation and manipulation (e.g., one or more origin of replication, genes imparting drug or antibiotic resistance, a multiple cloning site, operably linked promoter / enhancer elements which enable the expression of a cloned gene, etc.). Vectors are generally recombinant nucleic acid molecules, often denved from bacteriophages, or plant or animal viruses. Plasmids and cosmids refer to two such recombinant vectors. A “cloning vector” or “shuttle vector” or “subcloning vector” contain operably linked parts that facilitate subcloning steps (e.g., a multiple cloning site containing multiple restriction endonuclease target sequences). A nucleic acid vector can be a linear molecule, or in circular form, depending on type of vector or type of application. Some circular nucleic acid vectors can be intentionally linearized prior to delivery into a cell.

[0051] As used herein, the term “expression vector’7refers to a recombinant vector comprising operably linked polynucleotide elements that facilitate and optimize expression of a desired gene (e.g., a gene that encodes a protein) in a particular host organism (e.g., a bacterial expression vector or mammalian expression vector). Polynucleotide sequences that facilitate gene expression can include, for example, promoters, enhancers, transcription termination sequences, and ribosome binding sites.II. Overview of Therapeutic-Grade Plasmid DNA Purification Methods Using Magnetic Particles

[0052] In one aspect, the present invention comprises methods of purifying high yields of therapeutic-grade plasmid DNA (pDNA). In some embodiments, the purified therapeutic-grade pDNA has a high degree of purity with low levels of contaminants such as RNA. genomic DNA, or endotoxin. In some embodiments, the therapeutic-grade pDNA is primarily (e.g.. at least 80%) supercoiled in form.Generating a Cleared Lysate

[0053] This application describes the use of magnetic particles in therapeutic-grade pDNA purification for multi-liter E. coli cell growths. One of skill in the art will understand that plasmid amplification can be accomplished through a variety of means known in the art. For purposes of illustration, a culture of an E. coli strain (e.g. DH5ot) can be transformed with a plasmid of interest using any appropriate method (e.g, heat school or electroporation). Transformants can be selected for using an appropriate marker in the plasmid of interest (e.g, ampicillin resistance, chloramphenicol resistance, or kanamycin resistance), and then expanded via culturing in suitable liquid media conditions. For example, a 3000-ml cell growth could be obtained using LB broth medium with several passages until the optical densify at 600 nm reached ~1.4.

[0054] This application describes the use of a resuspension buffer resuspend bacterial cells with pDNA and chelate metal ions. One of skill in the art will understand that cell resuspension can be accomplished through a variety of means known in the art. For purposes of illustration, a resuspension buffer (also referred to as an LI buffer) can comprise glucose, Tris HC1 (pH 8.0), and EDTA (pH 8.0). Glucose concentration in the resuspension buffer can be, for example,from about 0 mM to about 100 mM. In some embodiments, resuspension buffer comprises about 0 mM glucose. In some embodiments, resuspension buffer comprises about 5 mM glucose. In some embodiments, resuspension buffer comprises about 10 mM glucose. In some embodiments, resuspension buffer comprises about 15 mM glucose. In some embodiments, resuspension buffer comprises about 20 mM glucose. In some embodiments, resuspension buffer comprises about 25 mM glucose. In some embodiments, resuspension buffer comprises about 30 mM glucose. In some embodiments, resuspension buffer comprises about 35 mM glucose. In some embodiments, resuspension buffer comprises about 40 mM glucose. In some embodiments, resuspension buffer comprises about 45 mM glucose. In some embodiments, resuspension buffer comprises about 50 mM glucose. In some embodiments, resuspension buffer comprises about 55 mM glucose. In some embodiments, resuspension buffer comprises about 60 mM glucose. In some embodiments, resuspension buffer comprises about 65 mM glucose. In some embodiments, resuspension buffer comprises about 70 mM glucose. In some embodiments, resuspension buffer comprises about 75 mM glucose. In some embodiments, resuspension buffer comprises about 80 mM glucose. In some embodiments, resuspension buffer comprises about 85 mM glucose. In some embodiments, resuspension buffer comprises about 90 mM glucose. In some embodiments, resuspension buffer comprises about 95 mM glucose. In some embodiments, resuspension buffer comprises about 100 mM glucose. Tris HC1 (pH 8.0) concentration in the resuspension buffer can be. for example, from about 10 mM to about 100 mM. In some embodiments, resuspension buffer comprises about 10 mM Tris HC1 (pH 8.0). In some embodiments, resuspension buffer comprises about 10 mM Tris HC1 (pH 8.0). In some embodiments, resuspension buffer comprises about 15 mM Tris HC1 (pH 8.0). In some embodiments, resuspension buffer comprises about 20 mM Tris HC1 (pH 8.0). In some embodiments, resuspension buffer comprises about 25 mM Tris HC1 (pH 8.0). In some embodiments, resuspension buffer comprises about 30 mM Tris HC1 (pH 8.0). In some embodiments, resuspension buffer comprises about 35 mM Tris HC1 (pH 8.0). In some embodiments, resuspension buffer comprises about 40 mM Tris HC1 (pH 8.0). In some embodiments, resuspension buffer comprises about 45 mM Tris HC1 (pH 8.0). In some embodiments, resuspension buffer comprises about 50 mM Tris HC1 (pH 8.0). In some embodiments, resuspension buffer comprises about 55 mM Tris HC1 (pH 8.0). In some embodiments, resuspension buffer comprises about 60 mM Tris HC1 (pH 8.0). In some embodiments, resuspension buffer comprises about 65 mM Tris HC1 (pH 8.0). In someembodiments, resuspension buffer comprises about 70 mM Tris HC1 (pH 8.0). In some embodiments, resuspension buffer comprises about 75 mM Tris HC1 (pH 8.0). In some embodiments, resuspension buffer comprises about 80 mM Tris HC1 (pH 8.0). In some embodiments, resuspension buffer comprises about 85 mM Tris HC1 (pH 8.0). In some embodiments, resuspension buffer comprises about 90 mM Tris HC1 (pH 8.0). In some embodiments, resuspension buffer comprises about 95 mM Tris HC1 (pH 8.0). In some embodiments, resuspension buffer comprises about 100 mM Tris HC1 (pH 8.0). EDTA (pH 8.0) concentration in the resuspension buffer can be, for example, from about 2 mM to about 25 mM. In some embodiments, resuspension buffer comprises about 2 mM EDTA (pH 8.0). In some embodiments, resuspension buffer comprises about 4 mM EDTA (pH 8.0). In some embodiments, resuspension buffer comprises about 6 mM EDTA (pH 8.0). In some embodiments, resuspension buffer comprises about 8 mM EDTA (pH 8.0). In some embodiments, resuspension buffer comprises about 10 mM EDTA (pH 8.0). In some embodiments, resuspension buffer comprises about 12 mM EDTA (pH 8.0). In some embodiments, resuspension buffer comprises about 14 mM EDTA (pH 8.0). In some embodiments, resuspension buffer comprises about 2 mM EDTA (pH 8.0). In some embodiments, resuspension buffer comprises about 16 mM EDTA (pH 8.0). In some embodiments, resuspension buffer comprises about 18 mM EDTA (pH 8.0). In some embodiments, resuspension buffer comprises about 20 mM EDTA (pH 8.0). In some embodiments, resuspension buffer comprises about 22 mM EDTA (pH 8.0). In some embodiments, resuspension buffer comprises about 24 mM EDTA (pH 8.0). In some embodiments, resuspension buffer comprises about 25 mM EDTA (pH 8.0). In one embodiment, the resuspension buffer comprises 50 mM glucose, 25 mM Tris HC1 (pH 8.0), and 10 mM EDTA (pH 8.0). In one embodiment, 5 ml of resuspension buffer is used per gram of bacterial wet cell mass.

[0055] This application describes the use of a lysis buffer to release pDNA from bacterial cells and denature proteins and chromosomal DNA. One of skill in the art will understand that cell lysis can be accomplished through a variety of means know n in the art. For purposes of illustration, a lysis buffer (also referred to as an L2 buffer) can comprise NaOH and SDS. NaOH concentration in the lysis buffer can be, for example, from about 10 mM to about 200 mM. In some embodiments, lysis buffer comprises about 10 mM NaOH. In some embodiments, lysisbuffer comprises about 20 mM NaOH. In some embodiments, lysis buffer comprises about 10 mM NaOH. In some embodiments, lysis buffer comprises about 30 mM NaOH. In some embodiments, lysis buffer comprises about 40 mM NaOH. In some embodiments, lysis buffer comprises about 50 mM NaOH. In some embodiments, lysis buffer comprises about 60 mM NaOH. In some embodiments, lysis buffer comprises about 70 mM NaOH. In some embodiments, lysis buffer comprises about 80 mM NaOH. In some embodiments, lysis buffer comprises about 90 mM NaOH. In some embodiments, lysis buffer comprises about 100 mM NaOH. In some embodiments, lysis buffer comprises about 110 mM NaOH. In some embodiments, lysis buffer comprises about 120 mM NaOH. In some embodiments, lysis buffer comprises about 130 mM NaOH. In some embodiments, lysis buffer comprises about 140 mM NaOH. In some embodiments, lysis buffer comprises about 150 mM NaOH. In some embodiments, lysis buffer comprises about 160 mM NaOH. In some embodiments, lysis buffer comprises about 170 mM NaOH. In some embodiments, lysis buffer comprises about 180 mM NaOH. In some embodiments, lysis buffer comprises about 190 mM NaOH. In some embodiments, lysis buffer comprises about 200 mM NaOH. SDS concentration in the lysis buffer can be, for example, from about 0.1% to about 2%. In some embodiments, lysis buffer comprises about 0.1% SDS. In some embodiments, lysis buffer comprises about 0.2% SDS. In some embodiments, lysis buffer comprises about 0.3% SDS. In some embodiments, lysis buffer comprises about 0.4% SDS. In some embodiments, lysis buffer comprises about 0.5% SDS. In some embodiments, lysis buffer comprises about 0.6% SDS. In some embodiments, lysis buffer comprises about 0.7% SDS. In some embodiments, lysis buffer comprises about 0.8% SDS. In some embodiments, lysis buffer comprises about 0.9% SDS. In some embodiments, lysis buffer comprises about 1.0% SDS. In some embodiments, lysis buffer comprises about 1.1 % SDS. In some embodiments, lysis buffer comprises about 1 .2% SDS. In some embodiments, lysis buffer comprises about 1.3% SDS. In some embodiments, lysis buffer comprises about 1.4% SDS. In some embodiments, lysis buffer comprises about 1.5% SDS. In some embodiments, lysis buffer comprises about 1.6% SDS. In some embodiments, lysis buffer comprises about 1.7% SDS. In some embodiments, lysis buffer comprises about 1.8% SDS. In some embodiments, lysis buffer comprises about 1.9% SDS. In some embodiments, lysis buffer comprises about 2.0% SDS. In some embodiments, the lysis buffer comprises about 200 mM NaOH and 1% SDS. In one embodiment, 10 ml of lysis buffer is used per gram of bacterial wet cell mass.

[0056] This application describes the use of a neutralization buffer to precipitate the chromosomalDNA and cellular debris. One of skill in the art will understand that neutralization can be accomplished through a variety of means know n in the art. For purposes of illustration, a neutralization buffer (also called a L3 buffer) can comprise potassium acetate, acetic acid, and calcium chloride. Potassium acetate concentration in the neutralization buffer can be, for example, from about 20 mM to about 200 mM. In some embodiments, neutralization buffer comprises about 20 mM potassium acetate, In some embodiments, neutralization buffer comprises about 30 mM potassium acetate, In some embodiments, neutralization buffer comprises about 40 mM potassium acetate, In some embodiments, neutralization buffer comprises about 50 mM potassium acetate, In some embodiments, neutralization buffer comprises about 60 mM potassium acetate, In some embodiments, neutralization buffer comprises about 70 mM potassium acetate, In some embodiments, neutralization buffer comprises about 80 mM potassium acetate, In some embodiments, neutralization buffer comprises about 90 mM potassium acetate. In some embodiments, neutralization buffer comprises about 100 mM potassium acetate, In some embodiments, neutralization buffer comprises about 110 mM potassium acetate, In some embodiments, neutralization buffer comprises about 120 mM potassium acetate, In some embodiments, neutralization buffer comprises about 130 mM potassium acetate, In some embodiments, neutralization buffer comprises about 140 mM potassium acetate, In some embodiments, neutralization buffer comprises about 150 mM potassium acetate, In some embodiments, neutralization buffer comprises about 160 mM potassium acetate. In some embodiments, neutralization buffer comprises about 170 mM potassium acetate. In some embodiments, neutralization buffer comprises about 180 mM potassium acetate. In some embodiments, neutralization buffer comprises about 190 mM potassium acetate. In some embodiments, neutralization buffer comprises about 200 mM potassium acetate. Acetic acid concentration in the neutralization buffer can be, for example, from about 10 mM to about 300 mM. In some embodiments, neutralization buffer comprises about 10 mM acetic acid. In some embodiments, neutralization buffer comprises about 20 mM acetic acid. In some embodiments, neutralization buffer comprises about 40 mM acetic acid. In some embodiments, neutralization buffer comprises about 60 mM acetic acid. In some embodiments, neutralization buffer comprises about 80 mM acetic acid. In some embodiments, neutralization buffer comprises about 100 mM acetic acid. In some embodiments, neutralization buffer comprises about 120 mM acetic acid. In someembodiments, neutralization buffer comprises about 140 mM acetic acid. In some embodiments, neutralization buffer comprises about 160 mM acetic acid. In some embodiments, neutralization buffer comprises about 180 mM acetic acid. In some embodiments, neutralization buffer comprises about 200 mM acetic acid. In some embodiments, neutralization buffer comprises about 220 mM acetic acid. In some embodiments, neutralization buffer comprises about 240 mM acetic acid. In some embodiments, neutralization buffer comprises about 260 mM acetic acid. In some embodiments, neutralization buffer comprises about 280 mM acetic acid. In some embodiments, neutralization buffer comprises about 300 mM acetic acid. CaCh concentration in the neutralization buffer can be. for example, from about 0. 1 mM to about 200 mM. CaCh is known to be useful for precipitating RNA out of solution while not precipitation DNA. In some embodiments, CaCh in the neutralization buffer is at a concentration sufficient to result in precipitation of at least some RNA. In some embodiments, lithium chloride or ammonium acetate are used as alternatives to CaCh. In some embodiments, neutralization buffer comprises about 0. 1 mM CaCh. In some embodiments, neutralization buffer comprises about 10 mM CaCh. In some embodiments, neutralization buffer comprises about 20 mM CaCh. In some embodiments, neutralization buffer comprises about 30 mM CaCh. In some embodiments, neutralization buffer comprises about 40 mM CaCh. In some embodiments, neutralization buffer comprises about 50 mM CaCh. In some embodiments, neutralization buffer comprises about 60 mM CaCh. In some embodiments, neutralization buffer comprises about 70 mM CaCh. In some embodiments, neutralization buffer comprises about 80 mM CaCh. In some embodiments, neutralization buffer comprises about 90 mM CaCh. In some embodiments, neutralization buffer comprises about 100 mM CaCh. In some embodiments, neutralization buffer comprises about 1 10 mM CaCh. In some embodiments, neutralization buffer comprises about 120 mM CaCh. In some embodiments, neutralization buffer comprises about 130 mM CaCh. In some embodiments, neutralization buffer comprises about 140 mM CaCh. In some embodiments, neutralization buffer comprises about 150 mM CaCh. In some embodiments, neutralization buffer comprises about 160 mM CaCh. In some embodiments, neutralization buffer comprises about 170 mM CaCh. In some embodiments, neutralization buffer comprises about 180 mM CaCh. In some embodiments, neutralization buffer comprises about 190 mM CaCh. In some embodiments, neutralization buffer comprises about 200 mM CaCh. In some embodiments, neutralization buffer comprises 120 mM potassium acetate. 200mM acetic acid, 100 mM CaCh. In one embodiment. 5 ml of neutralization buffer is used per gram of bacterial wet cell mass.

[0057] This application describes the use of a centrifugation to remove floc precipitate from one or more solutions, such as a neutralized lysate solution. In some embodiments, the neutralized lysate solution is generated from a bacterial pellet which has been resuspended in resuspension buffer, lysed in lysis buffer, and neutralized with neutralization buffer. Floc precipitate is a mixture of cell debris, proteins, genomic DNA, and RNA. including RNA bound to CaCh. One of skill in the art will understand that removing floc precipitate from a solution can be accomplished through a variety of means known in the art, including centrifugation, and that centrifugation speed, duration, and temperature can be varied as needed to remove floc precipitate. For purposes of illustration, centrifugation can be performed at about 6,000 ref for about 30 minutes at about 4°C. In some embodiments, centrifugation can be performed at about 4,000 ref In some embodiments, centrifugation can be performed at about 4,500 ref In some embodiments, centrifugation can be performed at about 5,000 ref In some embodiments, centrifugation can be performed at about 5,500 ref. In some embodiments, centrifugation can be performed at about 6,000 ref. In some embodiments, centrifugation can be performed at about 6,500 ref In some embodiments, centrifugation can be performed at about 7,000 ref In some embodiments, centrifugation can be performed for about 15 minutes. In some embodiments, centrifugation can be performed for about 20 minutes. In some embodiments, centrifugation can be performed for about 21 minutes. In some embodiments, centrifugation can be performed for about 30 minutes. In some embodiments, centrifugation can be performed for about 35 minutes. In some embodiments, centrifugation can be performed for about 40 minutes. In some embodiments, centrifugation can be performed at about 1°C. In some embodiments, centrifugation can be performed at about 2°C. In some embodiments, centrifugation can be performed at about 3°C. In some embodiments, centrifugation can be performed at about 4°C. In some embodiments, centrifugation can be performed at about 5°C. In some embodiments, centrifugation can be performed at about 6°C. In some embodiments, centrifugation can be performed at about 7°C. In some embodiments, centrifugation can be performed at about 8°C. Pall Supracap™ 50 Depth Filter capsules may be considered if no centrifugation step is performed. These capsules exhibit low hold-up volume, allowing for optimal product recovery for sample filtration of 1 - 3 L.

[0058] This application describes the use of a microfiltration to remove residual floc precipitate from one or more solutions, including neutralized lysate solutions. One of skill in the art will understand that residual floc precipitate can be removed from a solution through a variety of means known in the art, including microfiltration. For purposes of illustration, microfiltration can be performed once or twice using 0.2-pm filters. In some embodiments, the neutralized lysate solution has already been centrifuged, and microfiltration serves to remove any residual floc precipitation. In some embodiments, a 0.2-pm filter is used for microfiltration. In some embodiments, microfiltration is performed one or more times.

[0059] This application describes the use of an ultrafiltration and diafiltration step to concentrate a sample, remove RNA from the sample, and exchange the sample buffer. One of skill in the art will understand that ultrafiltration and diafiltration can be achieved through a variety' of means known in the art, including tangential flow filtration (TFF) capsules or ultrafiltration spin tubes and that the filter size can range from about 100 kDa to about 300 kDa. For purposes of illustration, ultrafiltration and diafiltration using a diafiltration buffer can be performed using a TFF capsule or ultrafiltration spin tubes to filter the solution to a low volume retentate, with RNA being extruded in the permeate. Alternative methods include centrifugation, use of micromolar filters, or use of depth filters. In some embodiments, the sample is filtered using the TFF multiple times or using multiple ultrafiltration spin tubes, wherein diafiltration buffer is used to bring the retentate up to a desired volume. In some embodiments, the diafiltration buffer comprises potassium acetate at about pH 4 to about pH 6.5 and EDTA. Potassium acetate concentration in the diafiltration buffer can be, for example, from about 10 mM to about 200 mM. In some embodiments, diafiltration buffer comprises about 20 mM potassium acetate. In some embodiments, diafiltration buffer comprises about 30 mM potassium acetate. In some embodiments, diafiltration buffer comprises about 40 mM potassium acetate. In some embodiments, diafiltration buffer comprises about 50 mM potassium acetate. In some embodiments, diafiltration buffer comprises about 60 mM potassium acetate. In some embodiments, diafiltration buffer comprises about 70 mM potassium acetate. In some embodiments, diafiltration buffer comprises about 80 mM potassium acetate. In some embodiments, diafiltration buffer comprises about 90 mM potassium acetate. In some embodiments, diafiltration buffer comprises about 100 mM potassium acetate. In some embodiments, diafiltration buffer comprises about 1 10 mM potassium acetate. In someembodiments, diafiltration buffer comprises about 120 mM potassium acetate. In some embodiments, diafiltration buffer comprises about 130 mM potassium acetate. In some embodiments, diafiltration buffer comprises about 140 mM potassium acetate. In some embodiments, diafiltration buffer comprises about 150 mM potassium acetate. In some embodiments, diafiltration buffer comprises about 160 mM potassium acetate. In some embodiments, diafiltration buffer comprises about 170 mM potassium acetate. In some embodiments, diafiltration buffer comprises about 180 mM potassium acetate. In some embodiments, diafiltration buffer comprises about 190 mM potassium acetate. In some embodiments, diafiltration buffer comprises about 200 mM potassium acetate. In some embodiments, the potassium acetate is at about pH 4.0. In some embodiments, the potassium acetate is at about pH 4.2. In some embodiments, the potassium acetate is at about pH 4.4. In some embodiments, the potassium acetate is at about pH 4.6. In some embodiments, the potassium acetate is at about pH 4.8. In some embodiments, the potassium acetate is at about pH 5.0. In some embodiments, the potassium acetate is at about pH 5.2. In some embodiments, the potassium acetate is at about pH 5.4. In some embodiments, the potassium acetate is at about pH 5.6. In some embodiments, the potassium acetate is at about pH 5.8. In some embodiments, the potassium acetate is at about pH 6.0. EDTA concentration in the diafiltration buffer can be, for example, from about 0 mM to about 30 mM. In some embodiments, diafiltration buffer comprises about 0 mM EDTA. In some embodiments, diafiltration buffer comprises about 6 mM EDTA. In some embodiments, diafiltration buffer comprises about 10 mM EDTA. In some embodiments, diafiltration buffer comprises about 15 mM EDTA. In some embodiments, diafiltration buffer comprises about 20 mM EDTA. In some embodiments, diafiltration buffer comprises about 25 mM EDTA. In some embodiments, diafiltration buffer comprises about 30 mM EDTA. In some embodiments, the diafiltration buffer comprises 120 mM potassium acetate at pH 5.5 and 25 mM EDTA. In this described mode of discontinuous diafiltration, the sample is first concentrated and then diluted back to its original volume with replacement buffer. However, using a buffer feed container, continuous diafiltration also may be considered.

[0060] After ultrafiltration and diafiltration have been performed the retentate comprises the pDNA and is referred to as the cleared lysate. The steps described in this section from growth through ultrafiltration generate a product that is henceforth described as cleared lysate. In someembodiments, the 0.2-pm filtration precedes the TFF (ultrafiltration concentration / diafiltration) step.Magnetic Beads

[0061] This application describes the use of magnetic beads to bind plasmid DNA in a cleared lysate. One of skill in the art will understand that binding of plasmid DNA using magnetic beads can be achieved through a variety of means known in the art, including a variety of different types of beads. For purposes of illustration, in some embodiments superparamagnetic siliceous oxide coated beads are used. Superparamagnetic siliceous oxide coated beads can be prepared by any appropriate method known in the art, including the following: (1) preparing magnetite core particles by aqueous precipitation of a mixture of FeC12 and FeC13 (2) depositing a siliceous oxide coating on the magnetite core particles by exposing a slurry of the particles to a mixture of SiO2 and Na2O for at least about 45 minutes at a temperature of at least 60° C. and then adding an acid solution to the mixture until the pH is lowered to a pH less than 9, (3) allowing the resulting slurry7to age for at least about 15 minutes, preferably while continuing to agitate the slurry, and (4) washing the particles. US Patent No. 8,093,373, which is hereby incorporated by reference, describes methods of making superparamagnetic siliceous oxide coated beads. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 20 wt. % silica to about 90 wt. % silica. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 20 wt. % silica. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 25 wt. % silica. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 30 wt. % silica. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 35 wt. % silica. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 40 wt. % silica. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 45 wt. % silica. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 50 wt. % silica. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 55 wt. % silica. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 60 wt. % silica. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 65 wt. % silica. In some embodiments, thesuperparamagnetic siliceous oxide coated beads comprise about 70 wt. % silica. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 75 wt. % silica. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 80 wt. % silica. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 85 wt. % silica. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 90 wt. % silica. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 10 wt. % superparamagnetic core material to about 80 wt. % superparamagnetic core material. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 10 wt. % superparamagnetic core material. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 20 wt. % superparamagnetic core material. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 30 wt. % superparamagnetic core material. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 40 wt. % superparamagnetic core material. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 50 wt. % superparamagnetic core material. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 60 wt. % superparamagnetic core material. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 70 wt. % superparamagnetic core material. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise about 80 wt. % superparamagnetic core material.

[0062] Particle surface area can be determined by any appropriate means in the art, such as the Brunauer-Emmett-Teller (BET). Nitrogen BET surface area is a measure of the surface area of a material, determined using the Brunauer-Emmett-Teller (BET) th eon of gas adsorption. The BET method involves the adsorption of nitrogen gas onto the surface of a material, and the subsequent analysis of the resulting adsorption isotherm to calculate the surface area. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise a surface area of about 5 m2 / g to about 100 m2 / g. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise a surface area of about 5 m2 / g. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise a surface area of about 20 m2 / g. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise a surface area of about 40 m2 / g. In some embodiments, the superparamagnetic siliceous oxide coatedbeads comprise a surface area of about 60 m2 / g. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise a surface area of about 80 m2 / g. In some embodiments, the superparamagnetic siliceous oxide coated beads comprise a surface area of about 100 m2 / g.

[0063] In some embodiments, the superparamagnetic siliceous oxide coated beads are about 1 pm to about 25 pm in diameter. In some embodiments, the superparamagnetic siliceous oxide coated beads are about 1 pm in diameter. In some embodiments, the superparamagnetic siliceous oxide coated beads are about 5 pm in diameter. In some embodiments, the superparamagnetic siliceous oxide coated beads are about 10 pm in diameter. In some embodiments, the superparamagnetic siliceous oxide coated beads are about 15 pm in diameter. In some embodiments, the superparamagnetic siliceous oxide coated beads are about 20 pm in diameter. In some embodiments, the superparamagnetic siliceous oxide coated beads are about 25 pm in diameter. In some embodiments, the superparamagnetic siliceous oxide coated beads leach less than about 15 parts per million from the core. In some embodiments, the superparamagnetic siliceous oxide coated beads leach less than about 10 parts per million from the core. In some embodiments, the superparamagnetic siliceous oxide coated beads leach less than about 5 parts per million from the core. In some embodiments, the superparamagnetic siliceous oxide coated beads leach less than about 1 parts per million from the core.

[0064] Magnetic bead response is defined in terms of ultraviolet transmitance through the sample, with greater transmitance indicating an improved magnetic response. UV-Vis transmitance is the ratio of the intensity of light that passes through a sample to the intensity of the light that is incident on the sample, as measured by a UV-Vis spectrophotometer. The transmitance is usually expressed as a percentage, with 100% transmitance meaning that all the light passes through the sample and 0% transmitance meaning that no light passes through the sample. For magnetic response, transmitance is an indication on how fast the resin will move towards a magnet, thus clearing the sample and allowing light to pass through. For purposes of illustration, in some embodiments the transmitance of the superparamagnetic siliceous oxide coated beads is about 10% to about 80% at 5 seconds of field exposure, about 45% to about 100% at 40 seconds of exposure, and about 80% to about 100% at 600 seconds of exposure. In some embodiments, the transmitance is about 10% at 5 seconds of field exposure. In some embodiments, the transmitance is about 20% at 5 seconds of field exposure. In someembodiments, the transmittance is about 30% at 5 seconds of field exposure. In some embodiments, the transmittance is about 40% at 5 seconds of field exposure. In some embodiments, the transmittance is about 50% at 5 seconds of field exposure. In some embodiments, the transmittance is about 60% at 5 seconds of field exposure. In some embodiments, the transmittance is about 70% at 5 seconds of field exposure. In some embodiments, the transmittance is about 80% at 5 seconds of field exposure. In some embodiments, the transmittance is about 45% at 40 seconds of field exposure. In some embodiments, the transmittance is about 55% at 40 seconds of field exposure. In some embodiments, the transmittance is about 65% at 40 seconds of field exposure. In some embodiments, the transmittance is about 75% at 40 seconds of field exposure. In some embodiments, the transmittance is about 85% at 40 seconds of field exposure. In some embodiments, the transmittance is about 95% at 40 seconds of field exposure. In some embodiments, the transmittance is about 100% at 40 seconds of field exposure. In some embodiments, the transmittance is about 80% at 600 seconds of field exposure. In some embodiments, the transmittance is about 85% at 600 seconds of field exposure. In some embodiments, the transmittance is about 90% at 600 seconds of field exposure. In some embodiments, the transmittance is about 95% at 600 seconds of field exposure. In some embodiments, the transmittance is about 100% at 600 seconds of field exposure.

[0065] One of skill in the art will understand that this disclosure is not limited to using magnetic beads made by the methods described herein. Alternative methods of making beads may be used, such as those described in U.S. Pat. No. 7.897,257 or U.S. Pat. No. 10,040,073, both of which are incorporated by reference in their entirety.Magnetic Bead Binding and Purification of the pDNA

[0066] The cleared lysate, can then be resuspended with a binding buffer for plasmid DNA purification. In some embodiments, the binding buffer comprises guanidine HO, potassium acetate, and EDTA. Alternatives for guanidine HC1 include sodium perchlorate, sodium trichloroacetate, guanidinium thiocyanate, and potassium acetate. Guanidine HC1 concentration in the binding buffer can be, for example, from about 0.5 M to about 6 M. In some embodiments, binding buffer comprises about 0.5 M guanidine HC1. In some embodiments, binding buffer comprises about 1.0 M guanidine HC1. In some embodiments, binding buffercomprises about 1.0 M guanidine HC1. In some embodiments, binding buffer comprises about1 .5 M guanidine HC1. In some embodiments, binding buffer comprises about 2.0 M guanidine HCL In some embodiments, binding buffer comprises about 2.5 M guanidine HC1. In some embodiments, binding buffer comprises about 3.0 M guanidine HC1. In some embodiments, binding buffer comprises about 3.5 M guanidine HC1. In some embodiments, binding buffer comprises about 4.0 M guanidine HC1. In some embodiments, binding buffer comprises about4.5 M guanidine HC1. In some embodiments, binding buffer comprises about 5.0 M guanidine HCL In some embodiments, binding buffer comprises about 5.5 M guanidine HC1. In some embodiments, binding buffer comprises about 6.0 M guanidine HCL In some embodiments, binding buffer comprises guanidine HC1 at a final concentration of 3M after accounting for the volume of the retentate. Potassium acetate at pH 5.5 concentration in the binding buffer can be, for example, from about 10 mM to about 200 mM. In some embodiments, binding buffer comprises about 20 mM potassium acetate at pH 5.5. In some embodiments, binding buffer comprises about 30 mM potassium acetate at pH 5.5. In some embodiments, binding buffer comprises about 40 mM potassium acetate at pH 5.5. In some embodiments, binding buffer comprises about 50 mM potassium acetate at pH 5.5. In some embodiments, binding buffer comprises about 60 mM potassium acetate at pH 5.5. In some embodiments, binding buffer comprises about 70 mM potassium acetate at pH 5.5. In some embodiments, binding buffer comprises about 80 mM potassium acetate at pH 5.5. In some embodiments, binding buffer comprises about 90 mM potassium acetate at pH 5.5. In some embodiments, binding buffer comprises about 100 mM potassium acetate a 1 pH 5.5. In some embodiments, binding buffer comprises about 110 mM potassium acetate at pH 5.5. In some embodiments, binding buffer comprises about 120 mM potassium acetate at pH 5.5. In some embodiments, binding buffer comprises about 130 mM potassium acetate at pH 5.5. In some embodiments, binding buffer comprises about 140 mM potassium acetate at pH 5.5. In some embodiments, binding buffer comprises about 150 mM potassium acetate at pH 5.5. In some embodiments, binding buffer comprises about 160 mM potassium acetate at pH 5.5. In some embodiments, binding buffer comprises about 170 mM potassium acetate at pH 5.5. In some embodiments, binding buffer comprises about 180 mM potassium acetate at pH 5.5. In some embodiments, binding buffer comprises about 190 mM potassium acetate at pH 5.5. In some embodiments, binding buffer comprises about 200 mM potassium acetate at pH 5.5. EDTA concentration in the binding buffer can be, for example, from about 0 mM to about 30 mM. In some embodiments, bindingbuffer comprises about 0 mM EDTA. In some embodiments, binding buffer comprises about 6 mM EDTA. In some embodiments, binding buffer comprises about 10 mM EDTA. In some embodiments, binding buffer comprises about 15 mM EDTA. In some embodiments, binding buffer comprises about 20 mM EDTA. In some embodiments, binding buffer comprises about 25 mM EDTA. In some embodiments, binding buffer comprises about 30 mM EDTA. In some embodiments, the binding buffer comprises 120 mM potassium acetate at pH 5.5 and 25 mM EDTA. In some embodiments, the binding buffer comprises a final composition of 3 M guanidine HC1, 120 mM potassium acetate at pH 5.5 and 25 mM EDTA after resuspension of the retentate.

[0067] Magnetic beads are then added to the binding buffer solution. For purposes of illustration, magnetic beads are added at about 0. 1 wt. % to about 3 wt. %. In some embodiments, the magnetic beads are added at about 0.1 wt. %. In some embodiments, the magnetic beads are added at about 0. 1 wt. %. In some embodiments, the magnetic beads are added at about 0. 1 wt. %. In some embodiments, the magnetic beads are added at about 0.1 wt. %. In some embodiments, the magnetic beads are added at about 0. 1 wt. %. In some embodiments, the magnetic beads are added at about 0.2 wt. %. In some embodiments, the magnetic beads are added at about 0.3 wt. %. In some embodiments, the magnetic beads are added at about 0.4 wt. %. In some embodiments, the magnetic beads are added at about 0.5 wt. %. In some embodiments, the magnetic beads are added at about 0.6 wt. %. In some embodiments, the magnetic beads are added at about 0.7 wt. %. In some embodiments, the magnetic beads are added at about 0.8 wt. %. In some embodiments, the magnetic beads are added at about 0.9 wt. %. In some embodiments, the magnetic beads are added at about 1.0 wt. %. In some embodiments, the magnetic beads are added at about 1.1 wt. %. In some embodiments, the magnetic beads are added at about 1.2 wt. %. In some embodiments, the magnetic beads are added at about 1.3 wt. %. In some embodiments, the magnetic beads are added at about 1.4 wt. %. In some embodiments, the magnetic beads are added at about 1.5 wt. %. In some embodiments, the magnetic beads are added at about 1.6 wt. %. In some embodiments, the magnetic beads are added at about 1.7 wt. %. In some embodiments, the magnetic beads are added at about 1.8 wt. %. In some embodiments, the magnetic beads are added at about 1.9 wt. %. In some embodiments, the magnetic beads are added at about 2.0 wt. %. In some embodiments, the magnetic beads are added at about 2. 1 wt. %. In some embodiments, themagnetic beads are added at about 2.2 wt. %. In some embodiments, the magnetic beads are added at about 2.3 wt. %. In some embodiments, the magnetic beads are added at about 2.4 wt. %. In some embodiments, the magnetic beads are added at about 2.5 wt. %. In some embodiments, the magnetic beads are added at about 2.6 wt. %. In some embodiments, the magnetic beads are added at about 2.7 wt. %. In some embodiments, the magnetic beads are added at about 2.8 wt. %. In some embodiments, the magnetic beads are added at about 2.9 wt. %. In some embodiments, the magnetic beads are added at about 3.0 wt. %.

[0068] This application describes the use of magnetic beads to bind and purify pDNA. One of skill in the art will understand that a variety of incubation and purification methods could be used. For purposes of illustration, beads and the cleared lysate are incubated for 30 minutes at 18°C with mixing using a revolver adjustable rotator at 40 RPM. Incubation times can vary based on method parameters, and one of skill in the art will appreciate that incubation times can be anywhere from about 5 minutes to about 12 hours. In some embodiments, the incubation is 5 minutes. In some embodiments, the incubation is 10 minutes. In some embodiments, the incubation is 15 minutes. In some embodiments, the incubation is 20 minutes. In some embodiments, the incubation is 25 minutes. In some embodiments, the incubation is 30 minutes. In some embodiments, the incubation is 35 minutes. In some embodiments, the incubation is 40 minutes. In some embodiments, the incubation is 45 minutes. In some embodiments, the incubation is 50 minutes. In some embodiments, the incubation is 55 minutes. In some embodiments, the incubation is 60 minutes. In some embodiments, the incubation is 2 hours. In some embodiments, the incubation is 3 hours. In some embodiments, the incubation is 4 hours. In some embodiments, the incubation is 5 hours. In some embodiments, the incubation is 6 hours. In some embodiments, the incubation is 7 hours. In some embodiments, the incubation is 8 hours. In some embodiments, the incubation is 9 hours. In some embodiments, the incubation is 10 hours. In some embodiments, the incubation is 11 hours. In some embodiments, the incubation is 12 hours. Any appropriate rotator can be used to gently mix the incubation. In some embodiments, the incubation is at about 1°C. In some embodiments, the incubation is at about 5°C. In some embodiments, the incubation is at about 10°C. In some embodiments, the incubation is at about 15°C. In some embodiments, the incubation is at about 18°C. In some embodiments, the incubation is at about 20°C. In some embodiments, the incubation is at about 25°C. In some embodiments, the incubation is at about30°C. In some embodiments, the incubation is at about 35°C. In some embodiments, the incubation is at about 40°C. In some embodiments, the incubation is at about 45°C.

[0069] This application describes the use of a magnetic field to separate pDNA bound to magnetic beads from solution, with subsequent wash steps. One of skill in the art will understand that a variety of magnetic field sources and applications could be used. For purposes of illustration, the solution with pDNA bound to magnetic beads can be placed in a Sepmag A200ml magnetic separation unit for a three-minute separation time. Any appropriate source of a magnetic field can be used to pull the beads to the sides or bottom of the tube, and any appropriate separation time can be used. Magnetic response is measured in terms of ultraviolet transmittance through the sample, with greater transmittance indicating an improved magnetic response. For example, in some embodiments the transmittances is about 10% to about 80% at 5 seconds of field exposure, about 45% to about 100% at 40 seconds of exposure, and about 80% to about 100% at 600 seconds of exposure. After the separation time the supernatant is aspirated using any appropriate means (e.g, a peristaltic pump) and a wash solution is added. Washes are performed to remove any final impurities prior to elution of the pDNA. For purposes of illustration, the wash buffer can comprise about 50% to about 90% ethanol or isopropanol and about 50% to about 10% of about a 0 mM to about 50 mM Tris-EDTA solution at about pH 7.5 to about pH 9.0. In some embodiments, the ethanol or isopropanol is 50% of the wash solution. In some embodiments, the ethanol or isopropanol is 50% of the wash solution. In some embodiments, the ethanol or isopropanol is 55% of the wash solution. In some embodiments, the ethanol or isopropanol is 60% of the wash solution. In some embodiments, the ethanol or isopropanol is 65% of the wash solution. In some embodiments, the ethanol or isopropanol is 70% of the wash solution. In some embodiments, the ethanol or isopropanol is 75% of the wash solution. In some embodiments, the ethanol or isopropanol is 80% of the wash solution. In some embodiments, the ethanol or isopropanol is 85% of the wash solution. In some embodiments, the ethanol or isopropanol is 90% of the wash solution. In some embodiments, the Tris-EDTA is at 0 mM concentration. In some embodiments, the Tris-EDTA is at 5 mM concentration. In some embodiments, the Tris-EDTA is at 10 mM concentration. In some embodiments, the Tris-EDTA is at 15 mM concentration. In some embodiments, the Tris- EDTA is at 20 mM concentration. In some embodiments, the Tris-EDTA is at 25 mM concentration. In some embodiments, the Tris-EDTA is at 30 mM concentration. In someembodiments, the Tris-EDTA is at 35 mM concentration. In some embodiments, the Tris- EDTA is at 40 mM concentration. In some embodiments, the Tris-EDTA is at 45 mM concentration. In some embodiments, the Tris-EDTA is at 50 mM concentration. In some embodiments, the solution pH is about 7.5. In some embodiments, the solution pH is about 7.6. In some embodiments, the solution pH is about 7.7. In some embodiments, the solution pH is about 7.8. In some embodiments, the solution pH is about 7.9. In some embodiments, the solution pH is about 8.0. In some embodiments, the solution pH is about 8.1. In some embodiments, the solution pH is about 8.2. In some embodiments, the solution pH is about 8.3. In some embodiments, the solution pH is about 8.4. In some embodiments, the solution pH is about 8.5. In some embodiments, the solution pH is about 8.6. In some embodiments, the solution pH is about 8.7. In some embodiments, the solution pH is about 8.8. In some embodiments, the solution pH is about 8.9. In some embodiments, the solution pH is about 9.0. In some embodiments, the wash buffer only comprises ethanol or isopropanol. In some embodiments where low volumes of ethanol or isopropanol are used the solution further comprises salts such as potassium acetate. In some embodiments the wash buffer is chilled before use (e.g., at 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, or 8°C). In some embodiments, the wash buffer comprises 70% ethanol or isopropanol and 30% 10 mM Tris-EDTA at pH 8.0 and is chilled to 4°C. Once the wash buffer is added to the beads the mixture is removed from the magnetic field and the solution is mixed for a period of time (e.g, at 20 RPM on a JOANLAB RM-6Pro tube roller mixer for 1 minute). The beads solution is then returned for another magnetic incubation as described above, and the solution is aspirated. This process, of magnetic field application, supernatant removal, wash buffer addition, mixing, magnetic field application, and supernatant removal, comprises a wash. One of skill in the art will appreciate that any suitable number of washes can be performed to remove supernatants. In some embodiments, one wash is performed. In some embodiments, two washes are performed. In some embodiments, three washes are performed. In some embodiments, four washes are performed. In some embodiments, five washes are performed. In some embodiments, two washes are performed.

[0070] This application describes the use of an elution buffer to elute pDNA from magnetic beads after a final wash step. One of skill in the art will understand that a variety of elution buffers and conditions could be used. For purposes of illustration, the elution buffer can comprisewater or a low-salt buffer, about 5 mM to about 100 mM Tris-HCl at a pH of about 7 to about 8.5, and about 0 mM EDTA to about 10 mM EDTA. In some embodiments, the elution buffer comprises about 5 mM Tris-HCl. In some embodiments, the elution buffer comprises about 10 mM Tris-HCl. In some embodiments, the elution buffer comprises about 15 mM Tris-HCl. In some embodiments, the elution buffer comprises about 20 mM Tris-HCl. In some embodiments, the elution buffer comprises about 25 mM Tris-HCl. In some embodiments, the elution buffer comprises about 30 mM Tris-HCl. In some embodiments, the elution buffer comprises about 35 mM Tris-HCl. In some embodiments, the elution buffer comprises about 40 mM Tris-HCl. In some embodiments, the elution buffer comprises about 45 mM Tris-HCl. In some embodiments, the elution buffer comprises about 50 mM Tris-HCl. In some embodiments, the elution buffer comprises about 55 mM Tris-HCl. In some embodiments, the elution buffer comprises about 60 mM Tris-HCl. In some embodiments, the elution buffer comprises about 65 mM Tris-HCl. In some embodiments, the elution buffer comprises about 70 mM Tris-HCl. In some embodiments, the elution buffer comprises about 75 mM Tris-HCl. In some embodiments, the elution buffer comprises about 80 mM Tris-HCl. In some embodiments, the elution buffer comprises about 85 mM Tris-HCl. In some embodiments, the elution buffer comprises about 90 mM Tris-HCl. In some embodiments, the elution buffer comprises about 95 mM Tris-HCl. In some embodiments, the elution buffer comprises about 100 mM Tris-HCl. In some embodiments, the Tris-HCl is at a pH of about 7.0. In some embodiments, the Tris-HCl is at a pH of about 7.1. In some embodiments, the Tris-HCl is at a pH of about 7.2. In some embodiments, the Tris-HCl is at a pH of about 7.3. In some embodiments, the Tris-HCl is at a pH of about 7.4. In some embodiments, the Tris-HCl is at a pH of about 7.5. In some embodiments, the Tris-HCl is at a pH of about 7.6. In some embodiments, the Tris-HCl is at a pH of about 7.7. In some embodiments, the Tris-HCl is at a pH of about 7.8. In some embodiments, the Tris-HCl is at a pH of about 7.9. In some embodiments, the Tris-HCl is at a pH of about 8.0. In some embodiments, the Tris-HCl is at a pH of about 8.1. In some embodiments, the Tris-HCl is at a pH of about 8.2. In some embodiments, the Tris-HCl is at a pH of about 8.3. In some embodiments, the Tris-HCl is at a pH of about 8.4. In some embodiments, the Tris-HCl is at a pH of about 8.5. In some embodiments, the elution buffer comprises about 0 mM EDTA. In some embodiments, the elution buffer comprises about 1 mM EDTA. In some embodiments, the elution buffer comprises about 2 mM EDTA. In some embodiments, the elution buffer comprises about 3mM EDTA. In some embodiments, the elution buffer comprises about 4 mM EDTA. In some embodiments, the elution buffer comprises about 5 mM EDTA. In some embodiments, the elution buffer comprises about 6 mM EDTA. In some embodiments, the elution buffer comprises about 7 mM EDTA. In some embodiments, the elution buffer comprises about 8 mM EDTA. In some embodiments, the elution buffer comprises about 9 mM EDTA. In some embodiments, the elution buffer comprises about 10 mM EDTA. In some embodiments, the elution buffer comprises water, 10 mM Tris-HCl at pH 8, and ImM EDTA. Once the elution buffer has been added to the solution it is exposed to a magnetic field, as described above, and the plasmid containing supernatant is removed from the beads. At this stage an optional polishing step can be performed (e.g, HIC chromatograph) to improve purity.

[0071] At various stages in the methods of the present technology a roller mixer may be used to mix solutions gently to minimize DNA damage. One of skill in the art will appreciate that steps, including cell lysis, neutralization of lysate, clearance of lysate, binding of magnetic beads, and washing of magnetic beads, can use a roller mixer at any appropriate speed. For example, in some embodiments a roller mixer can operate at about 10 RPM to about 200 RPM. In some embodiments, a roller mixer operates at about 10 RPM. In some embodiments, a roller mixer operates at about 20 RPM. In some embodiments, a roller mixer operates at about 30 RPM. In some embodiments, a roller mixer operates at about 40 RPM. In some embodiments, a roller mixer operates at about 50 RPM. In some embodiments, a roller mixer operates at about 60 RPM. In some embodiments, a roller mixer operates at about 70 RPM. In some embodiments, a roller mixer operates at about 80 RPM. In some embodiments, a roller mixer operates at about 90 RPM. In some embodiments, a roller mixer operates at about 100 RPM. In some embodiments, a roller mixer operates at about 110 RPM. In some embodiments, a roller mixer operates at about 120 RPM. In some embodiments, a roller mixer operates at about 130 RPM. In some embodiments, a roller mixer operates at about 140 RPM. In some embodiments, a roller mixer operates at about 150 RPM. In some embodiments, a roller mixer operates at about 160 RPM. In some embodiments, a roller mixer operates at about 170 RPM. In some embodiments, a roller mixer operates at about 180 RPM. In some embodiments, a roller mixer operates at about 190 RPM. In some embodiments, a roller mixer operates at about 200 RPM. In some embodiments, a roller mixer operates different RPM speeds for different steps of the method.

[0072] Plasmid yield can be calculated using any suitable method. Nucleic acid concentration can be given as, for example, pg of nucleic acids per gram of wet cell mass (pg / g WCM). This application describes methods of pDNA purification that result in high yields. One of skill in the art will understand that a variety of yields could be achieved. For purposes of illustration, the methods described herein can achieve a yield of about 200 pg / g WCM to about 650 pg / g WCM. In some embodiments, the methods achieve a yield of about 200 pg / g WCM. In some embodiments, the methods achieve a yield of about 200 pg / g WCM. In some embodiments, the methods achieve a yield of about 225 pg / g WCM. In some embodiments, the methods achieve a yield of about 250 pg / g WCM. In some embodiments, the methods achieve a yield of about 275 pg / g WCM. In some embodiments, the methods achieve a yield of about 300 pg / g WCM. In some embodiments, the methods achieve a yield of about 325 pg / g WCM. In some embodiments, the methods achieve a yield of about 350 pg / g WCM. In some embodiments, the methods achieve a yield of about 375 pg / g WCM. In some embodiments, the methods achieve a yield of about 400 pg / g WCM. In some embodiments, the methods achieve a yield of about 425 pg / g WCM. In some embodiments, the methods achieve a yield of about 450 pg / g WCM. In some embodiments, the methods achieve a yield of about 475 pg / g WCM. In some embodiments, the methods achieve a yield of about 500 pg / g WCM. In some embodiments, the methods achieve a yield of about 525 pg / g WCM. In some embodiments, the methods achieve a yield of about 550 pg / g WCM. In some embodiments, the methods achieve a yield of about 575 pg / g WCM. In some embodiments, the methods achieve a yield of about 600 pg / g WCM. In some embodiments, the methods achieve a yield of about 625 pg / g WCM. In some embodiments, the methods achieve a yield of about 650 pg / g WCM. A commercially available alternative bead-based DNA purification kit, identified as Comparative Trial 7. achieved yields ranging from 410 pg / g WCM to 522 pg / g WCM (FIG. 3).

[0073] Plasmid obtained after the elution step of the methods of the present disclosure is therapeutic-grade due to the high degree of purity and the primarily supercoiled form. Therapeutic-grade plasmid DNA refers to plasmid DNA that can be safely and effectively used in therapeutic applications. Therapeutic-grade plasmid DNA: (i) is free of RNase contamination and has never been exposed to bovine sourced RNases, (ii) is at least 80% supercoiled, (iii) has an A260 / A280 ratio of about 1.80 to about 1.97 (iv) has an A260 / A230 ratio greater than about 1.5 and (v) has less than 100 EU endotoxin per mg pDNA.

[0074] Plasmid purity can be calculated using any suitable methods, including the A260 / A280 ratio, the A260 / A230 ratio, and the endotoxin concentration. The A260 / A280 ratio is the ratio of a solution’s absorbance at 260nm and 280nm. An A260 / A280 ratio below 1.8 may indicate protein contamination of a DNA sample, while a ratio above 2.0 may indicate RNA contamination (Koetsier and Cantor, 2019). In some embodiments, the methods of the present technology generate purified pDNA with an A260 / A280 ratio between about 1.80 and about 2.0. In some embodiments, the methods of the present technology generate purified pDNA with an A260 / A280 ratio of about 1.80. In some embodiments, the methods of the present technology' generate purified pDNA with an A260 / A280 ratio of about 1.85. In some embodiments, the methods of the present technology generate purified pDNA with an A260 / A280 ratio of about 1.89. In some embodiments, the methods of the present technology generate purified pDNA with an A260 / A280 ratio of about 1.90. In some embodiments, the methods of the present technology7generate purified pDNA with an A260 / A280 ratio of about 1.95. In some embodiments, the methods of the present technology generate purified pDNA with an A260 / A280 ratio of about 1.97. In some embodiments, the methods of the present technology generate purified pDNA with an A260 / A280 ratio of about 2.00. The A260 / A230 ratio is the ratio of a solution’s absorbance at 260nm and 230nm. An A260 / A230 ratio below 1.5 may indicate contamination of nucleic acids with chaotropic salts, proteins, carbohydrates or phenol (Koetsier and Cantor, 2019). In some embodiments, the methods of the present technology generate purified pDNA with an A260 / A230 ratio of about 1.5 to about 2.4. In some embodiments, the methods of the present technology generate purified pDNA with an A260 / A230 ratio of about 1.5. In some embodiments, the methods of the present technology generate purified pDNA with an A260 / A230 ratio of about 1.5. In some embodiments, the methods of the present technology generate purified pDNA with an A260 / A230 ratio of about 1 .5. In some embodiments, the methods of the present technology generate purified pDNA with an A260 / A230 ratio of about 1.5. In some embodiments, the methods of the present technology generate purified pDNA with an A260 / A230 ratio of about 1.6. In some embodiments, the methods of the present technology generate purified pDNA with an A260 / A230 ratio of about 1.7. In some embodiments, the methods of the present technology generate purified pDNA with an A260 / A230 ratio of about 1.8. In some embodiments, the methods of the present technology generate purified pDNA with an A260 / A230 ratio of about 1.9. In some embodiments, the methods of the present technology generate purified pDNA with anA260 / A230 ratio of about 2.0. Endotoxin concentration can be calculated using a kineticchromogenic or turbidimetric test with Limulus amebocyte lysate (LAL). In some embodiments, the methods of the present technology generate purified pDNA with about 1 to about 200 EU endotoxin per mg pDNA. In some embodiments the purified pDNA has about 200 EU endotoxin per mg pDNA. In some embodiments the purified pDNA has about 200 EU endotoxin per mg pDNA. In some embodiments the purified pDNA has about 200 EU endotoxin per mg pDNA. In some embodiments the purified pDNA has about 175 EU endotoxin per mg pDNA. In some embodiments the purified pDNA has about 150 EU endotoxin per mg pDNA. In some embodiments the purified pDNA has about 125 EU endotoxin per mg pDNA. In some embodiments the purified pDNA has about 100 EU endotoxin per mg pDNA. In some embodiments the purified pDNA has about 75 EU endotoxin per mg pDNA. In some embodiments the purified pDNA has about 50 EU endotoxin per mg pDNA. In some embodiments the purified pDNA has about 25 EU endotoxin per mg pDNA. In some embodiments the purified pDNA has about 1 EU endotoxin per mg pDNA.

[0075] Purified pDNA can be present in different forms including supercoiled, open circular, and linear. Therapeutic-grade pDNA must be primarily supercoiled in nature. pDNA form can be determined by any appropriate means. For example, a sample of the pDNA can be applied to a gel e.g., agarose gel) and gel electrophoresis can be performed, allowing the different forms of pDNA to separate. Software can then be used to estimate the percentage of each form of pDNA visible on the gel. One of skill in the art will understand that the percentage of purified plasmids in the supercoiled form can vary. For purposes of illustration, the purified pDNA of the present disclosure can be about 80% to about 99% supercoiled. In some embodiments, the purified pDNA is about 80% supercoiled. In some embodiments, the purified pDNA is about 81% supercoiled. In some embodiments, the purified pDNA is about 82% supercoiled. In some embodiments, the purified pDNA is about 83% supercoiled. In some embodiments, the purified pDNA is about 84% supercoiled. In some embodiments, the purified pDNA is about 85% supercoiled. In some embodiments, the purified pDNA is about 86% supercoiled. In some embodiments, the purified pDNA is about 87% supercoiled. In some embodiments, the purified pDNA is about 88% supercoiled. In some embodiments, the purified pDNA is about 89% supercoiled. In some embodiments, the purified pDNA is about 90% supercoiled. In some embodiments, the purified pDNA is about 90% supercoiled. In some embodiments, the purified pDNA is about 91% supercoiled. In some embodiments, the purified pDNA is about92% supercoiled. In some embodiments, the purified pDNA is about 93% supercoiled. In some embodiments, the purified pDNA is about 94% supercoiled. In some embodiments, the purified pDNA is about 95% supercoiled. In some embodiments, the purified pDNA is about 96% supercoiled. In some embodiments, the purified pDNA is about 97% supercoiled. In some embodiments, the purified pDNA is about 98% supercoiled. In some embodiments, the purified pDNA is about 99% supercoiled.

[0076] The methods of the present technology are based on several surprising and unexpected discoveries, including the fact that increasing percentages of silica present in the superparamagnetic beads lead to an increased purity (as seen by the A260 / A280 ratios being greater than 2) and an increased percentage of supercoiled plasmid. The enhanced purity and super coiled yield, as described in the Examples below, are requirements for the therapeutic- grade plasmids produced by the methods of the present technology.

[0077] This application describes the methods wherein there is an initial volume of pDNA containing bacteria. One of skill in the art will understand that total bacterial load can vary based on method parameters and desired products. For purposes of illustration, in some embodiments the initial volume of pDNA containing bacteria comprises about 1 ml bacteria to about 3 L bacteria. In some embodiments, the initial volume of pDNA containing bacteria is about 1 ml. In some embodiments, the initial volume of pDNA containing bacteria is about 500 ml. In some embodiments, the initial volume of pDNA containing bacteria is about 1 L. In some embodiments, the initial volume of pDNA containing bacteria is about 1.5 L. In some embodiments, the initial volume of pDNA containing bacteria is about 2 L. In some embodiments, the initial volume of pDNA containing bacteria is about 2.5 L. In some embodiments, the initial volume of pDNA containing bacteria is about 3 L.

[0078] Porous superparamagnetic silica with a D50 6.5 pm has the DNA binding capacity equal to 0.8 pm non-porous particles but can have separation rates up to 60x higher for very dilute suspensions; and 3x higher for concentrated suspensions. Compared to chromatography, magnetic particles are a "mobile solid phase” with binding of nucleic acids in solution. Solid phase purification methods adsorb pDNA depending on the pH and salt content of the buffers. The interactions include metal ion bridging with a hydrophilic matrix under chaotropic conditions, ion exchange under aqueous conditions by means of an anion exchanger, andaffinity and size exclusion mechanisms. Chaotropic salts such as guanidine HC1 drive nucleic acid adsorption to the silica surface of each magnetic particle. Guanidine HC1 denatures biomolecules by disrupting the shell of hydration, allowing positively charged ions to form a salt bridge between negatively charged silica and negatively charged nucleic acids. For nonfunctionalized silica, the guanidine HC1 concentration may be reduced significantly or eliminated, when a high concentration of potassium acetate is used to neutralize the alkaline lysis of bacteria (Wang et al., 2000). Similar to liquid phase pDNA purification methods, solid phase pDNA isolation also involves four steps: cell lysis, pDNA adsorption, washing, and elution. Particles with superparamagnetic properties are removed by using a permanent magnet. Magnetic bead-based purification methods avoid centrifugation steps and offer the possibility of automation and / or high throughput extraction of therapeutic-grade pDNA. At the appropriate buffer pH and salt concentration, DNA will associate with the magnetic particle surface. After washing with 70% ethanol, adding an aqueous solution (e.g., deionized water or low ionic strength TE buffer) fully hydrates DNA, resulting in elution. Current methods of pDNA production have high operating costs and time-consuming downstream purification processes.

[0079] Methods using superparamagnetic silica offer an alternative, non-chromatographic approach to purify therapeutic-grade pDNA for emerging therapies. Superparamagnetic particle technology does not require column packing; does not generate shear forces like that can fragment larger pDNA; and binds nucleic acids, but not proteins under high salt conditions. Magnetic separation technologies can offer high recovery, selectivity and purity of pDNA in downstream purification, with the opportunity to replace one or more-unit operations (AEX and / or HIC), saving time and costs (reagents, consumables & hardware) and resulting in a therapeutic-grade product.

[0080] The present disclosure provides a method for purifying therapeutic-grade plasmid DNA (pDNA) comprising the use of a magnetic particle, wherein the magnetic particle comprises a siliceous oxide surface layer and a superparamagnetic core material. The present disclosure also provides an isolated therapeutic-grade plasmid DNA (pDNA) produced by a process comprising the use of a magnetic particle, wherein the magnetic particle comprises a siliceous oxide surface layer and a superparamagnetic core material.

[0081] The present disclosure also provides methods that do not comprise a chromatography step or use of a chromatography column.III. Kits for Therapeutic-Grade Plasmid DNA Purification Using Magnetic Particles

[0082] The present technology contemplates kits comprising magnetic particles for plasmid DNA purification. Kits may comprise any one or more of the materials needed for performing the methods of the present technology as described herein. Kits may also comprise instructions for performing methods of the present technology. Kits may comprise superparamagnetic siliceous oxide coated beads that are preformulated with guanidine HC1, or an equivalent functional agent as described herein. Typically, beads for use in purifying pDNA are formulated with agents to prevent microbial growth. However, such agents would represent undesirable contaminants in preparation of therapeutic grade DNA. Guanidine HC1 possesses antimicrobial activities and is present in the buffers used during bead incubation steps. Accordingly, preformulated beads with guanidine HC1 will remain free of microbes while avoiding introduction of additional contaminants during preparation of the therapeutic-grade plasmid DNA.EXAMPLES

[0083] The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results. The examples should in no way be construed as limiting the scope of the present technology7, as defined by the appended claims.

[0084] Herein, simple and efficient therapeutic-grade pDNA purification protocols are described using the superparamagnetic silica particles of the present disclosure. In this proof-of-concept study, it was demonstrated that the quality and integrity of the pDNA purified by the presently disclosed methods using Trial 1-5 beads was superior to that of pDNA samples cleaned up with commercial plasmid purification kits containing RNase A, for cell growths ranging from 50 to 3000 ml.Example 1: Materials and MethodsPlasmid.

[0085] A cell bank of a 6.1 kbp plasmid, pcDNA3-EGFP (Addgene plasmid number 13031), was prepared using E. coll strain DH5a and stored at -80°C. This plasmid was procured by the Keck Graduate Research Institute (Claremont, CA, USA) where the experiments were performed.Alkaline lysis pDNA purification.

[0086] Briefly, plasmid-containing bacterial cells were cultivated in LB broth containing ampicillin. Bacterial cells were pelleted in pre-weighed 50-ml Falcon tubes or 250-ml Nalgene centrifuge bottles, resuspended in ice-cold LI buffer (50 mM glucose, 25 mM Tris-HCl, 10 mM EDTA, pH 8.0) with incubation on ice for 15 min.; lysed in L2 buffer (0.2 N NaOH, 1% SDS, 18°C) and mixed gently by inversion four to five times before incubation on ice for up to 5 min.; then neutralized with L3 buffer (3 M potassium acetate. 2 M acetic acid, 1 M CaCh, pH 5.5) with gentle mixing by inversion four to five times. The neutralized lysate container was left undisturbed for 10 to 30 min. until three layers formed. The buffer volumes were relative to wet cell mass (wcm) as indicated: LI, Resuspension buffer ml = grams wcm x 5; L2, Lysis buffer ml = grams wcm x 10; L3, Neutralization buffer ml = grams wcm x 5. The final concentration of potassium acetate after neutralization was 0.75 M. Calcium chloride was included since it has been shown to be a precipitant with high RNA removal (A. Eon-Duval et al., 2003). Calcium chloride only was added to the L3 buffer on the day of use to avoid solubility issues.

[0087] Neutralized lysate was centrifuged at the top speed of the centrifuge units to pellet the floc precipitate because using magnetic beads immediately after neutralization may result in floc precipitate interfering with pDNA binding. The supernatant containing the pDNA was transferred to new tubes for the further processing via filtration (e.g., 0.2 pm filters, depth filters, ultrafiltration centrifuge tubes, and / or tangential flow filtration capsules) prior to bead addition. Ultrafiltration / diafiltration using ultrafiltration (UF) centrifuge tube units or tangential flow filtration (TFF) capsules is important to exchange the medium into a buffer suitable for binding to magnetic silica, and for the removal of RNA, small sized genomic DNA, and small proteins.DNA isolation using commercial DNA purification kits.

[0088] A commercially available Plasmid Mini Kit, hereafter referred to as Reference (e.g., Reference 1, 2, 3, 4), was used in pDNA isolation as a reference for yield comparison. Reference spin columns contain a unique silica membrane that binds up to 20 pg DNA in the presence of a high concentration of chaotropic salt and allows elution in a small volume of low salt buffer. The pDNA was isolated according to manufacturer’s instructions. This kit contains RNase A for digestion of RNA. Reference isolated pDNA was dissolved in 10 mM Tris-HCl, pH 8.0. 1 mM EDTA (hereafter referred to as Elution buffer) and kept at -20°C.Nucleic acid purity and concentration measurements.

[0089] A NanoQuant Infinite M200 multi-detection plate reader (Tecan) was used to determine nucleic acid qualify (A260 / A280 and A260 / A230 ratios) and concentration (ng / pl). Before running any samples, the plate was wiped clean with Kimwipes saturated with Milli-Q water, followed by 70% ethanol, and finally with Milli-Q water again. 2pl of Elution buffer was used to blank each of the wells. On the Infinite M200, an additional measurement at a reference wavelength (310 nm) is performed automatically with each measurement.Gel electrophoresis of nucleic acids.

[0090] pDNA samples and a 1-kb DNA ladder (New England Biolabs) were resolved on 1% agarose gels, with 12- or 22 -lanes, and visualized with GelGreen (Biotium) fluorescent dye, based on inclusion in the cast gel or post-staining. Up to ~100ng and 200 ng nucleic acid was loaded per well on the 22- and 12-lane agarose gel, respectively. An IVYX Scientific electrophoresis system was set at 50 V for 60 min. for each run.Plasmid supercoiled quantification.

[0091] To determine the percent of supercoiled purified plasmid DNA. densitometry measurements were performed on high-resolution (12 megapixels) digital images of the agarose gels, using Fiji or ImageJ2, a Java-based image processing program.Endotoxin quantification.

[0092] The Pierce Chromogenic Endotoxin Quant Kit (ThermoFisher Scientific) was used per manufacturer instructions. Standards and samples were transferred by pipette to a 96-well flat bottom plate (Coming Costar) with heating at 37°C using a Model HB120-S heating block incubator (ONiLAB Scientific). The plate was loaded into the NanoQuant Infinite M200 multidetection plate reader. A coefficient of determination, r2, >0.99 was obtained for all endotoxin standard curves.Magnetic response.

[0093] This method measures percent transmittance over time for the movement of suspended magnetic particles towards a permanent magnet pair at the bottom of a spectrophotometer cuvette holder. A Shimadzu UV-1800 spectrophotometer was turned on and allowed to warmup for one hour. Over 4 ml of a 1 wt.% final solution of magnetic beads was prepared in ASTM Type II water. The wavelength w as set to 590 nm and the instrument was zeroed using a cuvette vial filled with Type II water. The blank cuvette sample vial was then removed. A pair of 3 / 8 x 3 / 8 x 1 / 8” and 3 / 8 x 3 / 8 x 3 / 16” SmCo magnets (CMS Magnetics) was placed at the bottom of the sample cell holder. Samples were vigorously shaken until fully suspended before aliquoting a sample. 2 ml of the first magnetic sample was aliquoted into a clean cuvette vial and a 600- second transmittance scan was started on the spectrophotometer. Once the scan w as completed, the same sample procedure was repeated for remaining samples. Each sample was tested at least twice for reproducibility. The first sample tested has a known performance to verify the testing method.Iron leaching quantification.

[0094] The magnetic beads were filtered and washed with Type II water on a Whatman #41 filter paper placed in a Buchner funnel. The resulting filter cake was then placed in a vacuum oven (50°C, -25 mm Hg) for > 12 hours. The dried cake was removed from the oven and 0.33 g of material was weighed and placed in a bottle containing 20 ml 1.0 N HC1. The solution was mixed well and allowed to age for 15 minutes. After aging, the solution was filtered through a 0.45 pm filter unit (Nalgene). 3.0 g of filtrate was w eighed into a 15-ml Falcon tube and 10 ml40-ppm cobalt standard solution was added before testing on a Spectro Arcos 2 ICP-OES, with verification using a 4-ppm iron check standard.Surface area measurement.

[0095] The magnetic beads were filtered and washed with Type II water on a Whatman #41 filter paper. The resulting filter cake was placed in a vacuum oven (50°C, -25 mm Hg) for > 12 hours. The dried cake was then measured for surface area using a Micromeritics Gemini or TriStar II PLUS nitrogen porosimeter. The 5-point Brunauer-Emmett-Teller (BET) method was applied.Determination of SiCh wt. % / FezOs wt. %.

[0096] The magnetic beads were filtered and washed with Type II water on a Whatman #41 filter paper. The resulting filter cake was then placed in a vacuum oven (50°C, -25 mm Hg) for > 12 hours. Each 0.5 g dried sample was digested in a PTFE tube using reagent grade perchloric, hydrochloric, nitric, hydrofluoric acid (0.55:0.07:0.11:0.27), with addition of 1 ml 10,000 ppm cobalt standard (Sigma) to 49 ml of the reaction mixture. Samples were heated for 2 hours at 350°C in a Labconco Model 23012-25 block digestion system under a perchloric acid hood. Digested samples were analyzed using a Spectro Arcos 2 ICP-OES, with verification using a 500-ppm iron check standard. SiCh is determined by difference based on the summation of 15 elements, in weight percent oxide form, subtracted from 100 percent.Particle size measurement.

[0097] The magnetic slurry was shaken until fully dispersed and then added dropwise into a Malvern Panalytical Mastersizer 3000 or HORIBA LA-960 particle size analyzer until the obscuration was between 5-10 %. Once a stable obscuration was achieved, the particle size analysis was performed at 2500 rpm without sonication on. The analysis was then repeated at least twice after sonicating for 60 seconds. The average of the D50 measurements was used. A Duke 1.0 pm particle size standard was used to check the accuracy of the instrument before samples were run.Performance Analysis

[0098] The desired range for several magnetic bead performance attributes was determined as shown below in Table 1.Example 2: 150 mL Magnetic Bead Binding and Purification Assay with 3-L Cell GrowthFermentation, lysis, and microfiltration.

[0099] E. coli grow th and harvesting, resuspension, alkaline lysis, neutralization, and filtration / concentration w ere performed as follows. 3000-ml cell growth was obtained using LB broth medium with several passages until the optical density’ at 600 nm reached ~1.4. This yielded 300 ml lysate for two duplicate 150 ml volumes in pDNA binding experiments with Trial 6 superparamagnetic particles. Cells were centrifuged (Dupont Sorvall Instruments RC5Cfloor unit, 5000 ref, 15 min., 4°C) up to two times in the same pre-weighed 250-ml Nalgene PPCO centrifuge bottles (7 total used) to accumulate larger pellets before freezing the pellets at -80°C. On the day of the lysis, cell pellets from the 7 bottles were thawed and combined into one 250-ml bottle using resuspension buffer (previously autoclaved and stored at 4°C); followed by centrifugation to a single pellet of 17.6 g wcm. The supernatant was aspirated down to 88 ml before resuspension, by vortex mixing, for transferring to a new 500- ml Nalgene bottle.

[0100] A 0.4-ml aliquot (0.45%) of the 88-ml cell resuspension was saved for processing with the Reference 1 kit (non-magnetic, spin column kit). The small cell aliquot was centrifuged (Eppendorf 5415 D, 16000 ref, 5 min.. 18°C) to pellet cells and then exchanged to the Reference 1 resuspension buffer containing RNase A, with follow through using the kit’s lysisand neutralization buffers. The Reference 1 protocol states that the spin tube is for processing of 1 to 5 ml cell growth. (0.4 ml / 88 ml) (3000 ml cell growth) is equivalent to 13.6 ml growth. To obtain comparative yield data based on triplicate Reference 1 spin column samples, the resuspension was processed with Reference 1 volumes equivalent to 4.5 ml growth (13.6 ml / 3) per tube. The wash was accomplished with 70% ethanol prepared fresh; and elution was with Elution buffer as prepared with composition stated above and stored at 4°C.

[0101] The ~88-ml cell resuspension was further processed with addition of 176-ml L2 buffer (stored at 18°C). The bottle was rolled on a JOANLAB RM-6Pro tube roller mixer (30 rpm. 5 min., 18°C). 88 ml of L3 buffer (stored at 18°C with CaCb included immediately before use) was added to the bottle before rolling on the tube roller mixer (30 rpm) for 10 min. out of a 30- min. neutralization period at 18°C. LI, L2, and L3 buffer volumes were calculated as follows: LI, Resuspension buffer ml = grams wcm x 5 = 17.6 g x 5 = 88 ml; L2, Lysis buffer ml = grams wcm x 10 = 17.6 g x 10 = 176 ml; L3, Neutralization buffer ml = grams wcm x 5 = 17.6 g x 5 = 88 ml.

[0102] Application of the roller mixing during both lysis and neutralization steps produced a coconut-like consistency of the floc precipitate, allowing for easy centrifugation and microfiltration. -340 ml total lysate material was centrifuged (Dupont Sorvall Instruments RC5Cfloor unit, 6000 ref, 30 min., 4°C) in 2x 250-ml PPCO bottles to pellet the floc precipitate. Supernatant containing pDNA was filtered through a 250-ml Nalgene Rapid-Flow 0.2-pm filter; and then repeated with a new 0.2-pm filter to obtain -310 ml pre-TFF cleared lysate.Sample concentration using TFF.

[0103] After clarification, a TFF step may be performed to allow concentration and diafiltration to exchange the medium into a buffer suitable for binding to magnetic silica, and the removal of RNA, small sized genomic DNA, and small proteins. Sample concentration and diafiltration (desalting) can take place sequentially using the same TFF capsule. The Pall Minimate® PES 100 kDa TFF capsule was initially flushed with 450 ml of Milli-Q water, using a Kamoer KK300 peristaltic pump set at 30 ml / min., ensuring the passage of 150 ml and 300 ml through the retentate and permeate tubing, respectively. The capsule was then flushed with 150 mlConditioning buffer with chelator (120 mM potassium acetate pH 5.5, 25 mM EDTA) at 30 ml / min. until 30 ml was collected in the permeate container. The feed container was filled with the -310 ml cleared (centrifuged and 0.2 pm filtered) lysate sample to be concentrated. A 1000-ml crystallization dish filled with ice water at between 5 - 10°C was used to maintain a low temperature of the feed container. The peristaltic pump feed flow rate was set to 30 ml / min., with pressure monitoring using a PressureMAT digital monitor and three single-use pressure sensors (PendoTECH). Permeate (filtrate) flow was increased by tightening the retentate clamp slightly, with inlet pressure, upstream of the TFF capsule, maintained between 11 to 16 psi. Transmembrane pressure (TMP) was calculated to range from 12.5 to 14.2 psi. The solution was filtered to a low volume of 25 ml, while continuous mixing (100 rpm) with stir bar on an ONiLAB Scientific magnetic stirrer was maintained during the refill process with retentate fluid. This resulted in a volumetric concentration factor, VCF, of 12. Mixing was gentle to avoid creating nicked DNA or DNA with broken strands, affecting the overall integrity and quality of the purified pDNA. 200 ml of Conditioning buffer was added to the 25- ml retentate; and filtered to 25 ml at 40 ml / min. Another 150 ml of Conditioning buffer was added to the 25-ml retentate; with filtering to 25 ml at 40 ml / min. To maximize product recovery, the feed tubing was removed from the feed container and placed in a 50-ml Falcon tube filled with Conditioning buffer. With the retentate clamp loosened, -15 ml of liquid was pumped to the feed container. The feed tubing was removed from the feed container, and all upstream liquid was pumped into the feed container. The -50-ml retentate volume in the feed container was brought up to 300 ml with 3.6 M guanidine HC1, 120 mM potassium acetate pH 5.5. 25 mM EDTA. The final guanidine HC1 concentration was 3 M. After 14 diafiltration volumes. DF, the sample appeared as clear as water. The retentate was then split to 150 ml volume in duplicate 250-ml polypropylene bottles for overnight storage at 4°C before pDNA binding to Trial 6 particles.

[0104] Following sample concentration on the TFF capsule, the feed container was filled with 500 ml of 0.5 N NaOH for cleaning. With the retentate clamp loosened, the pump flow rate was maintained at 30 ml / min. until 250 ml of cleaning solution was collected in a waste container. The retentate tubing was placed in the feed container and slightly tightened with clamp to increase permeate flow rate. Recirculation with cleaning solution occurred for 60 minutes. TheTFF capsule was flushed with 450 ml of Milli-Q water. Finally. 100 ml of 0. 1 N NaOH was pumped through the capsule and tubing before capping the ports for long-term storage.Magnetic bead binding to pDNA.

[0105] To obtain 2% final solids content for Trial 6 testing, 30.3 ml of a fully resuspended Trial 6 particle batch with 11.9% solids, was pipetted into each 250-ml polypropylene bottle containing the TFF-processed pDNA in a 150-ml solution of 3 M guanidine HC1, 120 mM potassium acetate pH 5.5. 25 mM EDTA. Below is the calculation of the slurry volume from the Trial 6 bottle to use for 2% dosing:(x)(l 1.90% Trial 6 solids) = (x + 150 ml after TFF)(2% Trial 6 solids) 11.90x = 2x + 300 ml 9.9x = 300 ml x = 30.3 mlTotal Volume after Solids Addition = 180.3 ml

[0106] The bottles were placed on the JOANLAB RM-6Pro tube roller mixer for 20 min. at 20 rpm under controlled laboratory temperature (18°C) to bind pDNA. Based on a Redington 9200-HT tachometer, an actual rpm of 10 rpm was recorded for a 250-ml polypropylene bottle with 61 mm outer diameter. One bottle was then placed in the Sepmag A200ml magnetic separation unit for a three-minute separation time. Aspiration of the clear supernatant to a waste bottle was possible using a KK300 peristaltic pump with rigid PTFE tubing connected to the silicone pump tubing. This magnetic separation was performed for the duplicate bottle as well. The pDNA-bound magnetic particles were washed with 90 ml of 30:70 v / v 10 mM Tris-HCl pH 8.0, 1 mM EDTA: ethanol (hereafter referred to Wash buffer, pre-chilled 4°C) with mixing (20 rpm, 1 min.) on the JOANLAB RM-6Pro tube roller mixer, before placing the 250-ml bottle back in the A200ml unit. The supernatant was discarded using the peristaltic pump setup. The washing was repeated an additional two times, each with 90-ml Wash buffer. The 3X washed particles were dried at room temperature for 5 min. Each bottle was rotated horizontally to disperse the residual slurry for uniform drying. 66 ml of Elution buffer (pre-chilled 4°C) was added to each 250-ml bottle, and the particles were resuspended via mixing on the tube roller mixer (20 rpm, 10 min., 18°C). Each bottle was placed in the A200ml unit for 3 min., before using a sterile serological pipet to transfer supernatant as aliquots to 2x 50-ml Falcon tubes (~30 ml / tube).Samples were filtered through 0.2 pm syringe filters (Cobetter 33 mm hydrophilic PVDF) into new Falcon tubes to remove any residual magnetic particles before freezing aliquots at -80°C.Results.

[0107] Incorporating 25 mM EDTA in UF / DF - TFF buffer stabilized pDNA (pcDNA3-EGFP) from the 3-L grow th. Primarily supercoiled (SC) isoform was observed on the agarose gel, with densitometry indicating between 82-88% SC and an average of 85% SC (FIGs. 1A-1B and 3). The observed nucleic acid yield, A260 / A280, A260 / A230. and endotoxin level were 404 pg / g wcm, 1.97, 1.81, and 21.6 EU / mg DNA, respectively (See FIGs. 1A-1B and 3). A strong absorbance around 230 nm can indicate the presence of organic compounds or salts in the purified DNA. In general, it is desirable to have an A26o / A23o> 1.5; this was achieved with the 3X washes. Meeting the criteria listed in Table 1. the quick magnetic response (55% transmittance at 5 seconds) and low iron leaching (2.4 ppm) also likely contributed to the overall purity. The total yield for the 3-L growth was 7. 1 mg. Accordingly, these results demonstrate that methods of pDNA purification using the Trial 6 beads are effective for producing high yields of therapeutic-grade pDNA from large volumes of bacteria.Example 3: 1 mL studies

[0108] A total of 12 different magnetic beads were studied with dosing levels of 0.5. 1, 2, and 3 wt.% as shown in FIG. 3. All dosing is dependent on the initial solids content of the magnetic bead formulation as produced by W suppliers. For example, to obtain 2% final solids content, 197 pl of a fully resuspended Trial 1 batch with 12. 15% solids was pipetted into each of duplicate 1.5-ml microcentrifuge tubes containing UF / DF processed pDNA in 1 ml of Binding buffer. Below is the calculation of the slurry volume from the Trial 1 stock bottle to use for 2% dosing:(y)(12.15% Trial 1 solids) = (y + 1000 pl after UF / DF)(2% Trial 1 solids) 12.15y = 2y + 2000 pl 10.15y = 2000 pl y = 197 plTotal Volume after Solids Addition = 1197 pl

[0109] To ensure consistent volume additions across the same dosing level, supplier beads were 2X washed with Milli-Q water and concentrated or diluted to 12% solids. The bead washing and removal of rinse fluid were achieved by use of a 16-position magnetic rack, with removable magnets (Sergi Lab Supplies), with a 5-minute magnetic separation. Three studies were performed to test different dosing levels in duplicate for multiple magnetic bead types (Trial 1-5 and Comparative Trial 1-7) using a fixed set of magnetic racks and processing times.Reference (non-magnetic, spin column kit) runs were performed to ensure equivalent plasmid content, with quantitation using the NanoQuant Infinite M200; and Trial 2 beads were run in each study as a reference.Study 1

[0110] Fermentation, lysis, and filtration: A 500-ml cell growth was obtained using LB broth medium with passages until the optical density at 600 nm reached ~1.5. Cells were centrifuged (Dupont Sorvall Instruments RC5C floor unit, 5000 ref, 15 min., 4°C) in pre-weighed 250-ml Nalgene PPCO centrifuge bottles and the supernatant discarded, before freezing the pellets at - 80°C. A pellet mass of -1.8 grams would yield enough lysate for duplicate 1 ml volumes in pDNA binding experiments with Trial 1, 2, 3, and 4 beads at three dosing levels. On the day of the lysis, the pellet of 1.804 g wem, from a 150 ml centrifugation volume, was thawed and resuspended in 9.02 ml LI buffer (1.804 g wem x 5). LI buffer was previously autoclaved and stored at 4°C. The 250-ml bottle containing fully resuspended cells was placed in an ice bucket for 15 min.[OHl] A 0.5-ml aliquot (5.5%) of the 9.02-ml cell resuspension was saved for processing with the Reference 2 (non-magnetic, spin column) kit. The small cell aliquot was centrifuged (Eppendorf 5415 D, 16000 ref, 5 min.. 18°C) to pellet cells and then exchanged to the Reference 2 resuspension buffer containing RNase A, with follow through using the kit’s lysis and neutralization buffers. The Reference 2 kit protocol states that each spin tube is for the processing of 1 to 5 ml cell grow th. (0.5 ml / 9.02 ml) (150 ml cell growth) is equivalent to -8 ml growth. To obtain comparative yield data, the resuspension was processed with Reference 2 volumes equivalent to -3 ml growth (-8 ml / 3) by splitting to triplicate Reference 2 spin columns. The wash was accomplished with 70% ethanol prepared fresh; and elution was with Elution buffer as prepared with composition stated above and stored at 4°C.|0112] The remaining 8.52-ml cell resuspension was processed with addition of 17.04-ml L2 buffer (stored at 18°C). The bottle was rolled on a JOANLAB RM-6Pro tube roller mixer (30 rpm, 5 min., 18°C). 8.52-ml L3 buffer (stored at 18°C with CaChincluded immediately before use) was added to the bottle before rolling on the tube roller mixer (30 rpm) for 10 min. out of a 30- min. neutralization period at 18°C. The buffer volumes used were 0.9446 times less (8.52 ml / 9.02 ml) due to the small aliquot taken for the Reference 1 processing. LI, L2, and L3 buffer volumes were calculated as follows: LI, Resuspension buffer ml = 1.804 g wcm x 5 x 0.9446 = 8.52 ml; L2, Lysis buffer ml = 1.804 g wcm x 10 x 0.9446 = 17.04 ml; L3, Neutralization buffer ml = 1.804 g wcm x 5 x 0.9446 = 8.52 ml. The floc precipitate was centrifuged (Dupont Sorvall Instruments RC5C floor unit, 6000 ref, 15 min., 4°C) to obtain ~34 ml supernatant. The supernatant was filtered through a 0.2 pm 33 mm hydrophilic PVDF syringe filter (Cobetter); and then repeated through a new 0.2 pm hydrophilic PVDF filter to obtain -31 ml. Instead of a TFF capsule used in the larger scale pDNA preparation, ultrafiltration (UF) spin tubes were used for these studies. Two UF centrifuge tube units (Cobetter Briscale UF-15; 100 kDa regenerated cellulose; 50-ml centrifuge tube with 15 ml capacity) were rinsed with Milli-Q water to remove glycerol prior to use. The microfiltered pDNA liquid was then evenly split as 15.5 ml in each UF unit for processing using a centrifuge (ONiLAB Scientific. 1800 ref. 175 min., 18°C). The low volume retentate of ~0.5 ml (volumetric concentration factor of 31) in one UF centrifuge tube unit was brought up to 3 ml with 120 mM potassium acetate pH 5.5, 25 mM EDTA and centrifuged (ONiLAB, 1800 ref, 75 min., 18°C) to 0.3 ml for the buffer exchange (8 diafiltration volumes). This retentate was transferred to a single 50-ml Falcon tube using Binding buffer (3 M guanidine HC1, 120 mM potassium acetate pH 5.5, 25 mM EDTA) in repeat rinses. This was accomplished by having 12 ml total Binding buffer added to each UF unit, before combining to a final 24 ml in the same 50-ml Falcon tube. 1-ml aliquots were split to a total of 24 microcentrifuge tubes (1.5 ml capacity).

[0113] Magnetic bead binding to pDNA: All dosing is dependent on the initial solids content of the magnetic particle formulation as produced by suppliers. pDNA binding with magnetic silica particles was ensured through mixing (40 rpm, 30 min., 18°C) on the revolver adjustable rotator (ThermoFisher). Afterwards, each 1.5-ml microcentrifuge tube was placed in 2X 16- position magnetic racks (Sergi Lab Supplies) for a 5-minute magnetic separation. The clearsupernatant was aspirated by pipetting and then discarded. The pDNA-bound magnetic particles in each tube were washed with 600 pl Wash buffer with mixing (40 rpm, 1 min.) on the revolver adjustable rotator, before placing in the magnetic rack. Supernatant was aspirated by pipetting and then discarded. The washing was repeated two more times, for a total Wash buffer volume of 1.8 ml. The washed particles were dried at room temperature for 10 min. Each tube was rotated horizontally by hand to disperse the residual slurry for uniform drying. 450-pl Elution buffer (pre-chilled to 4°C) was added to each tube, and the particles were resuspended via mixing (40 rpm, 10 min., 18°C) on the revolver adjustable rotator. Each tube was placed in a Sergi Lab magnetic rack for 5 minutes, before using a pipet to transfer supernatant to microcentrifuge tubes for storage at -80°C.Study 2

[0114] Fermentation, lysis, and filtration: A 500-ml cell growth was obtained using LB broth medium with passages until the optical density at 600 nm reached ~1.2. Cells were centrifuged (Dupont Sorvall Instruments RC5C floor unit, 5000 ref, 15 min., 4°C) in two pre-weighed 250- ml Nalgene PPCO centrifuge bottles and the supernatant discarded, before freezing the pellets at -80°C. A combined pellet mass of -4.3 grams would yield enough lysate for duplicate 1 ml volumes in pDNA binding experiments with up to seven different magnetic particles at four dosing levels. On the day of the lysis, 4.337 g w'em w as thaw ed and resuspended in 21.69 ml LI buffer (4.337 g wem x 5). LI buffer was previously autoclaved and stored at 4°C. The 250- ml bottle containing fully resuspended cells was placed in an ice bucket for 15 min.

[0115] A 0.5-ml aliquot (2.3%) of the 21.69-ml cell resuspension was saved for processing with the Reference 3 (non-magnetic, spin column) kit. The small cell aliquot was centrifuged (Eppendorf 5415 D, 16000 ref, 5 min.. 18°C) in a microfuge to pellet cells and then exchanged to the Reference 3 resuspension buffer containing RNase A, with follow through using the kit’s lysis and neutralization buffers. The Reference 3 protocol states that the spin tube is for processing of 1 to 5 ml cell growth. (0.5 ml / 21.69 ml) (500 ml cell growth) is equivalent to -11 ml growth. To obtain comparative yield data, the resuspension was processed with Reference 3 volumes equivalent to -3 to 4 ml growth (-11 ml / 3) by splitting to triplicate Reference 3 spin columns.|0116] The remaining 21.19-ml cell resuspension was processed with addition of 42.37-ml L2 buffer (stored at 18°C). The bottle was rolled on a JOANLAB RM-6Pro tube roller mixer (30 rpm, 5 min., 18°C). 21.19-ml L3 buffer (stored at 18°C with CaCh included immediately before use) was added to the bottle before rolling on the tube roller mixer (30 rpm) for 10 min. out of a 30-min. neutralization period at 18°C. The buffer volumes used were 0.977 times less (21.19 ml / 21.69 ml) due to the small aliquot taken for the Reference 1 processing. LI, L2, and L3 buffer volumes were calculated as follows: LI, Resuspension buffer ml = 4.337 g wcm x 5 x 0.977 = 21.19 ml; L2, Lysis buffer ml = 4.337 g wcm x 10 x 0.977 = 42.37 ml; L3, Neutralization buffer ml = 4.337 g wcm x 5 x 0.977 = 21.19 ml. The floc precipitate was centrifuged (Dupont Sorvall Instruments RC5C floor unit, 6000 ref, 15 min., 4°C) to obtain ~78 ml supernatant. The supernatant was filtered through a 0.2 pm 33 mm hydrophilic PVDF syringe filter (Cobetter); and then repeated through a new 0.2 pm hydrophilic PVDF filter to obtain ~72 ml. Two ultrafiltration (UF) centrifuge units (Pierce Concentrator; 100 kDa PES; 250-ml bottles with 90 ml capacity) were each rinsed with 20 ml of Milli-Q water to remove glycerol prior to use. The microfiltered pDNA liquid was then evenly split as 37 ml in each UF unit for processing using a centrifuge (Dupont Sorvall Instruments RC5C, 1200 ref, 90 min., 4°C). The low volume retentate of ~4.5 ml (volumetric concentration factor of 8) in one UF centrifuge tube unit was brought up to 30 ml with 120 mM potassium acetate pH 5.5, 25 mM EDTA and centrifuged (Dupont Sorvall Instruments RC5C. 1200 ref. 60 min., 4°C) to ~2.5 ml for the buffer exchange (11 diafiltration volumes). This retentate was transferred to a separate 50-ml Falcon tubes using Binding buffer (3 M guanidine HC1, 120 mM potassium acetate pH 5.5. 25 mM EDTA) in repeat rinses. This was accomplished by having 31 ml total Binding buffer added to each UF unit, before mixing to final 62 ml using two 50-ml Falcon tubes. 1-ml aliquots were split to a total of 62 microcentrifuge tubes (1.5 ml capacity).

[0117] Magnetic bead binding to pDNA: All dosing / binding / washing / elution steps were performed as described in Study 1.Study 3

[0118] Fermentation, lysis, and filtration: A 500-ml cell growth was obtained using LB broth medium. Cells were centrifuged (Dupont Sorvall Instruments RC5C floor unit, 5000 ref, 15 min., 4°C) in pre-weighed 250-ml Nalgene PPCO centrifuge bottles and the supernatantdiscarded, before freezing the pellets at -80°C. A pellet mass of ~2. 1 grams would yield enough lysate for duplicate 1 ml volumes in pDNA binding experiments with four magnetic particles at four dosing levels. On the day of the lysis, the pellet of 2.145 g wcm was thawed and resuspended in 10.725 ml LI buffer (2.145 g wcm x 5). LI buffer was previously- autoclaved and stored at 4°C. The 250-ml bottle containing fully resuspended cells was placed in an ice bucket for 15 min.

[0119] A 0.6-ml aliquot (5.6%) of the 10.725-ml cell resuspension was saved for processing with the Reference 4 (non-magnetic, spin column) kit. The small cell aliquot was centrifuged (Eppendorf 5415 D, 16000 ref, 5 min., 18°C) in a microfuge to pellet cells and then exchanged to the Reference 4 resuspension buffer containing RNase A, with follow through using the kit’s lysis and neutralization buffers. The Reference 4 protocol states that the spin tube is for processing of 1 to 5 ml cell growth. (0.6 ml / 10.725 ml)(200 ml cell growth) is equivalent to ~11 ml growth. To obtain comparative yield data, the resuspension was processed with Reference 4 volumes equivalent to ~3 - 4 ml growth (~11 ml / 3) by splitting to triplicate Reference 4 spin columns.[01201 The remaining 10. 125-ml cell resuspension was processed with addition of 20.25-ml L2 buffer (stored at 18°C). The bottle was rolled on a JOANLAB RM-6Pro tube roller mixer (30 rpm, 5 min., 18°C). 10.125-ml L3 buffer (stored at 18°C with CaCbincluded immediately before use) w as added to the bottle before rolling on the tube roller mixer (30 rpm) for 10 min. out of a 30-min. neutralization period at 18°C. The buffer volumes used were 0.9441 times less (10.125 ml / 10.725 ml) due to the small aliquot taken for the Reference 1 processing. L1, L2, and L3 buffer volumes were calculated as follows: LI, Resuspension buffer ml = 2.145 g wcm x 5 x 0.9441 = 10.125 ml; L2, Lysis buffer ml = 2.145 g wcm x 10 x 0.9441 = 20.25 ml; L3, Neutralization buffer ml = 2. 145 g wcm x 5 x 0.9441 = 10. 125 ml. The floc precipitate was centrifuged (Eppendorf 5415 D, 6000 ref, 15 min., 4°C) to obtain ~36 ml supernatant. The supernatant was fdtered through a 0.2 pm 33 mm hydrophilic PVDF syringe filter (Cobetter); and then repeated through a new 0.2 pm hydrophilic PVDF filter to obtain ~31 ml. Two ultrafiltration (UF) centrifuge tube units (Cobetter Briscale UF-15; 100 kDa regenerated cellulose: 50-ml centrifuge tube with 15 ml capacity) were rinsed with Milli-Q water to remove glycerol prior to use. The microfiltered pDNA liquid was then evenly split as 15 + 3 ml in eachUF unit for processing using a ONiLAB DM4102 centrifuge (1800 ref. 175 min., 18°C). The low volume retentate of -0.75 ml (volumetric concentration factor of 24) in one UF centrifuge tube unit was brought up to 3 ml with 120 rnM potassium acetate pH 5.5, 25 rnM EDTA and centrifuged (ONiLAB, 1800 ref, 95 min., 18°C) to 0.25 ml for the buffer exchange (9 diafiltration volumes). This retentate was transferred to a single 50-ml Falcon tube using Binding buffer (3 M guanidine HC1, 120 mM potassium acetate pH 5.5, 25 mM EDTA) in repeat rinses. This was accomplished by having 14 ml total Binding buffer added to each UF unit, before combining to a final 28 ml in the same 50-ml Falcon tube. 1-ml aliquots were split to a total of 28 microcentrifuge tubes (1.5 ml capacity).

[0121] Magnetic bead binding to pDNA: All dosing / binding / washing / elution steps were performed as described in Study 1.Results for Studies 1-3

[0122] Comparative Trial 1, 2, and 3 beads were produced to assess the potential effect of bead composition on pDNA purification. Comparative Trial 1. 2, and 3 beads, with respective % SiO2 content of -5. 10. and 20, generally exhibited A260 / A280 > 2.0 at two to four dosing levels (FIG. 3). Exceeding the desired absorbance ratio upper limit suggests that impurities contributed to an artefact high nucleic acid yield, > 500 pg / g wcm. As a general comparison, the average nucleic acid yield and average A260 / A280 purity for Reference 2, 3, and 4 (nonmagnetic, spin column kit) was 290 ± 31 pg / g wcm and 1.98, respectively. Iron leaching under the binding / washing / elution conditions for these Comparative Trial 1, 2, and 3 beads may be a significant contributor to an elevated 260 nm measurement. The standard 1 N HC1 iron leaching test is only a general indicator of magnetic bead susceptibility to iron, from the core, diffusing into solution under highly acidic conditions.

[0123] Surprisingly, an % SiCh content < 20% for Comparative Trial 1 and 2 beads contributed to low % SC values. SiCh content was not previously known to affect the form of plasmid DNA during purification, however the present disclosure establishes a clear relationship between increasing particle SiCh content and increased supercoiling, which is highly desirable for therapeutic applications of plasmids.

[0124] Comparative Trial 6 beads, with 10% SiCh, and Comparative Trial 5 beads also contributed to artefact high nucleic acid yields at most or all dosing levels. For Comparative Trial 5 beads, % SC was 80% for the 0.5% dosing level, but % SC decreased to as low as 30% at 3% dosing (FIG. 3). At most dosing levels for the Comparative Trial 6 beads, the supercoiled isoform was non-detectable on the agarose gel (FIG. 2), rendering such purifications of little use for therapeutic applications.

[0125] pDNA purified with Trial 3 beads at 2% and 3% dosing exhibited A260 / A230 < 1.5 (FIG. 3), indicating insufficient removal of salts during the washing steps. The low yield of 210 pg / g wcm at 3% dosing was a consequence of the low bulk density, leading to a smaller elution volume.

[0126] Overall, the Trial 1, 2, 4, and 5 beads provided therapeutic-grade pDNA in terms of yield, purities, and % SC at three different dosing levels (FIG. 3). Importantly, methods of plasmid purification using these beads resulted in low levels of RNA contamination despite not incorporating an RNase enzymatic step. RNase contamination, ty pically bovine RNase, presents a substantial hurdle to therapeutic applications of plasmid DNA purified using most conventional methods. The discovery that the presently disclosed methods can achieve superior purity' and yield without using such an enzyme represents a significant improvement to not only efficiency and cost but also to therapeutic applicability. Trial 2 and 5 beads were the best performing at dosing levels of 1, 2, and 3%, meeting all criteria listed in Table 1, including iron leaching (< 10 ppm) and magnetic response (> 20% transmittance at 5 seconds).

[0127] Comparative Trial 4 silica beads appeared orange, which is indicative of oxidation, with high iron leaching observed using 1 N HC1 in the separate testing protocol (FIG. 3). The color did not affect the apparent nucleic acid purity and % SC, although yields were lower compared to a low iron leaching material such as the Trial 2 beads.

[0128] pDNA with high % SC values could be obtained using Comparative Trial 7 beads (FIG. 3). However, iron leaching values were 6.2 times higher compared to Trial 2 beads. In addition, A260 / A280 values were at the upper desired limit of 2.0 for three of the four dosing levels with the Comparative Trial 7 beads.Example 4: Reduced Volume. Magnetic Bead Binding and Purification Assay with 500 to 1000-ml Cell GrowthMaterials and MethodsPlasmid

[0129] Cell banks of a 6.1 kbp plasmid, pcDNA3-EGFP (Addgene plasmid number 13031), and a 7.5 kbp plasmid, pAAV2 / 2 (Addgene plasmid number 104963), were prepared using E. coll strain DH5a and stored at -80°C. The plasmids were procured by the Keck Graduate Research Institute (Claremont. CA, USA) where the experiments were performed.Alkaline lysis pDNA purification

[0130] Briefly, plasmid-containing bacterial cells were cultivated in LB broth containing ampicillin. Bacterial cells were pelleted in pre-weighed 50-ml Falcon tubes or 250-ml Nalgene centrifuge bottles, resuspended in ice-cold LI buffer (50 mM glucose, 25 mM Tris-HCl, 10 mM EDTA, pH 8.0) with incubation on ice for 15 min.; lysed in L2 buffer (0.2 N NaOH, 1% SDS, 18°C) and mixed gently by inversion four to five times before incubation on ice for up to 5 min.; then neutralized with L3 buffer (3 M potassium acetate, 2 M acetic acid, 1 M CaCh, pH 5.5) with gentle mixing by inversion four to five times. The neutralized lysate container was left undisturbed for 10 to 30 min. until three layers formed. The buffer volumes were relative to wet cell mass (wcm) as indicated: LI. Resuspension buffer ml = grams wcm x 5; L2, Lysis buffer ml = grams wcm x 10; L3, Neutralization buffer ml = grams wcm x 5. The final concentration of potassium acetate after neutralization was 0.75 M. Calcium chloride was included since it has been show n to be a precipitant with high RNA removal (A. Eon-Duval et al., 2003). Calcium chloride only was added to the L3 buffer on the day of use to avoid solubility issues.

[0131] Neutralized lysate was centrifuged at the top speed of the centrifuge units to pellet the floc precipitate because using magnetic beads immediately after neutralization may result in floc precipitate interfering with pDNA binding. The supernatant containing the pDNA was transferred to new tubes for the further processing via filtration (e.g., 0.2 pm filters, depthfilters, ultrafiltration centrifuge tubes, and / or tangential flow filtration capsules) prior to bead addition. Ultrafiltration / diafiltration using ultrafiltration (UF) centrifuge tube units or tangential flow filtration (TFF) capsules is important to exchange the medium into a buffer suitable for binding to magnetic silica, and for the removal of RNA, small sized genomic DNA, and small proteins.DNA isolation using commercial DNA purification kits

[0132] A commercially available Plasmid Mini Kit. hereafter referred to as Reference (e.g., Reference 5, 6, 7, 8), was used in each pDNA isolation experiment as a reference for yield comparison. Reference spin columns contain a unique silica membrane that binds up to 20 pg DNA in the presence of a high concentration of chaotropic salt and allows elution in a small volume of low salt buffer. The pDNA was isolated according to manufacturer's instructions. This kit contains RNase A for digestion of RNA. Reference isolated pDNA was dissolved in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA (hereafter referred to as Elution buffer) and kept at - 20°C.Nucleic acid purity and concentration measurements

[0133] A NanoQuant Infinite M200 multi-detection plate reader (Tecan) was used to determine nucleic acid quality (A260 / A280 and A260 / A230 ratios) and concentration (ng / pl). Before running any samples, the plate was wiped clean with Kimwipes saturated with Milli-Q water, followed by 70% ethanol, and finally with Milli-Q water again. 2pl of Elution buffer was used to blank each of the wells. On the Infinite M200, an additional measurement at a reference wavelength (310 nm) is performed automatically with each measurement.Gel electrophoresis of nucleic acids

[0134] pDNA samples and a 1-kb DNA ladder (New England Biolabs) were resolved on 1% agarose gels, with 12-lanes, and visualized with GelGreen (Biotium) fluorescent dye, based on inclusion in the cast gel or post-staining. Up to ~100ng and 200 ng nucleic acid was loaded per well on the 22- and 12-lane agarose gel, respectively. An IVYX Scientific electrophoresis system was set at 50 V for 60 min. for each run.Plasmid supercoiled quantification

[0135] To determine the percent of supercoiled purified pDNA, densitometry measurements were performed on high-resolution (12 megapixels) digital images of the agarose gels, using Fiji or ImageJ2, a Java-based image processing program.

[0136] The other method for percent supercoiled quantification was anion-exchange (AEX) chromatography of pDNA eluted from the magnetic beads. A Waters Protein-Pak Hi Res Q, 5pm, 4.6 x 100 mm column and a Thermo UltiMate 3000 RS UHPLC system were used, with gradient, column temperature, and buffers specified by Waters Corp. (H. Yang et al., 2021).Performance Analysis

[0137] The desired range for several magnetic bead performance attributes w as determined as shown below in Table 2.

[0138] Example 5: Reduced Volume, 25 ml Magnetic Bead Binding and Purification Assay with 500 to 1000-ml Cell Growth (pcDNA3-EGFP Plasmid)Study 1Fermentation, lysis, and microfiltration

[0139] E. coli growth and harvesting, resuspension, alkaline lysis, neutralization, and filtration / concentration w ere performed as follows. A 500-ml aliquot from a full 3-L growth was obtained using LB broth medium with several passages until the optical density at 600 nm reached ~2.3 during the late stationary phase. This yielded sufficient lysate for a 25 ml pDNA binding experiment with Trial 7 superparamagnetic particles. Cells were centrifuged (Dupont Sorvall Instruments RC5Cfloor unit, 5000 ref, 15 min., 4°C) two times in the same pre-w eighed 250-ml Nalgene PPCO centrifuge bottle to accumulate a larger pellet of 2.958 g wem before freezing the pellet at -80°C. On the day of the lysis, the pellet was thawed and resuspended using 14.79 ml resuspension buffer (previously autoclaved and stored at 4°C).

[0140] A 0.2-ml aliquot (1.35%) of the 14.79-ml cell resuspension was saved for processing with the Reference 5 (non-magnetic, spin column) kit. The small cell aliquot was centrifuged (Eppendorf 5415 D, 16000 ref, 5 min., 18°C) to pellet cells and then exchanged to the Reference 5 resuspension buffer containing RNase A, with follow' through using the kit’s lysis and neutralization buffers. The Reference 5 protocol states that the spin tube is for processing of 1 to 5 ml cell growth. (0.2 ml / 14.79 ml) (500 ml cell growth) is equivalent to 6.8 ml growth. To obtain comparative yield data based on triplicate Reference 5 spin column samples, the resuspension was processed with Reference 5 volumes equivalent to 2.3 ml growth (6.8 ml / 3) per tube. The wash was accomplished w ith 70% ethanol prepared fresh; and elution was w ith 10 rnM Tris-HCl, pH 8.0, 1 mM EDTA (hereafter referred to as Elution buffer) and stored at 4°C.

[0141] The remaining 14.59-ml cell resuspension was further processed with addition of 29.18-ml L2 buffer (stored at 18°C). The bottle was rolled on a JOANLAB RM-6Pro tube roller mixer (30 rpm, 5 min., 18°C). 14.59 ml of L3 buffer (stored at 18°C with CaChincluded immediately before use) was added to the bottle before rolling on the tube roller mixer (30 rpm) for 20 min. out of a 30-min. neutralization period at 18°C. LI, L2, and L3 buffer volumes were calculated as follows: LI, Resuspension buffer ml = 2.958 g x 5 x 0.9865 = 14.59 ml; L2, Lysis buffer ml = 2.958 g x 10 x 0.9865 = 29.18 ml; L3. Neutralization buffer ml = 2.958 g x 5 x 0.9865 = 14.59 ml. The buffer volumes used were 0.9865 times less (14.59 / ml / 14.79 ml) due to the small aliquot taken for the Reference 1 processing.

[0142] Application of the roller mixing during both lysis and neutralization steps produced a coconut-like consistency of the floc precipitate, allowing for easy centrifugation and microfiltration. ~55 ml total lysate material was centrifuged (Dupont Sorvall Instruments RC5Cfloor unit, 6000 ref, 30 min., 4°C) in a 250-ml PPCO bottle to pellet the floc precipitate. Supernatant containing pDNA was filtered through a 250-ml Nalgene Rapid-Flow 0.2-pm filter; and then repeated with a new 0.2-pm filter to obtain ~50 ml pre-TFF cleared lysate.Sample concentration using TFF

[0143] After clarification, a TFF step may be performed to allow concentration and diafiltration to exchange the medium into a buffer suitable for binding to magnetic silica, and the removal of RNA, small sized genomic DNA, and small proteins. Sample concentration and diafiltration (desalting) can take place sequentially using the same TFF capsule. The Pall Minimate® PES 100 kDa TFF capsule was initially flushed with 450 ml of Milli-Q water, using a Kamoer KK300 peristaltic pump set at 30 ml / min., ensuring the passage of 150 ml and 300 ml through the retentate and permeate tubing, respectively. The capsule was then flushed with 150 ml Conditioning buffer with chelator (120 rnM potassium acetate pH 5.5, 25 mM EDTA) at 30 ml / min. until 30 ml was collected in the permeate container. The feed container, a 50-ml Falcon tube, was filled with the ~50 ml cleared (centrifuged and 0.2 pm filtered) lysate sample to be concentrated. A 1000-ml crystallization dish filled with ice water at between 5 - 10°C was used to maintain a low temperature of the feed container. The peristaltic pump feed flow rate was set to 30 ml / min., with pressure monitoring using a PressureMAT digital monitor and three single-use pressure sensors (PendoTECH). Permeate (filtrate) flow was increased by tightening the retentate clamp slightly, with inlet pressure, upstream of the TFF capsule, maintained between 11 to 13 psi. Transmembrane pressure (TMP) was calculated to range from 7 to 8 psi. The solution was filtered to a low volume of 7.5 ml, while continuous mixing (100 rpm) with stir bar on an ONiLAB Scientific magnetic stirrer was maintained during the refill process with retentate fluid. This resulted in a volumetric concentration factor, VCF, of 7. Mixing was gentle to avoid creating nicked DNA or DNA with broken strands, affecting the overall integrity' and quality of the purified pDNA. 42.5 ml of Conditioning buffer was added to the 7.5-ml retentate; and filtered to 5 ml at 30 ml / min. Another 45 ml of Conditioning buffer was added to the 5-ml retentate; with filtering to ~2 ml at 30 ml / min. To maximize productrecovery, the feed tubing was removed from the feed container; the retentate clamp was loosened, and all liquid was pumped to the feed container. The retentate tubing was disconnected from the TFF capsule and a sterile disposable syringe containing Conditioning buffer was connected to the retentate port to wash remaining liquid into the feed container. A ~12.5-ml TFF concentrate was saved in a 30-ml PPCO tube at 4°C until pDNA binding with Trial 7 particles (Trial 6 particles mentioned in Example 2 above). The ~12.5-ml retentate volume in the feed container was brought up to 25 ml with 8 M guanidine HCL 120 mM potassium acetate pH 5.5, 25 mM EDTA. The final guanidine HC1 concentration was 4 M after the proportioning of 1 : 1 cleared lysate: binding buffer.

[0144] Following sample concentration on the TFF capsule, the feed container was filled with 500 ml of 0.5 N NaOH for cleaning. With the retentate clamp loosened, the pump flow rate was maintained at 30 ml / min. until 250 ml of cleaning solution was collected in a waste container. The retentate tubing was placed in the feed container and slightly tightened with clamp to increase permeate flow rate. Recirculation with cleaning solution occurred for 60 minutes. The TFF capsule was flushed with 450 ml of Milli-Q water. Finally, 100 ml of 0.1 N NaOH was pumped through the capsule and tubing before capping the ports for long-term storage.Magnetic bead binding to pDNA

[0145] To obtain 2% final solids content for Trial 7 testing, 5.05 ml of a fully resuspended Trial 6 particle batch with 11.9% solids, was pipetted into the 30-ml PPCO tube containing the TFF- processed pDNA in a 25-ml solution of 4 M guanidine HCL 120 mM potassium acetate pH 5.5, 25 mM EDTA. Below is the calculation of the slurry volume from the Trial 6 bottle to use for 2% dosing:(x)(l 1.90% Trial 6 solids) = (x + 25 ml after TFF)(2% Trial 7 solids) 11.90x = 2x + 50 ml9.9x = 50 ml x = 5.05 ml Total Volume after Solids Addition = 30.05 ml

[0146] The 30-ml PPCO tube was placed on the JOANLAB RM-6Pro tube roller mixer for 20 min. at 20 rpm under controlled laboratory temperature (18°C) to bind pDNA. The tube was thenplaced in the Sepmag A200ml magnetic separation unit for a three-minute separation time. Aspiration of the clear supernatant to a waste bottle was possible using a KK300 peristaltic pump with rigid PTFE tubing connected to the silicone pump tubing. The pDNA-bound magnetic particles were washed with 15 ml of 30:70 v / v 10 mM Tris-HCl pH 8.0, 1 mM EDTA: ethanol (hereafter referred to Wash buffer, pre-chilled 4°C) with mixing (20 rpm, 1 min.) on the JOANLAB RM-6Pro tube roller mixer, before placing the 30-ml tube back in the A200ml unit. Wash buffer volume was proportioned to wet cell mass in the 3-L E. coli growth (pcDNA3-EGFP plasmid) study of Example 2 above (17.6 g / 3.0 g = 5.9; 90 ml wash / 5.9= 15 ml). The supernatant was discarded using the peristaltic pump setup. The washing was repeated an additional two times, each with 15-ml Wash buffer. The 3X washed particles were dried at room temperature for 5 min. The 30-ml tube was rotated horizontally to disperse the residual slurry for uniform drying. 11 ml of Elution buffer (pre-chilled 4°C) was added to the 30-ml tube, and the particles were resuspended via mixing on the tube roller mixer (20 rpm, 10 min., 18°C). Elution buffer volume was proportioned to wet cell mass in the 3-L E. coli growth (pcDNA3-EGFP plasmid) of Example 2 above (17.6 g / 3.0 g = 5.9; 66 ml wash / 5.9 = 1 1 ml). The 30-ml tube was placed in the A200ml unit for 3 min., before using a sterile serological pipet to transfer supernatant to a disposable syringe for filtering through a 0.2 pm syringe filter (Cobetter 33 mm hydrophilic PVDF) into a new 15-ml PPCO tube to remove any residual magnetic particles before freezing aliquots at -80°C.Study 2Fermentation, lysis, and microfiltration

[0147] E. coli growth and harvesting, resuspension, alkaline lysis, neutralization, and filtration / concentration were performed as follows. A 1000-ml aliquot from a full 3-L growth was obtained using LB broth medium with several passages until the optical density at 600 nm reached ~3.0 during the late stationary phase. This yielded sufficient lysate for a 25 ml pDNA binding experiment with Trial 8 superparamagnetic particles. Cells w ere centrifuged (Dupont Sorvall Instruments RC5Cfloor unit. 5000 ref, 15 min., 4°C) two times in the same pre-w eighed 250-ml Nalgene PPCO centrifuge bottle to accumulate a larger pellet of 5. 186 g wem before freezing the pellet at -80°C. On the day of the lysis, the pellet was thawed and resuspended using 14.79 ml resuspension buffer (previously autoclaved and stored at 4°C).

[0148] A 0.3-ml aliquot (1.16%) of the 25.93-ml cell resuspension was saved for processing with the Reference 6 (non-magnetic, spin column) kit. The small cell aliquot was centrifuged (Eppendorf 5415 D, 16000 ref. 5 min., 18°C) to pellet cells and then exchanged to the Reference 6 resuspension buffer containing RNase A, with follow through using the kit’s lysis and neutralization buffers. The Reference 6 protocol states that the spin tube is for processing of 1 to 5 ml cell growth. (0.3 ml / 25.93 ml) (1000 ml cell growth) is equivalent to 11.6 ml growth. To obtain comparative yield data based on triplicate Reference 6 spin column samples, the resuspension was processed with Reference 6 volumes equivalent to 3.9 ml growth (11.6 ml / 3) per tube. The wash was accomplished with 70% ethanol prepared fresh; and elution was with Elution buffer as prepared with composition stated above and stored at 4°C.

[0149] The remaining 25.63-ml cell resuspension was further processed with addition of 51.26-ml L2 buffer (stored at 18°C). The bottle was rolled on a JOANLAB RM-6Pro tube roller mixer (30 rpm, 5 min., 18°C). 25.63 ml of L3 buffer (stored at 18°C with CaCh included immediately before use) was added to the bottle before rolling on the tube roller mixer (30 rpm) for 30 min. out of a 30-min. neutralization period at 18°C. LI, L2, and L3 buffer volumes w ere calculated as follows: LI, Resuspension buffer ml = 5.186 g x 5 x 0.9884 = 25.63 ml; L2, Lysis buffer ml = 5.186 g x 10 x 0.9884 = 51.25 ml; L3, Neutralization buffer ml = 5.186 g x 5 x 0.9884 = 25.63 ml. The buffer volumes used were 0.9884 times less (25.63 ml / 25.93 ml) due to the small aliquot taken for the Reference 1 processing.

[0150] Application of the roller mixing during both lysis and neutralization steps produced a coconut-like consistency of the floc precipitate, allowing for easy centrifugation and microfiltration. -100 ml total lysate material was centrifuged (Dupont Sorvall Instruments RC5Cfloor unit, 6000 ref, 30 min., 4°C) in a 250-ml PPCO bottle to pellet the floc precipitate. Supernatant containing pDNA was filtered through a 250-ml Nalgene Rapid-Flow 0.2-pm filter; and then repeated with a new 0.2-pm filter to obtain -90 ml pre-TFF cleared lysate.Sample concentration using TFF

[0151] After clarification, a TFF step may be performed to allow concentration and diafiltration to exchange the medium into a buffer suitable for binding to magnetic silica, and the removal of RNA, small sized genomic DNA, and small proteins. Sample concentration and diafiltration(desalting) can take place sequentially using the same TFF capsule. The Pall Minimate® PES 100 kDa TFF capsule was initially flushed with 450 ml of Milli-Q water, using a Kamoer KK300 peristaltic pump set at 30 ml / min., ensuring the passage of 150 ml and 300 ml through the retentate and permeate tubing, respectively. The capsule was then flushed with 150 ml Conditioning buffer with chelator (120 mM potassium acetate pH 5.5, 25 mM EDTA) at 30 ml / min. until 30 ml was collected in the permeate container. The feed container, a 50-ml Falcon tube, was filled with the ~50 ml cleared (centrifuged and 0.2 pm filtered) lysate sample to be concentrated. A 1000-ml crystallization dish filled with ice water at between 5 - 10°C was used to maintain a low temperature of the feed container. The peristaltic pump feed flow rate was set to 30 ml / min.. with pressure monitoring using a PressureMAT digital monitor and three single-use pressure sensors (PendoTECH). Permeate (filtrate) flow was increased by tightening the retentate clamp slightly, with inlet pressure, upstream of the TFF capsule, maintained between 11 to 13 psi. TMP was calculated to range from 7 to 8 psi. The solution was filtered to a low volume of 7.5 ml, while continuous mixing (100 rpm) with stir bar on an ONiLAB Scientific magnetic stirrer was maintained during the refill process with retentate fluid. This resulted in a volumetric concentration factor, VCF, of 7. Mixing was gentle to avoid creating nicked DNA or DNA with broken strands, affecting the overall integrity and quality of the purified pDNA. 42.5 ml of Conditioning buffer was added to the 7.5-ml retentate; and filtered to 5 ml at 30 ml / min. Another 45 ml of Conditioning buffer was added to the 5-ml retentate; with filtering to ~2 ml at 30 ml / min. To maximize product recovery, the feed tubing was removed from the feed container; the retentate clamp was loosened; and all liquid was pumped to the feed container. The retentate tubing was disconnected from the TFF capsule and a sterile disposable syringe containing Conditioning buffer was connected to the retentate port to wash remaining liquid into the feed container. A ~ 12.5-ml TFF concentrate was saved in a 30-ml PPCO tube at 4°C until pDNA binding with Trial 8 particles (Trial 6 particles mentioned in Example 2 above). The ~12.5-ml retentate volume in the feed container was brought up to 25 ml with 8 M guanidine HC1. 120 mM potassium acetate pH 5.5, 25 mM EDTA. The final guanidine HC1 concentration was 4 M after the proportioning of 1: 1 cleared lysate: binding buffer.

[0152] Following sample concentration on the TFF capsule, the feed container was filled with 500 ml of 0.5 N NaOH for cleaning as stated above for Example 5.Magnetic bead binding to pDNA

[0153] To obtain 2% final solids content for Trial 8 testing, 5.05 ml of a fully resuspended Trial 6 particle batch with 11.9% solids, was pipetted into the 30-ml PPCO tube containing the TFF- processed pDNA in a 25-ml solution of 4 M guanidine HC1, 120 mM potassium acetate pH 5.5, 25 mM EDTA. Below is the calculation of the slurry volume from the Trial 6 bottle to use for 2% dosing:(x)(l 1.90% Trial 6 solids) = (x + 25 ml after TFF)(2% Trial 8 solids) 11.90x = 2x + 50 ml9.9x = 50 ml x = 5.05 ml Total Volume after Solids Addition = 30.05 ml

[0154] The 30-ml PPCO tube was placed on the JOANLAB RM-6Pro tube roller mixer for 20 min. at 20 rpm under controlled laboratory temperature (18°C) to bind pDNA. The tube was then placed in the Sepmag A200ml magnetic separation unit for a three-minute separation time. Aspiration of the clear supernatant to a waste bottle was possible using a KK300 peristaltic pump with rigid PTFE tubing connected to the silicone pump tubing. The pDNA-bound magnetic particles were washed with 26 ml of Wash buffer (pre-chilled 4°C) with mixing (20 rpm, 1 min.) on the JOANLAB RM-6Pro tube roller mixer, before placing the 30-ml tube back in the A200ml unit. Wash buffer volume was proportioned to wet cell mass in the 3-L E. coli growth (pcDNA3-EGFP plasmid) study of Example 2 above (17.6 g / 5.2 g = 3.4; 90 ml wash / 3.4 = 26 ml). The supernatant was discarded using the peristaltic pump setup. The washing was repeated an additional two times, each with 26-ml Wash buffer. The 3X washed particles were dried at room temperature for 5 min. The 30-ml tube was rotated horizontally to disperse the residual slurry for uniform drying. 19 ml of Elution buffer (pre-chilled 4°C) was added to the 30-ml tube, and the particles were resuspended via mixing on the tube roller mixer (20 rpm, 10 min., 18°C). Elution buffer volume was proportioned to wet cell mass in the 3-L E. coli grow th (pcDNA3-EGFP plasmid) of Example 2 above (17.6 g / 5.2 g = 3.4; 66 ml wash / 3.4 = 19 ml). The 30-ml tube was placed in the A200ml unit for 3 min., before using a sterile serological pipet to transfer supernatant to a disposable syringe for filtering through a 0.2 pmsyringe filter (Cobetter 33 mm hydrophilic PVDF) into a new 50-ml Falcon tube to remove any residual magnetic particles before freezing aliquots at -80°C.Results for Studies 1 and 2

[0155] Example 2 above focused on the pcDNA3-EGFP “Gene of Interest” plasmid, with mixing of 1 :5 ratio of cleared lysate: guanidine binding buffer, 3 M final concentration (i.e., dilute binding conditions). The cleared lysate is the solution after the tangential flow filtration (TFF) cartridge step mentioned in Example 2 (i.e., for binding study Trial 6 magnetic beads shown in that FIG. 3). Studies 1 and 2 under Examples 4-6 included optimization of the binding conditions with a concentrated 1 : 1 ratio of cleared lysate: guanidine binding buffer, 4 M final concentration, for the pcDNA3-EGFP plasmid (i.e., concentrated binding conditions). The observed nucleic acid yield, A260 / A280. and A260 / A230 were astoundingly 1199 pg / g wcm, 1.94, and 2.34 for the Trial 7 magnetic beads in Study 1; and 891 pg / g wcm, 1.98, and 2.30 for the Trial 8 magnetic beads in Study 2, with a separate aliquot of a 3-L growth (FIG. 4). These absorbance ratios are indicative of pure DNA, which are comparable to those obtained with Reference 5 and Reference 6 (non-magnetic, spin column kit that contains RNase) in each of the studies. Furthermore, a quantitative (103 to 109%) plasmid yield relative to the Reference 5 and Reference 6 was observed for Studies 1 and 2, respectively (FIG. 4), supporting the value of superparamagnetic particle technology for purifying therapeutic grade pDNA.

[0156] The total pcDNA3-EGFP yield for the 500-ml growth aliquot of Study 1 was 3.5 mg, representing 7.1 mg / L for Trial 7 magnetic beads; pDNA concentration after guanidine binding buffer addition to the cleared lysate (1 : 1 ratio) was 0. 14 mg / ml. In the Example 2 above, Trial 6 magnetic beads were used to purify 2.3 mg / L; pDNA concentration after guanidine binding buffer addition to the cleared lysate (1 :5 ratio) was 0.023 mg / ml. Essentially, at 2% dosing level with the optimized binding conditions (higher pDNA concentration in contact with magnetic beads at the higher 4 M guanidine concentration), the binding capacity was demonstrated to increase six-fold, from 0.98 mg pDNA / g beads to 5.8 mg pDNA / g beads for magnetic material of 50% (wt%) silicon dioxide composition. 5.4pm particle size, and 37 m2 / g surface area.

[0157] E. coli cell growth conditions can influence how much plasmid is produced in the cell, i.e.. the plasmid yield on a per liter or wet cell mass basis. For example, altering the type of carbon and nitrogen source, such as specific sugar type and yeast extract, can increase yield. Secondly, batch conditions with fixed carbon and nitrogen source will limit the yield as the cells will eventually run out of the nutrients during the exponential phase of growth. A fed-batch approach can maximize cell and plasmid yield, since nutrients are added to the liquid medium continuously or intermittently after the start of cultivation. Third, the present inventors discovered that one way to obtain a higher pDNA content, for instance, in Example 5, Studies 1 and 2, was to harvest the cells during the late stationary phase of growth where the cell count had stayed flat for ~4 hours past the growth inflection point. This harvest approach was useful to determine whether the binding capacity and pDNA yield with the magnetic beads could be higher at the 2% dosing level. The Example 5, Studies 1 and 2, depicts pDNA yield (“Nucleic acid per wcm”) far exceeding 500 pg / g wcm. Surprisingly, pDNA yield was well over about 1,000 pg / g wcm. In fact. Trial 7 pDNA yield was remarkably 1199 pg / g wcm. Accordingly, these results demonstrate that optimized methods of pDNA purification using the Trial 7 and 8 beads are surprisingly effective for producing higher yields of therapeutic-grade pDNA from bacteria.

[0158] Primarily supercoiled (SC) isoform was observed on the agarose gel, with densitometry indicating between 86 and 82% SC for Trial 7 and 8 magnetic beads, respectively (FIG. 4). The AEX-HPLC testing provided higher values of 99 and 96%, respectively. As stated above, methods using superparamagnetic silica offer an alternative, non-chromatographic approach to purify therapeutic-grade pDNA for emerging therapies; superparamagnetic particle technology does not require column packing; does not generate shear forces like that can fragment larger pDNA.Example 6: Magnetic Bead Binding and Purification Assay with 500 to 1000-ml Cell Growth (p AAV 2 / 2 Plasmid)Study 1Fermentation, lysis, and microfiltration

[0159] E. coli growth and harvesting, resuspension, alkaline lysis, neutralization, and filtration / concentration were performed as follows. A 1000-ml aliquot from a full 3-L growth was obtained using LB broth medium with several passages until the optical density at 600 nm reached ~1.8 during the late stationary phase. This yielded sufficient ly sate for a 25 ml pDNA binding experiment with Trial 9 superparamagnetic particles at four different dosing levels. Cells were centrifuged (Dupont Sorvall Instruments RC5Cfloor unit, 5000 ref, 15 min., 4°C) two times in the same pre-weighed 250-ml Nalgene PPCO centrifuge bottle to accumulate a larger pellet of 5.483 g wem before freezing the pellet at -80°C. On the day of the lysis, the pellet was thawed and resuspended using 27.41 ml resuspension buffer (previously autoclaved and stored at 4°C).

[0160] A 0.3-ml aliquot (1.09%) of the 27.41-ml cell resuspension was saved for processing with the Reference 7 (non-magnetic, spin column) kit. The small cell aliquot was centrifuged (Eppendorf 5415 D, 16000 ref, 5 min.. 18°C) to pellet cells and then exchanged to the Reference 7 resuspension buffer containing RNase A, with follow through using the kit’s lysis and neutralization buffers. The Reference 7 protocol states that the spin tube is for processing of 1 to 5 ml cell growth. (0.3 ml / 27.41 ml) (1000 ml cell growth) is equivalent to 10.9 ml growth. To obtain comparative yield data based on triplicate Reference 7 spin column samples, the resuspension was processed with Reference 7 volumes equivalent to 3.6 ml growth (10.9 ml / 3) per tube. The wash was accomplished with 70% ethanol prepared fresh; and elution w as with Elution buffer and stored at 4°C.

[0161] The remaining 27.11 -ml cell resuspension was further processed with addition of 54.22-ml L2 buffer (stored at 18°C). The bottle w as rolled on a JOANLAB RM-6Pro tube roller mixer (30 rpm, 5 min., 18°C). 27.11 ml of L3 buffer (stored at 18°C with CaChincluded immediately before use) was added to the bottle before rolling on the tube roller mixer (30 rpm) for 30 min. out of a 30-min. neutralization period at 18°C. LI, L2, and L3 buffer volumes were calculated as follows: LI, Resuspension buffer ml = 5.483 g x 5 x 0.9890 = 27. 11 ml; L2, Lysis buffer ml = 5.483 g x 10 x 0.9890 = 54.23 ml; L3, Neutralization buffer ml = 5.483 g x 5 x 0.9890 = 27. 11 ml. The buffer volumes used were 0.9890 times less (27.11 ml / 27.41 ml) due to the small aliquot taken for the Reference 1 processing.

[0162] Application of the roller mixing during both lysis and neutralization steps produced a coconut-like consistency of the floc precipitate, allowing for easy centrifugation and microfiltration. -108 ml total lysate material was centrifuged (Dupont Son all Instruments RC5Cfloor unit, 6000 ref, 30 min., 4°C) in a 250-ml PPCO bottle to pellet the floc precipitate. Supernatant containing pDNA was filtered through a 250-ml Nalgene Rapid-Flow 0.2-pm filter; and then repeated with a new 0.2-pm filter to obtain -100 ml pre-TFF cleared lysate.Sample concentration using TFF

[0163] After clarification, a TFF step may be performed to allow concentration and diafiltration to exchange the medium into a buffer suitable for binding to magnetic silica, and the removal of RNA, small sized genomic DNA, and small proteins. Sample concentration and diafiltration (desalting) can take place sequentially using the same TFF capsule. The Pall Minimate® PES 100 kDa TFF capsule was initially flushed with 450 ml of Milli-Q water, using a Kamoer KK300 peristaltic pump set at 30 ml / min., ensuring the passage of 150 ml and 300 ml through the retentate and permeate tubing, respectively. The capsule was then flushed with 150 ml Conditioning buffer with chelator (120 rnM potassium acetate pH 5.5, 25 mM EDTA) at 30 ml / min. until 30 ml was collected in the permeate container. The feed container, a 50-ml Falcon tube, was filled with an initial 50 ml cleared (centrifuged and 0.2 pm filtered) lysate sample to be concentrated. A 1000-ml crystallization dish filled with ice water at between 5 - 10°C was used to maintain a low temperature of the feed container. The peristaltic pump feed flow rate was set to 20 ml / min., with pressure monitoring using a PressureMAT digital monitor and three single-use pressure sensors (PendoTECH). Permeate (filtrate) flow was increased by tightening the retentate clamp slightly, with inlet pressure, upstream of the TFF capsule, maintained between 8 to 9 psi. TMP was calculated to range from 5 to 6 psi. Additional cleared lysate volume was added to the Falcon tube feed container as appropriate. The solution was filtered to a low volume of 7.5 ml, while continuous mixing (100 rpm) with stir bar on an ONiLAB Scientific magnetic stirrer was maintained during the refill process with retentate fluid. This resulted in a volumetric concentration factor, VCF, of 13. Mixing was gentle to avoid creating nicked DNA or DNA with broken strands, affecting the overall integrity and quality of the purified pDNA. 42.5 ml of Conditioning buffer was added to the 7.5-ml retentate; and filtered to 7.5 ml at 20 ml / min. Another 42.5 ml of Conditioning buffer wasadded to the 7.5-ml retentate; with filtering to ~1 ml at 20 ml / min. To maximize product recovery, the feed tubing was removed from the feed container; the retentate clamp was loosened; and all liquid was pumped to the feed container. The retentate tubing was disconnected from the TFF capsule and a sterile disposable syringe containing Conditioning buffer was connected to the retentate port to wash remaining liquid into the feed container. An ~11-ml TFF concentrate was saved in a 30-ml PPCO tube at 4°C until pDNA binding with Trial 9 particles (Trial 6 particles mentioned in Example 2 above). The ~11-ml retentate volume in the feed container was brought up to 100 ml with 5.6 M guanidine HC1, 120 mM potassium acetate pH 5.5, 25 mM EDTA. The final guanidine HC1 concentration was 5 M after the proportioning of 1 :8 cleared lysate: binding buffer.

[0164] Following sample concentration on the TFF capsule, the feed container was filled with 500 ml of 0.5 N NaOH for cleaning as stated above for Example 5.Magnetic bead binding to pDNA

[0165] To obtain 0.5, 1, 2, and 3% final solids content Trial 9 testing at four parallel dosing runs, the 1 :8 cleared lysate: binding buffer was aliquoted evenly (25 ml each) into four Falcon tubes. To obtain the respective solids content, 1.1, 2.29, 5.05, and 8.43 ml of a fully resuspended Trial 6 particle batch with 11.9% solids, was pipetted into the appropriately labeled Falcon tubes containing the TFF-processed pDNA in a 25 -ml solution of 5 M guanidine HC1, 120 mM potassium acetate pH 5.5, 25 mM EDTA. Below is the example calculation of the slurry volume from the Trial 6 bottle to use for 0.5% dosing:(x)(l 1.90% Trial 6 solids) = (x + 25 ml after TFF)(0.5% Trial 9 solids) 11.90x = 0.5x + 12.5 ml11.4x = 12.5 ml x = 1.1 mlTotal Volume after Solids Addition = 26. 1 ml

[0166] The four Falcon tubes were placed on the JOANLAB RM-6Pro tube roller mixer for 20 min. at 20 rpm under controlled laboratory temperature (18°C) to bind pDNA. Each tube was then placed in the Sepmag A200ml magnetic separation unit for a three-minute separation time. Aspiration of the clear supernatant to a waste bottle was possible using a KK300 peristalticpump with rigid PTFE tubing connected to the silicone pump tubing. The pDNA-bound magnetic particles were washed with 28 ml of Wash buffer (pre-chilled 4°C) with mixing (20 rpm, 1 min.) on the JOANLAB RM-6Pro tube roller mixer, before placing the Falcon tubes back in the A200ml unit. Wash buffer volume was proportioned to wet cell mass in the 3-L E. coli growth (pcDNA3-EGFP plasmid) of Example 2 (17.6 g / 5.5 g = 3.2; 90 ml wash / 3.2 = 28 ml per wash replicate). The supernatant was discarded using the peristaltic pump setup. The washing was repeated an additional two times, each with 28-ml Wash buffer. The 3X washed particles were dried at room temperature for 5 min. Each tube was rotated horizontally to disperse the residual slurry for uniform drying. 21 ml of Elution buffer (pre-chilled 4°C) was added to each tube, and the particles were resuspended via mixing on the tube roller mixer (20 rpm, 10 min., 18°C). Elution buffer volume was proportioned to wet cell mass in the 3-L E. coli growth (pcDNA3-EGFP plasmid) of Example 2 (17.6 g / 5.5 g = 3.2; 66 ml wash / 3.2 = 21 ml per elution replicate). Each tube was placed in the A200ml unit for 3 min., before using a sterile serological pipet to transfer supernatant to a disposable syringe for filtering through a 0.2 pm syringe filter (Cobetter 33 mm hydrophilic PVDF) into a new Falcon tube to remove any residual magnetic particles before freezing aliquots at -80°C.Study 2Fermentation, lysis, and microfiltration

[0167] E. coli growth and harvesting, resuspension, alkaline lysis, neutralization, and filtration / concentration were performed as follows. A 500-ml aliquot from a full 3-L grow th was obtained using LB broth medium with several passages until the optical density at 600 nm reached ~2.5 during the late stationary phase. This yielded sufficient ly sate for a 37.5 ml pDNA binding experiment with Trial 10 superparamagnetic particles. Cells were centrifuged (Dupont Sorvall Instruments RC5Cfloor unit, 5000 ref, 15 min., 4°C) two times in the same pre-weighed 250-ml Nalgene PPCO centrifuge bottle to accumulate a larger pellet of 2.826 g wem before freezing the pellet at -80°C. On the day of the lysis, the pellet was thaw ed and resuspended using 14.13 ml resuspension buffer (previously autoclaved and stored at 4°C).

[0168] A 0.2-ml aliquot (1.41%) of the 14.13-ml cell resuspension w as saved for processing with the Reference 8 (non-magnetic, spin column) kit. The small cell aliquot was centrifuged(Eppendorf 5415 D, 16000 ref, 5 min.. 18°C) to pellet cells and then exchanged to the Reference 8 resuspension buffer containing RNase A, with follow through using the kit’s lysis and neutralization buffers. The Reference 8 protocol states that the spin tube is for processing of 1 to 5 ml cell growth. (0.2 ml / 14.13 ml) (500 ml cell growth) is equivalent to 7.1 ml growth. To obtain comparative yield data based on triplicate Reference 8 spin column samples, the resuspension was processed with Reference 8 volumes equivalent to 2.4 ml growth (7. 1 ml / 3) per tube. The wash was accomplished with 70% ethanol prepared fresh; and elution w as with Elution buffer and stored at 4°C.

[0169] The remaining 13.93-ml cell resuspension was further processed with addition of 27.86-ml L2 buffer (stored at 18°C). The bottle w as rolled on a JOANLAB RM-6Pro tube roller mixer (30 rpm, 5 min., 18°C). 13.93 ml of L3 buffer (stored at 18°C with CaCh included immediately before use) was added to the bottle before rolling on the tube roller mixer (30 rpm) for 30 min. out of a 30-min. neutralization period at 18°C. LI, L2, and L3 buffer volumes were calculated as follows: LI, Resuspension buffer ml = 2.826 g x 5 x 0.9858 = 13.93 ml; L2, Lysis buffer ml = 2.826 g x 10 x 0.9858 = 27.86 ml; L3, Neutralization buffer ml = 2.826 g x 5 x 0.9858 = 13.93 ml. The buffer volumes used were 0.9858 times less (13.93 ml / 14.13 ml) due to the small aliquot taken for the Reference 1 processing.

[0170] Application of the roller mixing during both lysis and neutralization steps produced a coconut-like consistency of the floc precipitate, allowing for easy centrifugation and microfiltration. ~55 ml total lysate material was centrifuged (Dupont Sorvall Instruments RC5Cfloor unit, 6000 ref, 30 min., 4°C) in a 250-ml PPCO bottle to pellet the floc precipitate. Supernatant containing pDNA was filtered through a 250-ml Nalgene Rapid-Flow 0.2-pm filter; and then repeated with a new 0.2-pm filter to obtain ~50 ml pre-TFF cleared lysate.Sample concentration using TFF

[0171] After clarification, a TFF step may be performed to allow concentration and diafiltration to exchange the medium into a buffer suitable for binding to magnetic silica, and the removal of RNA, small sized genomic DNA, and small proteins. Sample concentration and diafiltration (desalting) can take place sequentially using the same TFF capsule. The Pall Minimate® PES 100 kDa TFF capsule was initially flushed with 450 ml of Milli-Q water, using a KamoerKK300 peristaltic pump set at 30 ml / min.. ensuring the passage of 150 ml and 300 ml through the retentate and permeate tubing, respectively. The capsule was then flushed with 150 ml Conditioning buffer with chelator (120 mM potassium acetate pH 5.5, 25 mM EDTA) at 30 ml / min. until 30 ml was collected in the permeate container. The feed container, a 50-ml Falcon tube, was filled with the ~50 ml cleared (centrifuged and 0.2 pm filtered) lysate sample to be concentrated. A 1000-ml crystallization dish filled with ice water at between 5 - 10°C was used to maintain a low temperature of the feed container. The peristaltic pump feed flow rate was set to 20 ml / min., with pressure monitoring using a PressureMAT digital monitor and three single-use pressure sensors (PendoTECH). Permeate (filtrate) flow was increased by tightening the retentate clamp slightly, with inlet pressure, upstream of the TFF capsule, maintained between 9 to 10 psi. TMP was calculated to range from 6 to 7 psi. The solution was filtered to a low volume of 7.5 ml, while continuous mixing (100 rpm) with stir bar on an ONiLAB Scientific magnetic stirrer was maintained during the refill process with retentate fluid. This resulted in a volumetric concentration factor, VCF, of 7. Mixing was gentle to avoid creating nicked DNA or DNA with broken strands, affecting the overall integrity and quality of the purified pDNA. 42.5 ml of Conditioning buffer was added to the 7.5-ml retentate; and filtered to 5 ml at 20 ml / min. Another 45 ml of Conditioning buffer was added to the 5 -ml retentate; with filtering to ~1 ml at 20 ml / min. To maximize product recovery’, the feed tubing was removed from the feed container; the retentate clamp was loosened; and all liquid was pumped to the feed container. The retentate tubing was disconnected from the TFF capsule and a sterile disposable syringe containing Conditioning buffer was connected to the retentate port to wash remaining liquid into the feed container. A ~12.5-ml TFF concentrate was saved in a 50-ml Falcon tube at 4°C until pDNA binding with Trial 10 particles (Trial 6 particles mentioned in Example 2 above). The ~12.5-ml retentate volume in the feed container was brought up to 37.5 ml with 7.5 M guanidine HC1, 120 mM potassium acetate pH 5.5, 25 mM EDTA. The final guanidine HC1 concentration was 5 M after the proportioning of 1:2 cleared lysate: binding buffer.

[0172] Following sample concentration on the TFF capsule, the feed container was filled with 500 ml of 0.5 N NaOH for cleaning as stated above for Example 5.Magnetic bead binding to pDNA

[0173] To obtain 2% final solids content for Trial 10 testing, 7.57 ml of a fully resuspended Trial 6 particle batch with 1 1 .9% solids, was pipetted into the Falcon tube containing the TFF- processed pDNA in the 37.5-ml solution of 5 M guanidine HC1, 120 mM potassium acetate pH 5.5. 25 mM EDTA. Below is the calculation of the slurry volume from the Trial 6 bottle to use for 2% dosing:(x)(l 1.90% Trial 6 solids) = (x + 37.5 ml after TFF)(2% Trial 10 solids) 11.90x = 2x + 75 ml9.9x = 75 ml x = 7.57 ml Total Volume after Solids Addition = 45.07 ml

[0174] The Falcon tube was placed on the JOANLAB RM-6Pro tube roller mixer for 20 min. at 20 rpm under controlled laboratory temperature (18°C) to bind pDNA. The tube was then placed in the Sepmag A200ml magnetic separation unit for a three-minute separation time. Aspiration of the clear supernatant to a waste bottle was possible using a KK300 peristaltic pump with rigid PTFE tubing connected to the silicone pump tubing. The pDNA-bound magnetic particles were washed with 14 ml of Wash buffer (now prepared with 10 mM Tris-HCl, 1 mM EDTA, pH 8.77; pre-chilled 4°C) with mixing (20 rpm, 1 min.) on the JOANLAB RM-6Pro tube roller mixer, before placing the tube back in the A200ml unit. Wash buffer volume was proportioned to wet cell mass in the 3-L E. coll growth (pcDNA3-EGFP plasmid) study of Example 2 above (17.6 g / 2.8 g = 6.2; 90 ml wash / 6.2 = 14 ml). The supernatant was discarded using the peristaltic pump setup. The washing was repeated an additional two times, each with 14-ml Wash buffer. The 3X washed particles were dried at room temperature for 5 min. The tube was rotated horizontally to disperse the residual slurry for uniform drying. 10 ml of Elution buffer (pre-chilled 4°C) was added to the tube, and the particles were resuspended via mixing on the tube roller mixer (20 rpm. 10 min., 18°C). Elution buffer volume was proportioned to wet cell mass in the 3-L E. colt growth (pcDNA3-EGFP plasmid) of Example 2 above (17.6 g / 2.8 g = 6.3; 66 ml wash / 6.3= 10 ml). The tube was placed in the A200ml unit for 3 min., before using a sterile serological pipet to transfer supernatant to a disposable syringe for filtering through a 0.2 pm syringe filter (Cobetter 33 mm hydrophilic PVDF) into a new Falcon tube to remove any residual magnetic particles before freezing aliquots at -80°C.Results for Studies 1 and 2

[0175] In the case of the second plasmid, pAAV2 / 2 (a Rep / Cap plasmid), four different dosing levels were assessed at binding conditions of 1:8 cleared lysate: guanidine binding buffer, 5 M final concentration. As for the Example 5 study with pcDNA3-EGFP plasmid, the same magnetic material technology (i.e., 50 wt% silicon dioxide composition, 5.4pm particle size, and 37 m2 / g surface area) was used for the Example 6 Studies 1 and 2. The observed nucleic acid yield increased from 401 to 558 pg / g wcm, as the Trial 9 magnetic bead dosing level increased from 0.5 to 3.0% in Study 1 (FIG. 5). The A260 / A280 and A260 / A230 ratios met the criteria indicated in Table 2 at all dosing levels.

[0176] In Study 2, with binding conditions of 1:2 cleared lysate: guanidine binding buffer, 5 M final concentration, the observed nucleic acid yield, A260 / A280, and A260 / A230 were 464 pg / g wcm, 2.00, and 2.13 for the Trial 10 magnetic beads, with a separate aliquot of a 3-L growth (FIG. 5 A quantitative (98%) plasmid yield relative to the Reference 8 (non-magnetic, spin column kit that contains RNase) was observed for the Trial 10 magnetic beads at 2% dosing level in Study 2 (FIG. 5).

[0177] Primarily supercoiled (SC) isoform was observed on the agarose gel, with densitometry indicating between 85 to 93% SC for Trial 9 magnetic beads, at the different dosing levels in Study 1 (FIG. 5). For Study 2, densitometry' indicated 81% SC for Trial 10 magnetic beads. In both studies, the AEX-HPLC testing provided higher values of 97 to 98% SC. These unexpected results support the applicability of the superparamagnetic silica technology for purifying higher molecular mass pDNA.

[0178] These results show that the magnetic particle-based methods of the present disclosure were effective for purifying large amounts of therapeutic-grade plasmid DNA in a supercoiled form with high degrees of purify and no RNAse contamination. The present inventors discovered that plasmid yield can vary based on plasmid type, growth conditions, and superparamagnetic silica binding conditions. Accordingly, the methods of the present disclosure are useful for purifying therapeutic-grade plasmid DNA.EQUIVALENTS

[0179] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present technology is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0180] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0181] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as ‘ftp to.” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1. 2, 3, 4, or 5 cells, and so forth.

[0182] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

[0183] Other embodiments are set forth within the following claims.ReferencesN. Ali el al. Current nucleic acid extraction methods and their implications to point-of-care diagnostics. BioMed Res Int. 2017;2017:e9306564.S. Berensmeier. Magnetic particles for the separation and purification of nucleic acids. Appl. Microbiol. Biotechnol. 2006. 73:495-504.H.C. Bimboim and J. Doly. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979; 7(6): 1513-1523.A. Eon-Duval et al. Precipitation of RNA impurities with high salt in a plasmid DNA purification process: Use of experimental design to determine reaction conditions. Biotechnol Bioeng 83: 544-553, 2003.G. Koetsier and E. Cantor. A Practical Guide to Analyzing Nucleic Acid Concentration and Purity with Microvolume Spectrophotometers. Technical Note. New England Biolabs. 2019.P. Oberacker et al. Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation. PLOS Biology. 2019.J. N. Pryor. Silica Adsorbent on Magnetic Substrate. U.S. Pat. No. 6,296.937 B2. 2001.C. Smith et al. Mixed-Bed Solid Phase and Its Use in the Isolation of Nucleic Acids. U.S. Pat. No. 6,270,970. 2001.J. Stadler et al. Plasmid DNA purification. J Gene Med. 2004;6(Suppl l):S54-66.J. Van Alstine et al. Plasmid Purification. U.S. Pat. No. 8,093,373 B2. 2012.X. Wang et al. High-Throughput Method for Isolating Plasmid DNA with Reduced Lipopolysaccharide Content. BioTechniques. 2000. 28: 106-108.H. Yang et al. Plasmid isoform separation and quantification by anion-exchange chromatography (AEX). Waters Application Note. 2021.

Claims

1. What is claimed is:

1. A method for purifying therapeutic-grade plasmid DNA (pDNA) comprising the use of a magnetic particle, wherein the magnetic particle comprises a siliceous oxide surface layer and a superparamagnetic core material.

2. The method of claim 1 , wherein the particles have a nitrogen BET surface area of about 6 m2 / g to about 62 m2 / g.

3. The method of any one of claims 1-2, wherein the superparamagnetic core comprises about 15 wt.% to about 60 wt.% of the particles.

4. The method of any one of claims 1-3, wherein the particle is about 1 pm to about 15 pm in diameter.

5. The method of any one of claims 1-4, wherein the particle exhibits less than 10 parts per million of magnetic core leaching.

6. The method of any one of claims 1-5, wherein the particle comprises about 40% to about 85% siliceous oxide.

7. The method of any one of claims 1-6, wherein the method does not comprise use of an RNase enzyme.

8. The method of any one of claims 1-7, wherein the method further comprises a step (i) of concentrating a volume of bacteria, wherein the bacteria comprise the pDNA.

9. The method of claim 8, wherein the method further comprises a step (ii) of resuspending the concentrated bacteria in a resuspension buffer.

10. The method of claim 9, wherein the method further comprises a step (iii) of adding a lysis buffer to the bacteria resuspended in resuspension buffer to generate a lysate.1 1. The method of claim 10, wherein the method further comprises a step (iv) of adding a neutralization buffer to the lysate.

12. The method of claim 11. wherein the method further comprises a step (v) of performing one or more centrifugation and / or filtration steps on the lysate to generate a cleared lysate, wherein the cleared lysate comprises a portion of the pDNA.

13. The method of claim 12. wherein the method further comprises a step (vi) of adding the magnetic particles to the cleared lysate and incubating the mixture, wherein the portion of the pDNA binds to the magnetic particles.

14. The method of claim 13. wherein the method further comprises a step (vii) of performing one or more wash steps.

15. The method of claim 14, wherein the method further comprises a step (viii) of performing an elution step, wherein the elution step releases the bound pDNA from the magnetic particles.

16. The method of claim 13 further comprising a magnetic separation step of the solution comprising the magnetic particles and the cleared lysate, wherein the magnetic separation step comprises applying a magnetic field to the solution and wherein the magnetic separation step results in about 10% to about 80% transmittance through the solution after 5 seconds of magnetic field exposure, about 45% to about 100 % transmittance through the solution after 40 seconds of magnetic field exposure, and about 80% to about 100 % transmittance through the solution after 600 seconds of magnetic field exposure.

17. The method of any one of claims 1-16, wherein the volume of bacteria comprises about 4ml bacteria to about 3L bacteria.

18. The method of any one of claims 9-17, wherein the resuspension buffer comprises about 50 mM glucose, about 25 mM Tris-HCl pH 8.0, and about 10 mM EDTA pH 8.0.

19. The method of any one of claims 10-18, wherein the lysis buffer comprises about 0.2 M NaOH and about 0.5% to about 2% SDS.

20. The method of any one of claims 11-19. wherein the neutralization buffer comprises about 2 M to about 4 M potassium acetate, about 1 M to about 3 M acetic acid, andabout 0.5M to about 2 M CaCh. and wherein the neutralization buffer is at about pH4.5 to about 6.5.

21. The method of any one of claims 12-20, wherein step (v) comprises one or more of centrifugation, use of micromolar filters, use of depth filters, use of ultrafiltration centrifuge tubes, or use of tangential flow filtration capsules.

22. The method of claim 21, wherein step (v) comprises use of ultrafiltration centrifuge tubes in an ultrafiltration centrifuge step, and wherein the ultrafiltration centrifugation step comprises a buffer exchange step, wherein a new' buffer replaces the buffer of the lysate and w'herein the new buffer comprises 120 mM potassium acetate at pH 5.5 and 25 mM EDTA.

23. The method of claim 21, wherein step (v) comprises use of tangential flow filtration capsules in a tangential flow' filtration step, and wherein the tangential flow' filtration step comprises a buffer exchange step wherein a new buffer replaces the buffer of the lysate and wherein the new buffer comprises about 100 mM to about 10 mM potassium acetate at about pH 4.5 to about pH 6.5 and about 20 mM to about 30 mM EDTA.

24. The method of any one of claims 13-23, wherein step (vi) further comprises adding guanidine hydrochloride to the cleared lysate to achieve a final concentration of about 1 M to about 5 M prior to the addition of the magnetic particles.

25. The method of any one of claims 10-24, w herein a roller mixer is used to mix solutions comprising the pDNA during one or more of steps (iii), (iv), (vi), (vii), and (viii).

26. The method of any one of claims 1-25, wherein the method does not comprise a chromatography step or use of a chromatography column.

27. The method of any one of claims 1-26, wherein the purified therapeutic-grade pDNA is at a concentration of about 300 pg / g w et cell mass to about 1200 pg / g wet cell mass.

28. The method of any one of claims 1-27, wherein the purified therapeutic-grade pDNA is at a concentration of about 400 pg / g w et cell mass to about 1100 pg / g wet cell mass.

29. The method of any one of claims 1-27, wherein the purified therapeutic-grade pDNA is at a concentration of about 300 pg / g wet cell mass to about 500 pg / g wet cell mass.

30. The method of any one of claims 1-29, wherein the purified therapeutic-grade pDNA has an A260 / A280 ratio of about 1.80 to about 1.97.

31. The method of any one of claims 1-30, wherein the purified therapeutic-grade pDNA has an A260 / A230 ratio of about 1.5 to about 2.4.

32. The method of any one of claims 1-31, wherein the purified therapeutic-grade pDNA is about 80% to about 99% supercoiled.

33. The method of any one of claims 1-32, wherein the purified therapeutic-grade pDNA comprises about 1 to about 200 EU endotoxin per mg pDNA.

34. An isolated therapeutic-grade plasmid DNA (pDNA) produced by a process comprising the use of a magnetic particle, wherein the magnetic particle comprises a siliceous oxide surface layer and a superparamagnetic core material.

35. The isolated pDNA of claim 34, wherein the particles have a nitrogen BET surface area of about 6 m2 / g to about 62 m2 / g.

36. The isolated pDNA of any one of claims 34-35, wherein the superparamagnetic core comprises about 15 wt.% to about 60 wt.% of the particles.

37. The isolated pDNA of any one of claims 34-36, wherein the particle is about 1 pm to about 15 pm in diameter.

38. The isolated pDNA of any one of claims 34-37. wherein the particle exhibits less than 10 parts per million of magnetic core leaching.

39. The isolated pDNA of any one of claims 34-38, wherein the particle comprises about 40% to about 85% siliceous oxide.

40. The isolated pDNA of any one of claims 34-39, wherein the method does not comprise use of an RNase enzy me.

41. The isolated pDNA of any one of claims 34-40. wherein the method further comprises a step (i) of concentrating a volume of bacteria, wherein the bacteria comprise the pDNA.

42. The isolated pDNA of claim 41, wherein the method further comprises a step (ii) of resuspending the concentrated bacteria in a resuspension buffer.

43. The isolated pDNA of claim 42, wherein the method further comprises a step (iii) of adding a lysis buffer to the bacteria resuspended in resuspension buffer to generate a lysate.

44. The isolated pDNA of claim 43, wherein the method further comprises a step (iv) of adding a neutralization buffer to the lysate.

45. The isolated pDNA of claim 44, wherein the method further comprises a step (v) of performing one or more centrifugation and / or filtration steps on the lysate to generate a cleared lysate, wherein the cleared lysate comprises a portion of the pDNA.

46. The isolated pDNA of claim 45, wherein the method further comprises a step (vi) of adding the magnetic particles to the cleared lysate and incubating the mixture, wherein the portion of the pDNA binds to the magnetic particles.

47. The isolated pDNA of claim 46, wherein the method further comprises a step (vii) of performing one or more wash steps.

48. The isolated pDNA of claim 47. wherein the method further comprises a step (viii) of performing an elution step, wherein the elution step releases the bound pDNA from the magnetic particles.

49. The isolated pDNA of claim 46 further comprising a magnetic separation step, wherein the magnetic separation step of the solution comprising the magnetic particles and the cleared lysate, wherein the magnetic separation step comprises applying a magnetic field to the solution and wherein the magnetic separation step results in about 10% to about 80% transmittance through the solution after 5 seconds of magnetic field exposure, about 45% to about 100 % transmittance through the solution after 40seconds of magnetic field exposure, and about 80% to about 100 % transmittance through the solution after 600 seconds of magnetic field exposure.

50. The isolated pDNA of any one of claims 34-49, wherein the volume of bacteria comprises about 4ml bacteria to about 3L bacteria.

51. The isolated pDNA of any one of claims 42-50, wherein the resuspension buffer comprises about 50 mM glucose, about 25 mM Tris-HCl pH 8.0, and about 10 mM EDTA pH 8.0.

52. The isolated pDNA of any one of claims 43-51, wherein the lysis buffer comprises about 0.2 M NaOH and about 0.5% to about 2% SDS.

53. The isolated pDNA of any one of claims 44-52, wherein the neutralization buffer comprises about 2 M to about 4 M potassium acetate, about 1 M to about 3 M acetic acid, and about 0.5M to about 2 M CaCh, and wherein the neutralization buffer is at about pH 4.5 to about 6.5.

54. The isolated pDNA of any one of claims 45-53, wherein step (v) comprises one or more of centrifugation, use of micromolar filters, use of depth filters, use of ultrafiltration centrifuge tubes, or use of tangential flow filtration capsules.

55. The isolated pDNA of claim 54, wherein step (v) comprises use of ultrafiltration centrifuge tubes in an ultrafiltration centrifuge step, and wherein the ultrafiltration centrifugation step comprises a buffer exchange step, wherein a new buffer replaces the buffer of the lysate and wherein the new buffer comprises 120 mM potassium acetate at pH 5.5 and 25 mM EDTA.

56. The isolated pDNA of claim 54, wherein step (v) comprises use of tangential flow filtration capsules in a tangential flow filtration step, and wherein the tangential flow filtration step comprises a buffer exchange step wherein a new buffer replaces the buffer of the lysate and wherein the new buffer comprises about 100 mM to about 10 mM potassium acetate at about pH 4.5 to about pH 6.5 and about 20 mM to about 30 mM EDTA.

57. The isolated pDNA of any one of claims 46-56. wherein step (vi) further comprises adding guanidine hydrochloride to the cleared lysate to achieve a final concentration of about 1 M to about 5 M prior to the addition of the magnetic particles.

58. The isolated pDNA of any one of claims 43-57. wherein a roller mixer is used to mix solutions comprising the pDNA during one or more of steps (iii), (iv), (vi), (vii), and (viii).

59. The isolated pDNA of any one of claims 34-58. wherein the method does not comprise a chromatography step or use of a chromatography column.

60. The method of any one of claims 34-59, wherein the purified therapeutic-grade pDNA is at a concentration of about 300 pg / g wet cell mass to about 1200 pg / g wet cell mass.

61. The method of any one of claims 34-60, wherein the purified therapeutic-grade pDNA is at a concentration of about 400 pg / g wet cell mass to about 1100 pg / g wet cell mass.

62. The isolated pDNA of any one of claims 34-60, wherein the purified therapeutic-grade pDNA is at a concentration of about 300 pg / g wet cell mass to about 500 pg / g wet cell mass.

63. The isolated pDNA of any one of claims 34-62, wherein the purified therapeutic-grade pDNA has an A260 / A280 ratio of about 1.80 to about 1.97.

64. The isolated pDNA of any one of claims 34-63. wherein the purified therapeutic-grade pDNA has an A260 / A230 ratio of about 1.5 to about 2.4.

65. The isolated pDNA of any one of claims 34-64, wherein the purified therapeutic-grade pDNA is about 80% to about 99% supercoiled.

66. The isolated pDNA of any one of claims 34-65, wherein the purified therapeutic-grade pDNA comprises about 1 to about 200 EU endotoxin per mg pDNA.

Images