Metal affinity extraction of host cell DNA

Abstract

A method for removing host cell DNA from a sample containing a species of interest, protein, virus or extracellular vesicle, comprising the steps of: - loading a matrix bearing anionic metal affinity ligands with metal ions, - equilibrating said matrix with a buffer having a pH in the range of pH 6 to pH 10 and a salt concentration in the range of concentrations up to 1 M, wherein the salt does not form a chemical complex with said anionic metal affinity ligand, - contacting said sample with said metal loaded anionic metal affinity matrix, - separating said negative from said sample, wherein the contaminating DNA content of said sample is reduced.

Description

[0001] This invention relates to a method for removing host cell DNA from a sample containing desired types of proteins, viruses, or extracellular vesicles.

[0002] background

[0003] Host cell-derived DNA is a ubiquitous contaminant in all biological products derived from cell culture. Regulatory agencies require that host DNA contamination in biotherapies be reduced to extremely low levels. They do this to minimize the possibility of unintentionally transmitting pathogenic viruses or carcinogenic DNA sequences to patients receiving treatment.

[0004] Although host cell DNA is thought to exist as a separate category of contaminants in cell cultures, it is always closely associated with proteins, primarily in well-defined compound structures. Host cell DNA in cell culture harvests is a remnant of the cellular chromosomal mass used by the cells to produce biotherapies of interest. This mass is called chromatin. Chromatin primarily consists of chromosomal DNA and histones that compress DNA and regulate transcription in the cell nucleus. In cell culture harvests, chromatin is degraded into linear arrays of 1 to approximately 30 nucleosomes, ranging in size from about 12–400 nm, and smaller histone-associated DNA fragments, ranging in size from about 2–12 nm. A subset of these fragments, along with the nucleosome array, forms a collection of compounds.

[0005] Chromatin contamination of cell culture harvests and cell lysates is also important because chromatin interacts nonspecifically with all known purification methods and media [1-3]. It has been documented to reduce volume, increase host protein contamination, and increase aggregate content, but it can also lead to excess levels of DNA persisting during multi-step purification processes when logically it is to be removed.

[0006] In multi-step chromatographic purification, host DNA levels can be sufficiently reduced in some cases, but in all cases, DNA reduction is enhanced if some chromatin load is removed before chromatographic purification begins. Many methods for pre-removing chromatin have been described in the field of IgG purification, including coprecipitation with positively charged particles, flocculation with positively charged polymers, flocculation with positively charged organic matter, and removal using positively charged depth filtration media [1-4]. However, for large-scale DNA removal, binding DNA to positively charged anion exchange chromatography columns is impractical because they are contaminated and clogged by the large amounts of chromatin in cell culture harvests and cell lysates.

[0007] Negatively charged organic reagents (fatty acids) exhibit flocculation specifically targeting histones, but may co-precipitate their associated host DNA. Combinations of fatty acids with positively charged flocculants are more effective than either alone. Their effectiveness is further enhanced when combined with allantoin, particularly for removing large molecules such as high molecular weight aggregates [1,2].

[0008] Extracting DNA from certain cell culture products is simpler than from others because the properties of different product classes overlap with DNA to varying degrees. The chemical properties of IgG monoclonal antibodies are fundamentally different from those of DNA. This makes it feasible to selectively remove DNA from antibody preparations using a variety of chemical methods. Most of these treatments do not interact with IgG, so IgG remains in solution and can be recovered in high yields.

[0009] Reducing DNA from viral and extracellular vesicle preparations is more challenging because they share many chemical similarities, including a net negative charge conferred at least partially by the presence of phosphate groups. This renders positively charged DNA extraction methods unsuitable, as the positive charge would remove the virus and vesicles along with the DNA. It also means that methods utilizing affinity chromatography on phosphate residues will be affected.

[0010] Fatty acid flocculation is also unsuitable for lipid-enveloped viruses and vesicles because fatty acids disrupt the stability of their lipid membranes. Allantoin is similarly unsuitable because it indiscriminately co-precipitates large species. Viruses and vesicles occupy the same size range as chromatin in the cell harvest, therefore allantoin removes them along with the chromatin.

[0011] The lack of methods for removing DNA in viral and vesicle preparations has led to reliance on enzymatic cleavage to reduce DNA. This method is partially successful but cannot remove all host cell DNA because it is protected by strong associations with histones.

[0012] Immobilized metal affinity chromatography (IMAC) is known for purifying proteins, viruses, exosomes, and DNA, in which metal-binding sites in the form of multi-histidine (histidine-tagged) compounds have been recombinantly introduced. Such columns are most commonly eluted with imidazole; less frequently by lowering the pH. RNA is known to be purified using IMAC columns loaded with iminodiacetic acid containing copper, nickel, zinc, and cobalt (from strongest to weakest) [5]. RNA binds to these metals by forming coordinate bonds with their nitrogenous bases, with purine bases binding more strongly than pyrimidine bases.

[0013] DNA exhibits low affinity for IMAC because its nitrogenous bases are involved in interstrand base pairing and are spatially inaccessible. This results in most DNA passing through the column. The problem of IMAC columns not binding DNA can be overcome by thermally or alkalily dissociating the DNA into single strands, so that the metal ions on the surface of the IMAC medium can spatially approach its nitrogenous bases [6].

[0014] Anion exchange chromatography and salt gradient elution are known to be used to separate empty and intact adeno-associated virus capsids [7-9]. Invention Summary

[0015] In one aspect, the method of the present invention can be used to remove host cell DNA from samples containing desired types of proteins, viruses, or extracellular vesicles. The method of the present invention includes the steps of:

[0016] - Use metal ions to load a matrix (subtract) with anionic metal affinity ligands.

[0017] - The matrix is ​​equilibrated with a buffer solution having a pH value in the range of pH 4 to pH 10 and a salt concentration in the range of up to 1 M, wherein the salt does not form a chemical complex with the anionic metal affinity matrix ligand of the loaded metal.

[0018] - The sample is brought into contact with the anionic metal affinity matrix of the loaded metal.

[0019] - Isolate the matrix from the sample, wherein the sample has a reduced content of host cell DNA.

[0020] The matrix is ​​equilibrated with a balancing buffer, typically by adjusting the buffer conditions to a combination of pH and salt conditions that prevent viral or extracellular vesicle binding but allow contaminating DNA binding. The specific conditions to be selected can be readily determined by those skilled in the art. As an indication, the pH value can be adjusted within the range of pH 6 to pH 10, and the salt concentration can be up to 1 M.

[0021] To improve readability, the technically correct but rather cumbersome expression "a salt that does not form a chemical complex with the anionic metal affinity matrix of the supported metal" has been replaced by the informal term "non-chelating salt." It is believed that those skilled in the art will understand that the use of the informal term "non-chelating salt" in the passages refers to the more technical expression "a salt that does not form a chemical complex with the anionic metal affinity matrix of the supported metal." Therefore, for simplicity, the expression "a salt that does not form a chemical complex with the anionic metal affinity matrix of the supported metal" will also be referred to as "non-chelating salt" in the following text.

[0022] It should be understood that the terms "host DNA" or "contaminated DNA" refer to DNA that is not encapsulated in or adsorbed onto particles, such as viruses, virus-like particles, capsids, vesicles, exosomes, liposomes, etc.

[0023] In a second aspect, the present invention relates to a two-step method for efficiently extracting chromatin from cell culture-derived biological products, but uniquely characterized by its ability to selectively remove host DNA from viral particles and extracellular vesicle products. The first step comprises treating a sample containing desired proteins, viruses, or extracellular vesicles with excess host cell DNA using an anionic metal affinity matrix loaded with metal ions. The second step comprises treating the metal affinity-treated sample by anion exchange chromatography.

[0024] In one embodiment of the method of the present invention, the anionic metal affinity ligand may be selected from the group consisting of aminodicarboxylic acids and aminotricarboxylic acids.

[0025] In another embodiment of the method of the present invention, the anionic metal affinity ligand may be iminodiacetic acid (IDA) or hyponitroacetic acid (NTA).

[0026] In another embodiment of the method of the present invention, the matrix with anionic metal affinity ligands can be in the form of particles, nanowires, porous membranes, bulk materials, hydrogels, depth filtration media, or soluble polymer media. In particular, the matrix with anionic metal affinity ligands can be in the form of a flow chromatography device.

[0027] In another embodiment of the method of the present invention, the matrix and / or the sample can be equilibrated using a buffer solution having a pH range of 7.0 to 9.5, 7.0 to 9.0, 7.5 to 9.0, or 8.0 to 9.0.

[0028] In another embodiment of the method of the present invention, the equilibration of the matrix and / or the sample can be performed using a buffer solution having a salt concentration range of up to 1 M, or 50 mM to 750 mM, or 100 mM to 500 mM, or 125 mM to 250 mM.

[0029] In one embodiment of the method of the present invention, the buffer solution can be adjusted by a salt that does not form a chemical complex with the anionic metal affinity matrix loaded with the metal, the salt being selected from: inorganic salts, such as sodium chloride, potassium chloride, sodium acetate, potassium acetate, etc.; organic salts, such as arginine-HCl, lysine-HCl, or salts based on imidazole, histidyl, or histamine cations; and ionizing salts, such as those containing guanidine cations or thiocyanate anions, or both; and combinations thereof. Those skilled in the art can readily adjust the salt conditions in a manner that prevents viral or extracellular vesicle binding but allows DNA binding.

[0030] In another embodiment of the method of the present invention, the anionic metal affinity matrix for loading the metal can be loaded with metal ions having at least two positive charges, preferably selected from the group consisting of: calcium, magnesium, copper, iron, manganese, zinc, barium, nickel, cobalt and combinations thereof.

[0031] In another embodiment of the method of the present invention, the virus and extracellular vesicle samples may comprise cell harvest, cell lysate, or entities selected from the group consisting of non-lipid-enveloped protein-capsulated virus particles, such as AAV capsids; lipid-enveloped viruses or virus-like particles, such as influenza viruses or coronaviruses; bacteriophages, extracellular vesicles, such as exosomes; and combinations thereof. Specifically, the AAV capsid is selected from the group consisting of: AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11; recombinant hybrid serotypes such as AAV2 / 8 or AAV2 / 9; synthetic recombinant serotypes; and combinations thereof.

[0032] In one embodiment of the method of the present invention, the sample may be treated before or after the method for removing contaminating DNA by bioaffinity chromatography, cation exchange after metal affinity chromatography, hydrophobic interaction chromatography, and / or tangential flow filtration. Bioaffinity chromatography refers to chromatography using ligands that have affinity for molecules or molecular structures, typically with protein properties, such as antibodies, antibody fragments like Fc or Fab fragments; lectins, protein A. However, other ligands are also known to those skilled in the art, such as biotin / avidin.

[0033] In another embodiment of the method of the present invention, the tangential flow filtration can use membranes with a pore size cutoff range of up to 1 MDa, particularly 200 kDa to 700 kDa.

[0034] In a preferred embodiment, the sample containing the desired substance is a cell harvest or cell lysate. In this embodiment, the method of the present invention is applied directly to the clarified or clarified sample after harvesting or lysis. This clarification may include a filtration step. In this embodiment, no ion exchange chromatography step is used prior to the method of the present invention. Preferably, no chromatography step is used prior to the method of the present invention.

[0035] In a preferred embodiment, the pH and salt concentration used in the method of the present invention prevent the desired substance from binding to the anionic metal affinity matrix of the loaded metal.

[0036] In another embodiment of the method of the present invention, the sample with reduced DNA content is further reduced by anion exchange chromatography to further reduce the level of contaminating DNA.

[0037] Another aspect of the invention is the use of a matrix with anionic metal affinity ligands in a method for removing contaminating DNA from viral and extracellular vesicle samples under alkaline conditions.

[0038] The practicality of this method stems from a series of unexpected findings. First, viruses and vesicles have a natural tendency to bind to a matrix containing anionic metal-affinity ligands that complex with certain metals, even if they lack genetic modifications (e.g., His tags) that mediate metal-affinity binding. Their natural metal affinity leads to partial binding to the metal-affinity matrix, resulting in the loss of the binding product. It has been found that this binding can decrease with alkaline pH. This is surprising because metal-affinity methods are known to elute IgG and His-tag proteins with acidic pH, not the other way around—that is, increasing pH can induce elution or prevent binding. It was expected that increasing pH would maintain or increase binding.

[0039] Even more surprisingly, an inverse relationship was found between pH and non-chelating salt concentration, where increasing the amount of non-chelating salt overcomes the stronger binding of viruses to extracellular vesicles at neutral and acidic conditions. This is unexpected because it is well known in the field of immobilized metal affinity chromatography (IMAC) that salts that do not form chemical complexes with the metal-loaded anionic metal affinity matrix (non-chelating salts) generally do not affect metal affinity binding. There are no known examples in the art of eluting proteins bound by metal affinity by increasing the concentration of non-chelating salts. Even more unexpected is the inverse relationship between pH and salt concentration.

[0040] Surprisingly, however, the discovery that conditions weakening viral and extracellular vesicle binding allowed for host cell DNA binding, despite existing techniques indicating poor DNA binding [5,6]. RNA is known to bind immobilized metals, but only copper, nickel, zinc, and cobalt, in descending order of strength. This method provides the strongest DNA binding with iron and manganese, but also applies to magnesium, calcium, and barium, further emphasizing that this mechanism differs from known methods of binding nucleic acids to immobilized metal matrices.

[0041] The mechanism by which DNA binding occurs remains unknown. Since DNA does not denature into its constituent single strands upon heating or sodium hydroxide, it appears that binding cannot occur through its nitrogen bases. This leaves the possibility that DNA binds to metals through its phosphate residues. However, this highlights a drawback, as the same metal is expected to bind to phosphate residues on both viruses and vesicles. Under the specific conditions under which this method was practiced, neither viruses nor vesicles bound.

[0042] These surprising findings collectively lay the foundation for new methods for removing host DNA, particularly from preparations containing viruses and extracellular vesicles. Experimental results demonstrate that this method somehow "threads the needle," with binding conditions sufficient to reduce the metal's affinity for proteins, viruses, and extracellular vesicles while preserving the metal's ability to bind DNA to the matrix. Subsequently, DNA-deficient post-processed samples were applied to anion exchangers and subjected to gradient elution to release the desired products while still retaining the DNA.

[0043] In summary, the method consists of a series of steps: a matrix with anionic metal affinity ligands is loaded with metal ions. The substrate is equilibrated to a combination of pH and salt conditions to prevent the binding of desired proteins, viruses, or extracellular vesicles, but allow DNA binding. A sample contaminated with DNA and containing the desired proteins, viruses, or extracellular vesicles is equilibrated to the same conditions and contacted with the metal-loaded anionic metal affinity matrix. The matrix is ​​then separated from the sample, leaving the sample lacking DNA but still containing most of the desired proteins, viruses, or extracellular vesicles. The metal affinity-treated sample is then treated with anion exchange chromatography to further reduce the level of contaminating DNA.

[0044] The application of this method in preparations containing adeno-associated virus (AAV) and host cell DNA is described in a non-limiting example:

[0045] - A solid phase with anionic metal affinity ligands is supported by a metal, such as iron.

[0046] The solid phase is equilibrated to a pH of approximately 9. This is considered highly unusual in the field of metal affinity chromatography, as sample applications are typically performed at neutral pH.

[0047] - Equilibrate the cell lysate containing host cell DNA and AAV to a pH of approximately 9.

[0048] - The sample is brought into contact with the metal-affinity solid phase. Most AAVs do not bind. Host cell DNA is bound.

[0049] - The anionic metal affinity solid phase is separated from the sample, allowing the DNA to bind to the solid.

[0050] - The AAV is separated by anion exchange chromatography to further reduce the amount of host cell DNA.

[0051] In another non-limiting example, the conditions and steps of the preceding example are repeated by separating the anionic metal affinity matrix from the sample. Thereafter:

[0052] - The sample was processed by tangential flow filtration using a membrane with a pore size cutoff of 300 kDa to concentrate the AAV and reduce protein contamination.

[0053] - The AAV is separated by anion exchange chromatography to further reduce the amount of host cell DNA.

[0054] In another non-limiting example, the conditions and steps of the first example were repeated by separating the anionic metal affinity matrix from the sample. Thereafter:

[0055] - The sample was subjected to affinity chromatography.

[0056] - The AAV is separated by anion exchange chromatography to further reduce the amount of host cell DNA.

[0057] The method of the present invention is also applicable to proteins, including antibodies, in which it may prove superior to known methods that utilize other chemical mechanisms to reduce the amount of host cell DNA.

[0058] The method of the present invention is in Figure 1 The method is described in the text. Further details and variations illustrating the full scope of this method are provided in the following sections. Brief description of the attached diagram

[0060] Figure 1 A diagram depicting the method of the present invention is provided.

[0061] Figure 2 The study described how the AAV capsid does not bind to magnesium-loaded anionic metal affinity ligands at pH 9.0, while DNA binding is strong and requires elution with sodium hydroxide.

[0062] Figure 3The binding of the AAV capsid to magnesium-loaded anionic metal affinity ligands was compared in independent experiments at pH 7.0 and pH 9.0. The curves were plotted at 280 nm.

[0063] Figure 4 The method described involves removing DNA and separating empty and intact AAV capsids by eluting with a quaternary ammonium anion exchanger using a salt gradient.

[0064] Figure 5 The removal of DNA and separation of empty and intact AAV capsids by eluting with a primary amine anion exchanger using a pH gradient is described.

[0065] Figure 6 Size exclusion chromatography was used to depict cell cultures containing extracellular vesicles, including exosomes.

[0066] Figure 7 Size exclusion chromatography of cell cultures containing extracellular vesicles after DNA removal by the method of the present invention is described.

[0067] Figure 8 Depicting Figure 7 and Figure 8 The size exclusion elution curves are overlapped to highlight the reduction of common contaminants, particularly DNA.

[0068] Figure 9 The removal of DNA and the separation of partially purified extracellular vesicles by elution with a quaternary ammonium anion exchanger using a salt gradient are described.

[0069] Figure 10 The flow of bacteriophage T4 was described as a monolith loaded with ferric iminodiacetic acid.

[0070] Figure 11 The secondary removal of DNA and the separation of bacteriophage T4 by chromatography using a salt gradient elution of a quaternary ammonium anion exchanger are described.

[0071] Figure 12 The secondary removal of DNA by chromatography and the separation of bacteriophage T4 by eluting primary amine anion exchangers with salt gradients are described. Invention Details

[0073] The sample comprises a preparation containing desired types of proteins, viral particles, or extracellular vesicles generated from cell culture, and also contains host cell-derived DNA. In one embodiment, the sample comprises a cell culture harvest. In one such embodiment, the cell culture harvest contains antibodies. In another such embodiment, the cell culture harvest contains viruses or virus-like particles. In another such embodiment, the cell culture harvest contains extracellular vesicles. In another such embodiment, the sample comprises cell lysates. In another such embodiment, the sample comprises a cell culture harvest or cell lysate that has been treated with nucleases to reduce the host cell DNA content. In another such embodiment, the sample comprises a partially purified preparation that still contains more host cell DNA than expected or permitted in the final product. In one such embodiment, the sample is a product fraction eluted from a chromatographic apparatus. In one such embodiment, the sample is a product eluted from an affinity chromatography column. In another such embodiment, the sample is an elution product from a size exclusion chromatography column. In another such embodiment, the sample is a product eluted from a hydrophobic interaction chromatography column. In another such embodiment, the sample is an elution product from a cation exchange column. In another such embodiment, the sample is an elution product from an immobilized metal affinity column. In another such embodiment, the sample is an elution product from an apatite column. In yet another such embodiment, the sample is a concentrated and / or percolated product from tangential flow filtration.

[0074] In one embodiment, the method of the present invention is used to treat a sample containing desired non-lipid envelope protein-capsid virus particles contaminated with host cell DNA. AAV has multiple serotypes. In one embodiment, the desired AAV serotype treated by the method of the present invention may be AAV1, or AAV2, or AAV3, or AAV4, or AAV5, or AAV6, or AAV7, or AAV8, or AAV9, or AAV10, or AAV11, or another serotype. In another embodiment, the AAV serotype treated by the method of the present invention may be a recombinant hybrid serotype, such as AAV2 / 8, or AAV2 / 9, or another hybrid serotype. In yet another embodiment, the AAV serotype treated by the method of the present invention may be a synthetic recombinant serotype. In any of these embodiments, the anion exchange step may be performed to separate the empty capsid from the intact capsid, while further reducing the amount of contaminating DNA. In one such embodiment, the anion exchanger is a strong anion exchanger (quaternary ammonium) eluted with salt. In another such embodiment, the anion exchanger is a weak anion exchanger (primary amine) eluted with an ascending pH gradient.

[0075] In another embodiment, the sample contains desired lipid-enveloped virus or virus-like particles contaminated with host cell DNA. In one such embodiment, the anion exchange step can separate non-infectious viral particles from infectious viral particles. In one such embodiment, the virus is an influenza virus. In another such embodiment, the virus is a coronavirus.

[0076] In another embodiment, the sample contains the desired phage contaminated with host cell DNA.

[0077] In another embodiment, the method of the present invention is used to process a sample containing extracellular vesicles contaminated with host cell DNA. In one such embodiment, the extracellular vesicles are exosomes contaminated with host cell DNA.

[0078] In one embodiment of the method of the present invention, the sample may have been previously partially purified, including by methods that reduce the amount of host cell DNA.

[0079] Anionic metal affinity matrices suitable for carrying out the methods of the present invention comprise immobilized amino-carboxylic acids. In one embodiment, the immobilized amino-carboxylic acid may be a dicarboxylic acid, such as iminodiacetic acid (IDA). In another embodiment, the amino-carboxylic acid may be a immobilized tricarboxylic acid, such as hypozinoacetic acid (NTA). In yet another embodiment, a mixture of IDA and NTA matrices may be used. Anionic metal affinity matrices are commercially available in a variety of physical forms and can be synthesized in any desired form. They may be particles, insoluble nanowires, porous membranes, bulk materials, hydrogels, depth filtration media, soluble polymer media, or other forms. In many cases, such matrices are provided in the form of flow chromatography devices to facilitate their use.

[0080] In some implementations, the choice of anionic metal affinity ligands can help prevent the retention of desired proteins, viruses, or extracellular vesicle products. In some such implementations, NTA may be superior to IDA because NTA carries three negative charges, while IDA carries only two. Complexes of divalent metal cations with IDA will produce a net charge of zero via the ligand-metal complex, but complexes of divalent metal cations with NTA will produce a net charge of -1 (1-), which may hinder the binding of the desired product. Complexes of trivalent metal cations with IDA will produce a net charge of +1 (1+) via the ligand-metal complex, which may endow the complex with anion exchange properties, favoring the binding of desired proteins, viruses, or extracellular vesicles. Complexes of trivalent metal cations with NTA will produce a net charge of zero, which does not endow the complex with significant anion exchange capacity. It is recommended that IDA and NTA be evaluated for routine application of this method.

[0081] Those skilled in the art will understand that protein-capped viruses without lipid envelopes tend to be robust and generally tolerate pH 9 over a wide range of salt concentrations. This will simplify carrying out one or two steps of the method of the present invention at a pH of approximately 9. It will also be understood that lipid-enveloped viruses, virus-like particles, bacteriophages, and extracellular vesicles are less stable and may require pH adjustment to maintain product stability. In one such embodiment, the metal affinity step can be carried out at a pH of approximately 8 and a sodium chloride concentration of approximately 250 mM. Less tolerant species may require lowering the pH to slightly above neutral and to salt concentrations close to 100 mM. More robust species can tolerate pH 8.75 and salt concentrations up to 375 mM or higher. Generally, the lowest salt concentration required to prevent the target product from binding to the product in the metal affinity step will be advantageous, as it will minimize the amount of the treated sample that must be diluted to bind with the anion exchanger in the final step of the method. Furthermore, generally, the closer the pH is to neutral, the more likely unstable products, such as those with lipid membranes, are to be tolerated.

[0082] Depending on the required stability of the protein, virus, or extracellular vesicle, the pH of the buffer solution can be in the range of pH 4.0 to pH 10.0, or 5.0 to 9.5, or 6.0 to 9.0, or 6.5 to 8.5, or 7.0 to 8.0, or 6.5 to 7.5, or different or narrower ranges. Those skilled in the art will recognize that some buffers can interact with metals

[10] and can be used to modulate the performance of the methods of the present invention.

[0083] In some embodiments, a salt (non-chelating salt) that does not form a chemical complex with the anionic metal affinity matrix of the loaded metal may be present at concentrations ranging from 0.1 mM to 1.0 M, or 50 mM to 750 mM, or 100 mM to 500 mM, or 125 mM to 250 mM. In some embodiments, the presence of the salt may help stabilize viral particles or extracellular vesicles. In some embodiments, extracellular vesicles or lipid-enveloped viruses should be transiently exposed to salt concentrations greater than 500 mM to minimize damage to the product. In some embodiments, the metal affinity step of the present invention binds chromatin even at salt concentrations of 1 M, or 2 M, or 3 M, or 4 M, or 5 M, or at saturation concentrations of non-chelating salts. It will be appreciated that such high concentrations are rarely or never beneficial to the overall implementation of the method of the present invention, especially when the desired product is unstable under such conditions, but such conditions will still support the selective removal of chromatin.

[0084] In some embodiments, the salt (non-chelating salt) used to maintain product stability and not form a chemical complex with the anionic metal affinity matrix of the supported metal can be an inorganic salt, such as sodium chloride, or potassium chloride, or sodium acetate, or potassium acetate, or another salt.

[0085] In closely related embodiments, the non-chelating salt can be an organic salt, such as arginine-HCl, lysine-HCl, or a salt based on imidazole, histidine, or histamine cations.

[0086] In another closely related embodiment, the non-chelating salt may be a liquid-free salt comprising a guanidine salt cation or a thiocyanate anion, or both, or other liquid-free ions. However, those skilled in the art will recognize that the use of such salts will be limited to proteins and protein-protein capsid viruses, as such salts may disrupt products with lipid membranes.

[0087] Anions with strong metal-binding capabilities often remove metals bound to solid-phase anionic ligands and impair the ability of anionic metal affinity matrix to bind chromatin. Known anions with strong metal-binding capabilities include citrate, phosphate, pyrophosphate, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), aspartic acid, glutamic acid, and glutamine.

[0088] Suitable polyvalent metal cations for implementing this method include, in particular, ferric and manganese. Copper, zinc, magnesium, calcium, and barium can also be used. Heavy metal ions such as nickel and cobalt can also mediate DNA reduction, but their use is discouraged due to their toxicity.

[0089] In some implementations, the choice of metal ions can also help to avoid retaining desired viral or extracellular vesicle products. Metal ions with significantly high affinity for phosphate residues tend to bind all phosphorylated substances more strongly compared to metal ions with weaker phosphate affinity. Metal ions with high affinity for phosphate residues particularly include ferric iron and manganese. Experimental data indicate that metals such as calcium and magnesium have lower affinity for phosphates. Metals such as divalent copper mediate moderate affinity for phosphate groups. In implementations where the desired protein, viral, or vesicle species exhibits inherently low affinity for metal ions bound to anionic metal-affinity solids, the use of iron or manganese may be advantageous for maximizing chromatin binding. In implementations where the desired protein, viral, or vesicle species are highly phosphorylated, the use of copper, calcium, or magnesium may be beneficial for maximizing the recovery of the desired product.

[0090] Many types of anion exchangers are commercially available worldwide. In one such embodiment, the anion exchanger is a quaternary ammonium anion exchanger, also known as a strong anion exchanger. In another such embodiment, the anion exchanger is a tertiary anion exchanger, also known as a weak anion exchanger. Anion exchangers can also use primary amines, secondary amines, and combinations of primary, secondary, tertiary, and quaternary amines. One such material is N,N-bis(2-aminoethyl)-1,2-ethylenediamine, commonly referred to as TREN. Other mixed-component anion exchangers employ ligands composed of polyallylamine, polyethyleneimine, and ethylenediamine, etc. Anion exchangers suitable for carrying out this method are also understood to include positively charged amine derivatives that include additional residues to impart excess hydrophobicity, hydrogen bonding, or both. Anion exchangers that include additional residues to impart excess hydrophobicity and / or hydrogen bonding are generally referred to as multimode or mixed-mode exchangers. Throughout this specification, all the aforementioned materials are referred to as anion exchangers. All of these are commercially available worldwide in various physical forms, including particles, insoluble nanowires, porous membranes, bulk materials, hydrogels, depth filtration media, or other forms. In many cases, they are provided as flow chromatography or filtration devices for ease of use. In one embodiment, both the metal affinity matrix and the anion exchanger are in the form of chromatographic devices, initially plumbed in sequence with the metal affinity device. In one such embodiment, they are equilibrated, loaded, washed, eluted, and cleaned in series. In another such embodiment, they are equilibrated, loaded, washed, and then the metal affinity is removed from the flow solution, and the anion exchanger is eluted independently. Those skilled in the art will recognize that sequential vertical setups can be attractive to industrial users because they significantly reduce the amount of water, buffers and salts, buffer preparation, processing time, equipment, and personnel required to process the desired product. All these benefits contribute to increased facility capacity, ultimately translating to increased productivity at a lower cost.

[0091] In some embodiments, the metal-affinity-treated sample can be applied to the anion exchanger without concern for residual free metal ions in the sample, as these positively charged metal ions will be repelled by the surface of the anion exchanger and eliminated during sample application. In other embodiments, metal ions may be intentionally added to the sample and the anion exchange buffer. In such embodiments, their presence can beneficially alter the surface charge or morphology of the virus or vesicle. In one such embodiment, the presence of calcium and / or magnesium ions in the buffer helps improve the separation of empty AAV capsids from intact AAV capsids.

[0092] In some embodiments, anion exchange chromatography is performed directly after the metal affinity step. In one embodiment, the metal affinity chromatography and anion exchange chromatography steps are performed in an uninterrupted sequence, with both media used in the form of a chromatography apparatus or a filtration apparatus. In another embodiment, the metal affinity chromatography and anion exchange chromatography steps are performed in an uninterrupted sequence, wherein the metal affinity step is performed by adding an insoluble metal affinity matrix to the sample, allowing it to bind and co-precipitate the DNA, which is then removed to apply the DNA-deficient supernatant to the anion exchanger. In yet another embodiment, the metal affinity chromatography and anion exchange chromatography steps are performed in an uninterrupted sequence, wherein the metal affinity step is performed by adding a soluble polymer matrix containing the metal affinity ligand to the sample, causing it to crosslink and precipitate chromatin, followed by removal of the precipitate by centrifugation and / or filtration to obtain a DNA-deficient supernatant, which is then processed by anion exchange chromatography.

[0093] In one related embodiment, at a salt concentration of up to 400 mM sodium chloride, the metal affinity chromatography matrix can be added to the sample along with positively charged particles or polymers. These particles or polymers will co-crosslink with the DNA associated with the metal affinity chromatography medium and further promote DNA reduction. Simultaneously, the increased salt concentration inhibits the binding of desired proteins, viruses, or vesicles to the matrix or positively charged matrix of the present invention. Generally, the salt concentration does not need to be higher than that necessary to prevent the binding of desired proteins, viruses, or vesicles to the matrix or positively charged matrix of the present invention. After treatment, if necessary, the salt concentration can be reduced to allow the sample to be processed by anion exchange chromatography.

[0094] In another closely related embodiment, the method of the present invention can be combined with fatty acid therapy. In some such embodiments, the fatty acid can be heptanoic acid, octanoic acid, or nonanoic acid at a concentration in the range of 0.01% to 1.0% and a pH in the range of 4 to 6. In some such embodiments, the fatty acid can coexist with particles or polymers carrying an anionic metal affinity matrix of the loaded metal. In some such embodiments, the fatty acid treatment can be performed before or after the method of the present invention. It will be apparent to those skilled in the art that treatment including fatty acids will not be suitable for viruses and extracellular vesicles with lipid membranes.

[0095] In another closely related embodiment, the method of the present invention can be combined with allantoin treatment. In some such embodiments, the allantoin may be present in an amount ranging from 2% to 10%. It will be apparent to those skilled in the art that treatments including allantoin are not suitable for certain viruses and extracellular vesicles.

[0096] In another embodiment, one or more additional processing steps may be inserted after the metal affinity step and before the anion exchange chromatography step.

[0097] In one such embodiment, the sample is processed after metal affinity chromatography. In one such embodiment, the affinity ligand is a biological ligand specific to one or more AAV serotypes. In another such embodiment, the affinity ligand is an antibody-specific biological ligand. In one such embodiment, the affinity ligand is protein A or a variant thereof.

[0098] In one relevant embodiment, the sample is treated by cation exchange chromatography followed by anion exchange chromatography after the metal affinity step. In one such embodiment, the cation exchanger is used to capture AAV. In another embodiment, the cation exchanger is used to capture antibodies.

[0099] In another related embodiment, the sample is treated by hydrophobic interaction chromatography after metal affinity chromatography, and then by anion exchange chromatography.

[0100] In another related embodiment, the sample is treated with anion-immobilized metal affinity chromatography after the anion-metal affinity DNA removal step, followed by anion exchange chromatography. In one such embodiment, this variant is applied to biomolecules naturally containing histidine clusters or produced by recombinant gene constructs to have artificial histidine clusters, tails, or tags. In one such embodiment, IgG, which naturally contains histidine clusters in the hinge region of the desired product. In one such embodiment, the anion-metal affinity ligand used in the DNA removal step is ferric and the anion-metal affinity ligand used in the subsequent purification step is nickel. This step binds IgG. The IgG can be eluted by competition with imidazole, by lowering the pH, or by both. The eluted IgG is then purified by anion exchange chromatography. In one such embodiment, the anion exchanger is a multimode anion exchanger. In one variant of the above embodiment, ferric iron is replaced by manganese. In another variant of the above embodiment, nickel is replaced by copper, zinc, or cobalt. In another variation of the above embodiment, the two metal affinity steps are performed using a pair of vertically (plumbed) columns, the first being an IDA column loaded with ferric iron and the second an IDA column loaded with zinc. In one such embodiment, the filtered cell culture harvest containing IgG monoclonal antibodies is passed through two columns, the first removing DNA and the second capturing IgG. The columns are washed, and then IgG is eluted from the second column using a buffer, while DNA remains bound to the first column until it is later removed with NaOH. In another variation of this method, the product of interest is not an antibody, but a His-labeled protein, or a His-labeled exosome, or a His-labeled viral particle.

[0101] In another relevant embodiment, the sample is treated by tangential flow filtration after metal affinity chromatography and before anion exchange chromatography. Since viral particles and extracellular vesicles are characterized by large, complex assemblages, typically ranging in size from 20 nm to over 200 nm, many such embodiments will benefit from the concentrated processing of tangential flow filtration, which retains the product of interest through the largest pore size. In some such embodiments, this would involve a TFF membrane with a pore size cutoff value in the range of 200 kDa to 700 kDa, and in some cases, a very large pore size class, such as 1 MDa. This filter allows smaller contaminants to be removed through the membrane pores. In one such embodiment, magnesium-loaded metal affinity particles or polymers are mixed with lysates of cell culture harvest at an alkaline pH to bind the DNA. The solids are then removed by centrifugation and / or membrane filtration, and the clarified supernatant is concentrated and / or percolated to concentrate and / or buffer-exchange the sample to prepare it for anion exchange chromatography. In relevant embodiments where the desired product is an IgG antibody, the pore size cutoff value of the membrane may be 30-50 kDa. In one such embodiment, the subsequent anion exchange chromatography step is performed using a multimode anion exchanger. In a closely related embodiment, where the desired product is an IgM antibody, the pore size cutoff of the membrane can be 30-100 kDa, and the subsequent anion exchange chromatography step is performed using a strong anion exchanger such as a quaternary ammonium anion exchanger.

[0102] In one embodiment, the method of the present invention is used to treat adeno-associated virus, wherein the anion exchanger performs the additional function of separating empty capsids from intact capsids. The term "intact capsid" is understood to refer to a capsid containing the intended payload of therapeutic plasmid DNA. The term "empty capsid" is understood to refer to a capsid lacking intact therapeutic plasmid DNA. In one such embodiment, the anion exchanger is a strong anion exchanger eluted with an increasing salt gradient. In another such embodiment, the anion exchanger is a primary amine anion exchanger eluted with an increasing pH gradient. In yet another such embodiment, the anion exchanger is a mixed amino anion exchanger. In one such embodiment, the anion exchanger is TREN.

[0103] In one embodiment, the method of the present invention is used to process extracellular vesicles, including exosomes. In one such embodiment, the anion exchanger is a strong anion exchanger eluted with an increasing salt gradient. In another such embodiment, the anion exchanger is a tertiary amine (weak) anion exchanger eluted with a salt gradient.

[0104] In one embodiment, the method of the present invention is used instead of treating a sample with a nuclease to reduce DNA content. In another embodiment, the method of the present invention is used to increase the degree of DNA reduction achieved by treatment with a nuclease. In one such embodiment, the metal affinity step of the present invention is performed before treatment with a nuclease. In another such embodiment, the method of the present invention is performed after treatment with a nuclease, wherein it provides additional utility by binding the nuclease through its associated metal ion cofactor. In one such embodiment, the anionic metal affinity ligand is loaded with the same metal ion species used as a cofactor for the nuclease. In one such embodiment, the metal ion is magnesium. In another such embodiment, the metal ion is calcium. In one embodiment, wherein the metal affinity step is performed concurrently with nuclease treatment of the sample, the metal ion used to load the affinity matrix is ​​different from the metal ion species of the enzyme cofactor. For example, if the enzyme cofactor is magnesium, the metal affinity ligand may be loaded with ferric iron, such that the metal affinity matrix does not bind to the enzyme during DNA cleavage. In any of the foregoing embodiments, the metal affinity ligand may be one of a variety of ligands covalently fixed to a variety of soluble polymers. In another such implementation, the metal affinity ligand can be covalently immobilized onto multiple insoluble solid particles.

[0105] In one embodiment, the metal-affinity DNA reduction step is performed using loose particles or soluble polymers with ligand-metal complexes, which can form precipitates and co-precipitates. These solids can be removed before further processing of the supernatant containing the desired viral or vesicle species. In one embodiment, they can be removed by membrane filtration, centrifugation, or a combination of both. After solid removal, the sample can be treated by tangential flow filtration (TFF). In one such embodiment, TFF is performed using a membrane with the largest possible pore size, which retains the virus or vesicle of interest while allowing smaller contaminants to pass through and be eliminated. In one such embodiment, the pore size cutoff level can be 100 kDa, or 300 kDa, or 500 kDa, or 700 kDa, or 1 MDa, or a larger or medium molecular weight cutoff (MWCO). In most or all of the foregoing embodiments, the TFF step is particularly effective in eliminating histones released via cleavage associated with the host cell DNA. In any of the foregoing embodiments, the TFF step can also be used to concentrate the sample and / or percolate the sample into a buffer suitable for performing the chromatography step.

[0106] In any of the foregoing embodiments, treating the sample via the metal affinity step can remove large aggregates and cellular debris to some extent, making the sample more filterable and easier to process by TFF or chromatography. In one such embodiment, TFF can be used to concentrate the sample and percolate it to conditions for enzymatic digestion by nucleases to further reduce DNA levels. In one embodiment, the sample treated by metal affinity chromatography can be further treated with TFF to remove histones before being processed by anion exchange chromatography or an intermediate chromatography step prior to anion exchange chromatography.

[0107] In some embodiments, secondary additives may be included in product preparation to inhibit nonspecific interactions between the desired product and the processed surface or to stabilize the desired product. Such additives may include nonionic or zwitterionic surfactants, such as octagoglucoside, poloxamer 188, Pluronic F68, CHAPS, or CHAPSO. These stabilizing compounds may alternatively or additionally include sugars, such as sucrose, sorbitol, xylose, mannitol, or trehalose. Such stabilizing compounds may alternatively or additionally include amino acids such as betaine, taurine, arginine, histidine, or lysine. All of these agents are known in the biopharmaceutical field because they tend to improve solubility and / or stabilize product recovery. In some cases, they also facilitate the separation of the desired product from unwanted substances.

[0108] It will be recognized that both steps of the method of the present invention have the potential to remove other phosphorylated contaminants, which are byproducts of chromatin removal. Other phosphorylated contaminants may include RNA, endotoxins, phosphoproteins, and phospholipids. Example

[0109] Example 1

[0110] Host cell DNA in adeno-associated virus (AAV) preparations was removed in advance by anionic metal affinity chromatography containing magnesium.

[0111] Magnesium was loaded onto the bulk sample containing iminodiacetic acid (IDA) chelating residues and equilibrated to pH 9.0. A cation-exchange purified capsid sample was equilibrated to the same conditions and loaded onto the column. The AAV capsid passed through the column without binding. Host cell DNA bound and was subsequently removed with 1M NaOH. Results are as follows. Figure 2 As shown.

[0112] Example 2

[0113] Host cell DNA was removed from AAV preparations in advance using magnesium-containing anion metal affinity chromatography.

[0114] Magnesium was loaded onto the monolithic feed containing IDA chelating residues and equilibrated to pH 7.0. The cation-exchange purified capsid sample was equilibrated to the same conditions and loaded onto the column. The AAV capsid binds to the DNA moiety (…). Figure 3 and Figure 2 (Comparison). Although the AAV capsids partially bound at pH 7.0, the results showed that they eluted at approximately 250 mM NaCl. This means that including this amount of salt in the equilibrated sample and column would prevent their binding. Figure 4 The separation of empty and intact AAV capsids was demonstrated, while DNA was removed by anion exchange chromatography with a sodium chloride gradient elution using a strong (quaternary ammonium) anion exchanger. Figure 5 This demonstrates the separation of empty and intact AAV capsids, while simultaneously removing DNA by eluting a weak (primary amine) anion exchanger using an anion exchange chromatography process with a pH gradient.

[0115] Example 3

[0116] DNA removal in advance from extracellular vesicle preparations

[0117] The bulk material containing IDA chelate residues was loaded with ferric iron and equilibrated with 50 mM Hepes, 50 mM NaCl, and pH 7.0. Clarified mammalian cell culture harvested fluid was then circulated through the bulk material. Figure 6 The analytical size exclusion chromatography (SEC) curve of the sample prior to application to the IDA-Fe bulk material is shown. Note the excess UV absorption at 260 nm from approximately 10 min to approximately 23 min. This indicates the presence of nucleic acids and corresponds to regions where chromatin would normally be eluted. Figure 7 The analytical size exclusion chromatograms of the sample after application to the IDA-Fe bulk material are shown. Note that excess absorption was eliminated at 260 nm, and the UV signal generally decreased after 10–23 minutes. This is consistent with chromatin removal. Figure 8The results before and after analytical size exclusion chromatography (SUSLC) are illustrated by multi-angle light scattering (MALS, LS) and immunofluorescence (IFL) monitoring. Light scattering selectively amplifies the optical detection of large solutes, such as extracellular vesicles, including exosomes, microvesicles, apoptotic bodies, chromatin, and cell debris. Immunofluorescence (IFL) is performed in conjunction with SEC by adding a fluorescently labeled antibody to the sample prior to chromatography and then monitoring the run using a fluorescence detector. It detects only solutes carrying the specific immunolabel targeted by the antibody. In this experiment, the antibody targets CD63, a known signature marker for exosomes. The MALS and IFL signal intensities of the “post-treatment” sample were up-adjusted by 5-fold to compensate for the 5-fold sample dilution during the early treatment phase of the present invention. It is noted that the IFL / MALS ratio increases with treatment. This indicates that the metal affinity step of the method selectively removes sample components lacking the exosome label, resulting in a more enriched exosome fraction. This is indicated by elution from 10–12 minutes and… Figure 6 Most of the solutes detected by MALS are related to chromatin. Figure 9 The processing procedure for a partially purified extracellular vesicle preparation is described. This preparation was loaded onto a strong anion exchanger (quaternary ammonium), equilibrated to 50 mM Hepes, 50 mM NaCl, pH 7.0, and then eluted with a linear gradient to 2 M NaCl before washing with 1 M NaOH. Extracellular vesicles primarily eluted in NaCl solutions smaller than 1 M. Chromatin primarily required NaOH for elution.

[0118] Example 4

[0119] Pre-removal of host DNA from phage T4 preparation

[0120] The bulk material containing IDA chelating residues is loaded with ferric iron and equilibrated to pH 7.0. Filtered cell culture harvest is then passed through the bulk material to remove DNA. Figure 10 The elution curves are shown. The bacteriophages flowed through the feedstock. Some contaminants were bound and eluted with NaCl. DNA was then removed with 1M NaOH. Figure 11 Fine purification by anion exchange chromatography on a strong anion exchanger (quaternary ammonium) with a sodium chloride gradient at pH 7 is shown. Figure 12 Fine purification was demonstrated by anion exchange chromatography over a weak anion exchanger (primary amine) using a sodium chloride gradient at pH 7.

[0121] Example 5

[0122] Removal of host DNA from preparations containing IgG monoclonal antibodies

[0123] The IDA-Fe matrix is ​​loaded with ferric iron, and excess iron is washed away with 1M NaCl. The IDA-Fe matrix is ​​washed with water to remove excess salt. Filtered cell culture harvest containing IgG monoclonal antibodies is passed through the matrix under near-physiological conditions. Physiological conditions are understood to include a pH of approximately 6.5 to 7.5 and a salt concentration corresponding to a conductivity of 50–200 mS / cm. The antibodies flow through. Chromatin is bound. The matrix is ​​washed to recover all of the antibodies. The antibodies are then treated with multimodal anion exchange chromatography to further reduce DNA content.

[0124] Example 6

[0125] Purification of IgG monoclonal antibodies

[0126] The method of Example 5 was repeated, except that a TFF step was inserted after metal affinity removal of chromatin. TFF was performed using a membrane with a molecular weight cutoff (MWCO) of 30 kDa to retain the IgG while reducing the levels of lower molecular weight proteins and low molecular weight contaminants prior to the anion exchange chromatography step. In a variation of this process, metal affinity removal of DNA can be performed on a large volume of the harvest using IDA-Fe particles instead of the flow chromatography apparatus described in Example 5.

[0127] Example 7

[0128] Removal of host DNA from preparations containing IgM monoclonal antibodies

[0129] The IDA substrate is loaded with ferric iron, and excess iron is washed away with 1M NaCl. The IDA-Fe matrix is ​​washed with water to remove excess salt. Filtered cell culture harvest containing IgM monoclonal antibodies is passed through the substrate under near-physiological conditions. The antibodies flow through. Chromatin is bound. The substrate is rinsed to recover all the antibodies. The antibodies are then treated with a strong anion exchanger and eluted with a salt gradient to further reduce DNA content.

[0130] Example 8

[0131] Purification of IgM monoclonal antibodies

[0132] The method of Example 7 was repeated, except that a TFF step was inserted after metal affinity removal of chromatin. TFF was performed using a membrane with an MWCO of 100 kDa to retain the IgM while reducing the levels of lower molecular weight proteins and low molecular weight contaminants prior to the anion exchange chromatography step. In a variation of this process, metal affinity removal of DNA can be performed on large-volume harvests using IDA-Fe particles instead of the flow chromatography apparatus described in Example 7.

[0133] Reference List

[0134] All references cited in this article are incorporated by way of citation, provided that their incorporation does not contradict the explicit teachings of this article.

[0135] [1] R Nian, W Zhang, L Tan, J Lee, X Bi, YS Yang, HT Gan, P Gagnon, Pre-chromatin extraction improves the capture performance of protein A affinity chromatography, Journal of Chromatography A (J Chromatogr A) 1431 (2016) 1-7.

[0136] [2]. R Nian, P Gagnon, Advance: Pre-extraction of chromatin improves the performance and productivity of cation exchange chromatography-based capture of immunoglobulin G monoclonal antibodies. Chromatography Journal A 1453(2016)54-61.

[0137] [3] P Gagnon, R Nian, L Tan, J Cheong, V Yeo, YS Yang, HT Gan, Inhibition of chromatin-mediated electronegative multi-peak chromatography media for the prevention and purification of immunoglobulin G branching, Chromatography Journal 1374(2014)145-155.

[0138] [4]T McNerney, A Thomas, A Senczuk, K Petty, X Zhao, R Piper, J Carvalho, M.H. Ammond, S. Sawant, J. Bussiere, pDADMAC flocculation of Chinese hamster ovary cells: a centrifugation-free harvesting process for monoclonal antibodies, mAbs 7 (2015) 413-427.

[0139] [5] J Murphy, D Jewell, K White, G Fox, R Wilson, Separation of nucleic acids by immobilized metal affinity chromatography, Progress in Biotechnology 19(2003)982-986.

[0140] [6] T Cano, J Murphy, G Fox, R Wilson, Isolation of genomic DNA from plasmid DNA by selective renaturation of immobilized metal affinity capture, Progress in Biotechnology 21(2005)1472-1477.

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[0144]

[10] C Ferreira, I Pinto, E Soares, H Soares, (in)applicability of pH buffers in biological, biochemical and environmental studies and their interactions with metal ions—a review, RSC Adv 5 (2015) 30989-31003.

Claims

1. A method for removing host cell DNA from a sample containing a desired type of virus or extracellular vesicle, comprising the steps of: - A matrix loaded with anionic metal affinity ligands using metal ions. - Equilibrate the matrix with a buffer solution with a pH range of 4 to 10 and a salt concentration up to 1 M, wherein the salt does not form a chemical complex with the anionic metal affinity ligand. - The sample is brought into contact with the anionic metal affinity matrix of the loaded metal. - Separate the matrix from the sample, wherein the content of host cell DNA in the sample is reduced. in, The DNA will not denature into its single strands due to heating or sodium hydroxide.

2. The method of claim 1, wherein the anionic metal affinity ligand is selected from the group consisting of aminodicarboxylic acids and aminotricarboxylic acids.

3. The method of claim 2, wherein the anionic metal affinity ligand is iminodiacetic acid (IDA) or hyponitroacetic acid (NTA).

4. The method according to any one of claims 1 to 3, wherein the matrix with anionic metal affinity ligands is in the form of particles, nanowires, porous membranes, solid materials, hydrogels, depth filtration media, or soluble polymer media.

5. The method of claim 4, wherein the matrix containing the anionic metal affinity ligand is in the form of a flow chromatography device.

6. The method of claim 1, wherein the matrix is ​​equilibrated using a buffer solution with a pH in the range of 7.0 to 9.

5.

7. The method of claim 1, wherein the matrix is ​​equilibrated using a buffer solution with a pH range of 8.0 to 9.

0.

8. The method of claim 1, wherein the matrix is ​​equilibrated using a buffer solution with a salt concentration ranging from 50 mM to 750 mM.

9. The method of claim 1, wherein the matrix is ​​equilibrated using a buffer solution with a salt concentration ranging from 100 mM to 500 mM.

10. The method of claim 1, wherein the matrix is ​​equilibrated using a buffer solution with a salt concentration ranging from 125 mM to 250 mM.

11. The method of claim 1, wherein the buffer solution for balancing the matrix provides salt conditions that prevent viral or extracellular vesicle binding but allow DNA binding, and is regulated with salts selected from the group consisting of inorganic salts, organic salts, and liquid salts, and combinations thereof.

12. The method of claim 1, wherein the buffer solution for balancing the matrix provides salt conditions that prevent viral or extracellular vesicle binding but allow DNA binding, and is regulated with salts selected from the group consisting of: sodium chloride, potassium chloride, sodium acetate, potassium acetate; arginine-HCl, lysine-HCl, or salts based on imidazole, histidine, or histamine cations; and salts comprising guanidine cations and / or thiocyanate anions; and combinations thereof.

13. The method of claim 1, wherein the metal-loaded anionic metal affinity matrix is ​​loaded with metal ions having at least two positive charges.

14. The method of claim 1, wherein the metal ion is selected from the group consisting of iron (II), manganese (II), calcium (II), magnesium (II), copper (II), zinc (II), barium (II), nickel (II), cobalt (II), and combinations thereof.

15. The method of claim 1, wherein the sample of the desired type is a cell harvest, cell lysate, or partially purified preparation, and the desired type is selected from the group consisting of: non-lipid-enveloped protein-capped viral particles; lipid-enveloped viruses or virus-like particles; bacteriophages, extracellular vesicles; and combinations thereof.

16. The method of claim 1, wherein the viral particle with a non-lipid-enveloped protein capsid is an AAV capsid; the lipid-enveloped virus or virus-like particle is an influenza virus or a coronavirus; and / or the extracellular vesicle is an exosome.

17. The method of claim 16, wherein the AAV capsid is selected from the group consisting of: AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, recombinant hybrid serotypes, synthetic recombinant serotypes, and combinations thereof.

18. The method of claim 17, wherein the recombinant hybrid serotype is AAV2 / 8 or AAV2 / 9.

19. The method of claim 1, wherein the sample is treated by bioaffinity chromatography after the metal affinity step, cation exchange after metal affinity, hydrophobic interaction chromatography after the metal affinity step, and / or tangential flow filtration after the metal affinity step.

20. The method of claim 19, wherein the pore size cutoff value of the membrane used in the tangential flow filtration ranges up to 1 MDa.

21. The method of claim 19, wherein the pore size cutoff value of the membrane used in the tangential flow filtration is in the range of 30-50 kDa.

22. The method of claim 19, wherein the pore size cutoff value of the membrane used in the tangential flow filtration is in the range of 30-100 kDa.

23. The method of claim 19, wherein the pore size cutoff value of the membrane used in the tangential flow filtration is in the range of 200 kDa to 700 kDa.

24. The method of claim 1, wherein the sample with reduced DNA content is further treated by anion exchange chromatography.

25. The method of claim 1, wherein the sample of the desired type is a cell harvest or cell lysate.

26. The method of claim 25, wherein the sample is not subjected to any chromatographic steps prior to the step of contacting the sample with the anionic metal affinity matrix of the loaded metal.

27. Use of a matrix containing anionic metal affinity ligands for use in the method of claim 1.

28. The use of claim 27, wherein the sample containing the desired type is a cell harvest or cell lysate.

29. The use according to claim 28, wherein the sample is not subjected to any chromatographic steps prior to the step of contacting the sample with the matrix.

30. The use according to claim 27, wherein the sample is under alkaline conditions.

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