METAL-AFFINITY EXTRACTION OF HOST CELL DNA

Abstract

A method for removal of host cell DNA from a sample containing a species of desired protein, virus, or extracellular vesicle comprising the steps of: Loading a substrate bearing an anionic metal affinity ligand with a metal ion, Equilibrating the substrate with a buffer having a pH in the range of pH 6 to pH 10, and a salt concentration in a concentration range up to 1 M which salt is not forming a chemical complex with the anionic metal affinity ligand, Contacting the sample with the metal-loaded anionic metal affinity substrate, Separating the substrate from the sample, wherein the sample has reduced content of contaminating DNA.

Claims

1. A method for removal of host cell DNA from a sample containing a desired species of a protein, a virus, or an extracellular vesicle comprising the steps of: Loading a substrate bearing an anionic metal affinity ligand with a metal ion, Equilibrating the substrate with a buffer having a pH in the range of pH 4 to pH 10, and a salt concentration in a concentration range up to 1 M which salt is not forming a chemical complex with the anionic metal affinity ligand, Contacting the sample with the metal-loaded anionic metal affinity substrate, Separating the substrate from the sample, wherein the sample has a reduced content of host cell DNA.

2. The method of claim 1 wherein the anionic metal affinity ligand is selected from the group consisting of amino-dicarboxylic acids and amino-tricarboxylic acids.

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

4. The method of claim 1 wherein the substrate bearing an anionic metal affinity ligand is in the form of particles, nanofilaments, porous membranes, monoliths, hydrogels, depth filtration media, or soluble polymer media.

5. The method of claim 4 wherein the substrate bearing an anionic metal affinity ligand is in the form of a flow-through chromatography device.

6. The method claim 1 wherein equilibrating the substrate is performed by means of a buffer having a pH in the range of pH 7.0 to 9.5, or 8.0 to 9.0.

7. The method of claim 1 wherein equilibrating the substrate is performed by means of a buffer having a concentration of the salt in the range of up to 1 M.

8. The method of claim 1 wherein the buffer used for equilibrating the substrate provides salt conditions that prevent binding of a virus or extracellular vesicle but permit binding of DNA and are adjusted with the salt selected from the group consisting of an inorganic salt, an organic salt, and a chaotropic salt, and combinations thereof.

9. The method of claim 1 wherein the metal-loaded anionic metal affinity substrate is loaded with metal ions having at least two positive charges.

10. The method of claim 1 wherein the sample containing the desired species is selected from the group consisting of a cell harvest, cell lysate, and a partially purified preparation thereof and the desired species is selected from the group consisting of non-lipid-enveloped protein capsid virus particles, lipid-enveloped virus, virus-like particles, bacteriophages, extracellular vesicles, proteins, and combinations thereof.

11. The method of claim 10 wherein the AAV capsid is selected from the group consisting of AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, a recombinant hybrid serotype like AAV2/8, or AAV2/9, a synthetic recombinant serotype and combinations thereof.

12. The method of claim 1 wherein the sample is processed by biological affinity chromatography after the metal affinity step of claim 1, cation exchange after the metal affinity step of claim 1, hydrophobic interaction chromatography after the metal affinity step of claim 1, tangential flow filtration after the metal affinity step of claim 1 or combinations thereof.

13. The method of claim 12 wherein the tangential flow filtration is using a membrane with pore size cutoffs in the range of up to 1 MDa.

14. The method of claim 1 wherein the sample having a reduced content of DNA is further processed by anion exchange chromatography.

15. The method of claim 1 wherein the sample containing the desired species is a cell harvest or cell lysate.

16. The method of claim 15 wherein the sample did not undergo any chromatographic step prior to the step of contacting the sample with the metal-loaded anionic metal affinity substrate.

17-19. (canceled)

20. The method of claim 1 wherein the sample has alkaline conditions.

21. The method of claim 1 wherein equilibrating the substrate is performed by means of a buffer having a concentration of the salt in the range of 125 mM to 250 mM.

22. The method of claim 8 wherein the inorganic salt is selected from the group consisting of sodium chloride, potassium chloride, sodium acetate, and potassium acetate, the organic salt is selected from the group consisting of arginine-HCl, lysine-HCl, a salt based on an imidazolium, histidyl, and histaminyl cation, and the chaotropic salt is selected from a guanidinium cation and a thiocyanate anion.

23. The method of claim 9 wherein the metal ions having at least two positive charges are 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.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0055] FIG. 1 depicts a diagram of the method of the invention.

[0056] FIG. 2 depicts non-binding of AAV capsids to an anionic metal affinity ligand loaded with magnesium at pH 9.0, while DNA binds strongly and requires sodium hydroxide for elution.

[0057] FIG. 3 depicts a comparison of AAV capsid binding to an anionic metal affinity ligand loaded with magnesium in separate experiments at pH 7.0 and pH 9.0. Profiles at 280 nm.

[0058] FIG. 4 depicts DNA removal and fractionation of empty and full AAV capsids by a quaternary amine anion exchanger eluted with a salt gradient.

[0059] FIG. 5 depicts DNA removal and fractionation of empty and full AAV capsids by a primary amine anion exchanger eluted with a pH gradient.

[0060] FIG. 6 depicts size exclusion chromatography of cell culture containing extracellular vesicles, including exosomes.

[0061] FIG. 7 depicts size exclusion chromatography of cell culture containing extracellular vesicles after removal of DNA by the method of the invention.

[0062] FIG. 8 depicts the size exclusion elution profiles of FIGS. 7 and 8 overlaid to highlight the reduction of contaminants in general, particularly including DNA.

[0063] FIG. 9 depicts DNA removal and fractionation of partially purified extracellular vesicles with a quaternary amine anion exchanger eluted with a salt gradient.

[0064] FIG. 10 depicts bacteriophage T4 flowing through an iminodiacetic acid monolith loaded with ferric iron.

[0065] FIG. 11 depicts secondary removal of DNA and fractionation of bacteriophage T4 by chromatography on a quaternary amine anion exchanger eluted with a salt gradient.

[0066] FIG. 12 depicts secondary removal of DNA and fractionation of bacteriophage T4 by chromatography on a primary amine anion exchanger eluted with a salt gradient.

DETAILED DESCRIPTION OF THE INVENTION

[0067] The sample consists of a preparation containing a desired species of proteins, virus particles, or extracellular vesicles produced by cell culture, and also containing host cell-derived DNA. In one embodiment, the sample consists of a cell culture harvest. In one such embodiment, the cell culture harvest contains an antibody. In another such embodiment, the cell culture harvest contains a virus or virus-like particle. In another such embodiment, the cell culture harvest contains extracellular vesicles. In another embodiment, the sample consists of a cell lysate. In another embodiment, the sample consists of a cell culture harvest or cell lysate that has been treated with nuclease enzymes to reduce host cell DNA content. In another embodiment, the sample consists of a partially purified preparation still containing more host cell DNA than is desired or permitted in the final product. In one such embodiment, the sample is a product fraction eluted from a chromatography device. In one such embodiment, the sample is the eluted product from an affinity chromatography column. In another such embodiment, the sample is the eluted product from a size exclusion chromatography column. In another such embodiment, the sample is the eluted product from a hydrophobic interaction chromatography column. In another such embodiment, the sample is the eluted product from a cation exchange chromatography column. In another such embodiment, the sample is the eluted product from an immobilized metal affinity chromatography column. In another such embodiment, the sample is the eluted product from an apatite chromatography column. In another such embodiment, the sample is concentrated and/or diafiltered product from tangential flow filtration.

[0068] In one embodiment, the method of the invention is used to process a sample that contains a desired non-lipid-enveloped protein-capsid virus particles contaminated with host cell DNA. AAV has many serotypes. In one embodiment, the desired AAV serotype processed by the method of the 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 processed by the method of the invention may be a recombinant hybrid serotype like AAV2/8, or AAV2/9, or another hybrid serotype. In another embodiment, the AAV serotype processed by the method of the invention may be a synthetic recombinant serotype. In any of these embodiments, the anion exchange step may be performed to separate empty capsids from full capsids while further reducing the content of contaminating DNA. In one such embodiment, the anion exchanger is a strong anion exchanger (quaternary amine) eluted with salt. In another such embodiment the anion exchanger is a weak anion exchanger (primary amine) eluted with an ascending pH gradient.

[0069] In another embodiment, the sample contains a desired lipid-enveloped virus or virus-like particles contaminated with host cell DNA. In one such embodiment, the anion exchange step may separate non-infective virus particles from infective virus particles. In one such embodiment, the virus is an influenza virus. In another such embodiment, the virus is a corona virus.

In another embodiment, the sample contains a desired bacteriophage contaminated with host cell DNA.

[0070] In another embodiment, the method of the invention is used to process a sample that contain an extracellular vesicle contaminated with host cell DNA. In one such embodiment, the extracellular vesicles are exosomes contaminated with host cell DNA.

[0071] In one embodiment of the method of the invention, a sample may previously have been partially purified, including by methods that have the effect of reducing the content of host cell DNA.

[0072] Anionic metal affinity substrates suitable to practice the method of the invention include immobilized amino-carboxylic acids. In one embodiment, an immobilized amino-carboxylic acid may be a dicarboxylic acid such as iminodiacetic acid (IDA). In another embodiment, the amino-carboxylic acid may be an immobilized tricarboxylic acids such as nitriloacetic acid (NTA). In another embodiment, a mixture of IDA and NTA substrates may be employed. Anionic metal affinity substrates are available commercially in a variety of physical forms and may be synthesized in any format desired. They may be in the form of particles, insoluble nanofilaments, porous membranes, monoliths, hydrogels, depth filtration media, soluble polymer media, or other formats. In many cases, such substrates are available in the form of a flow-through chromatography device to facilitate their use.

[0073] In some embodiments, the choice of anionic metal affinity ligand can contribute to non-retention of the desired protein, virus, or extracellular vesicle product. In some such embodiments, NTA may be preferred over IDA in some embodiments because NTA carries three negative charges where IDA carries only two. Complexes of divalent metal cations with IDA will produce a net charge of zero by the ligand-metal complex but complexes of divalent metal cations with NTA will produce a net charge of minus one (1−), which may tend to discourage binding of the desired product. Complexes of trivalent metal cations with IDA will produce a net charge of plus one (1+) by the ligand-metal complex, which may endow the complex with anion exchange characteristics that favor binding by the desired protein, virus, or extracellular vesicle. Complexes of trivalent metal cations with NTA will produce a net charge of zero, which will not endow the complex with significant anion exchange capability. It will be advisable to evaluate both IDA and NTA for routine application of the method.

[0074] It will be understood by persons of experience in the art that non-lipid-enveloped protein-capsid viruses tend to be robust and often tolerate pH 9 over a wide range of salt concentrations. This will make it a simple matter to conduct either or both steps of the method of the invention at a pH of about 9. It will be equally understood that lipid-enveloped viruses, virus-like particles, bacteriophages, and extracellular vesicles are more labile and may require moderation of pH to maintain product stability. In one such embodiment, the metal affinity step may be conducted at a pH of about 8 and a sodium chloride concentration of about 250 mM. Less tolerant species may require reduction to a pH slightly above neutral and a salt concentration close to 100 mM. More robust species may tolerate a pH of 8.75 and a concentration of salt up to 375 mM or more. As a general matter, the lowest concentration of salt required to prevent product binding of the target product during the metal affinity step will be advantageous because it will minimize the degree to which the processed sample must be diluted to bind to the anion exchanger in the final step of the method. Also, in general, the nearer the pH is to neutral, the more likely it will be tolerated by labile products such as those with lipid membranes.

[0075] Buffer pH may 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 a different or narrower range, according to the stability requirements of the desired protein, virus, or extracellular vesicle. It will be recognized by persons of knowledge in the art that some buffering agents may interact with metals [10] and may be exploited to modulate the performance of the method of the invention.

[0076] In some embodiments, salts which is not forming a chemical complex with the metal-loaded anionic metal affinity substrate (non-chelating salts) may be present at a concentration in the range of 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 salt may help to stabilize the virus particles or extracellular vesicles. In some embodiments, exposure of the extracellular vesicles or lipid-enveloped viruses to salt concentrations greater than 500 mM should be brief to minimize damage to the product. In some embodiments, the metal affinity step of the invention will bind chromatin even at salt concentrations of 1 M, or 2 M, or 3 M, or 4 M, or 5 M, or in saturating concentrations of non-chelating salts. It will be recognized that such high concentrations will be seldom or never beneficial to the overall practice of the method of the invention, and especially not when the desired product is labile to such conditions, but such conditions will still support selective removal of chromatin.

[0077] In some embodiments, the species of salt which is not forming a chemical complex with the metal-loaded anionic metal affinity substrate (non-chelating salt) employed to conserve stability of the product may be an inorganic salt such as sodium chloride, or potassium chloride, or sodium acetate, or potassium acetate, or another salt.

[0078] In closely related embodiments, the non-chelating salt may be an organic salt, such as arginine-HCl, lysine-HCl, or a salt based on an imidazolium, histidyl, or histaminyl cation.

[0079] In another closely related embodiment, the non-chelating salt may be a chaotropic salt comprising a guanidinium cation or a thiocyanate anion, or both, or other chaotropic ions. It will be recognized by persons of knowledge in the art however, that the use of such salts will be restricted to proteins and protein-protein capsid viruses since such salts are likely to damage products that possess a lipid membrane.

[0080] Anions with strong capacity to bind metal cations will tend to remove the metals bound to the anionic solid phase ligand and will compromise the ability of the anionic metal affinity substrate to bind chromatin. Anions known to have strong metal-binding ability include citrates, phosphates, pyrophosphates, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid also known as egtazic acid (EGTA), aspartic acid, glutamic acid, and glutamine.

[0081] Multivalent metal cations suitable to practice the method particularly include ferric iron and manganese. Cupric copper, zinc, magnesium, calcium, and barium may also be employed. Heavy metal ions such as nickel and cobalt, among others, also mediate DNA reduction but their use is discouraged by their toxicity.

[0082] In some embodiments, the choice of metal ion can also contribute to non-retention of the desired virus or extracellular vesicle product. Metal ions with notably high affinity for phosphate residues will tend to bind all phosphorylated species more strongly than 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 phosphate. Metals such as cupric copper mediate intermediate affinity for phosphate groups. In embodiments where the desired protein, virus, or vesicle species exhibits inherently low affinity for metal ions bound to an anionic metal affinity solid phase, it may be advantageous to use iron or manganese for the purpose of maximizing chromatin binding. In embodiments where the desired protein, virus, or vesicle species is highly phosphorylated, it may be beneficial to employ copper, calcium, or magnesium for the purpose of maximizing recovery of the desired product.

[0083] Many types of anion exchangers are commercially available worldwide. In one such embodiment, the anion exchanger is a quaternary amine 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 may also employ primary amino groups, secondary amino groups, and combinations of primary, secondary, tertiary, and quaternary amino groups. One such material is N,N-Bis(2-aminoethyl)-1,2-ethanediamine more commonly referred to as TREN. Other anion exchangers of mixed composition employ ligands consisting of polyallylamine, polyethyleneimine, and ethylenediamine, among others. Anion exchangers suitable to practice the method are also understood to include positively charged amine derivatives that include additional residues to confer excess hydrophobicity, hydrogen bonding, or both. Anion exchangers including additional residues to confer excess hydrophobicity and/or hydrogen bonding are commonly referred to as multimodal or mixed-mode exchangers. Throughout this specification, all of the foregoing materials are referred to as anion exchangers. All of them are commercially available worldwide in a variety of physical forms, including particles, insoluble nanofilaments, porous membranes, monoliths, hydrogels, depth filtration media, or other formats. In many cases they are available in the form of a flow-through chromatography or filtration device to facilitate their use. In one embodiment, the metal affinity substrate and the anion exchanger are both in the form of chromatography devices, plumbed in sequence with the metal affinity device first. In one such embodiment they are equilibrated in tandem, loaded in tandem, washed in tandem, eluted in tandem, and cleaned in tandem. In another such embodiment, they are equilibrated in tandem, loaded in tandem, washed in tandem, then the metal affinity is removed from the flow stream and the anion exchanger is eluted independently. It will be recognized by persons of knowledge in the art that plumbing devices in sequence can be attractive for industrial users because it substantially reduces the amount of water, buffers and salts, buffer preparation, process time, equipment, and staffing required to process a desired product. All of these benefits contribute to higher facility capacity, which ultimately translates to higher productivity at lower expense.

[0084] In some embodiments, the metal affinity-treated sample may be applied to the anion exchanger without concern for residual free metal ions in the sample since such metal ions, being positively charged, will be repelled by the surface of the anion exchanger and eliminated during sample application. In other embodiments, metal ions may be deliberately added to the sample and the anion exchange buffers. In such embodiments, their presence may modify the surface charge or surface topography of viruses or vesicles in ways that are beneficial. In one such embodiment, the presence of calcium and or magnesium ions in the buffers contributes to improved separation of empty AAV capsids from full AAV capsids.

[0085] In some embodiments, anion exchange chromatography is performed directly following the metal affinity step. In one embodiment where the metal affinity chromatography step and the anion exchange chromatography step are performed in uninterrupted sequence, both media are used in the form of a chromatography device or a filtration device. In another embodiment where the metal affinity chromatography step and the anion exchange chromatography step are performed in uninterrupted sequence, the metal affinity step is performed by adding an insoluble metal affinity substrate to the sample and permitting it to bind and co-precipitate the DNA, then removed so the DNA-deficient supernatant can be applied to the anion exchanger. In another embodiment where the metal affinity chromatography step and the anion exchange chromatography step are performed in uninterrupted sequence, the metal affinity step is performed by adding soluble polymer substrate bearing the metal affinity ligand to the sample, allowing it to cross-link and precipitate the chromatin, then removing the precipitate by centrifugation and/or filtration to yield a DNA-deficient supernatant to be processed by anion exchange chromatography.

[0086] In a related embodiment, performed at a salt concentration up to 400 mM sodium chloride, the metal affinity chromatography substrate may be added to a sample in combination with positively charged particles or polymers, which will co-crosslink to the DNA associated with the metal affinity chromatography media and further contribute to DNA reduction. Elevated salt will meanwhile discourage binding of the desired protein, virus, or vesicle with either the substrate of the invention or the positively charged substrate. As a general matter, the concentration of salt need be no higher than necessary to prevent binding the desired protein, virus, or vesicle to the substrate of the invention or to the positively charged substrate. After treatment, the salt concentration may be reduced, if necessary, to enable the sample to be processed by anion exchange chromatography.

[0087] In another closely related embodiment, the method of the invention may be combined with treatment by a fatty acid. In some such embodiments, the fatty acid may be heptanoic acid, or octanoic acid, or nonanoic acid at a concentration in the range of 0.01% to 1.0%, and at a pH in the range of 4 to 6. In some such embodiments, the fatty acid may be present at the same time that particles or polymers bearing the metal-loaded anionic metal affinity substrate is present. In some such embodiments, the fatty acid treatment may be conducted before or after the method of the invention. It will be apparent to persons of knowledge in the art that treatments including fatty acids will be unsuitable for viruses and extracellular vesicles with lipid membranes.

[0088] In another closely related embodiment, the method of the invention may be combined with treatment with allantoin. In some such embodiments, the allantoin may be present in an amount ranging from 2% to 10%. It will be apparent to persons of knowledge in the art that treatments including allantoin will be unsuitable for some viruses and extracellular vesicles.

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

[0089] In one such embodiment, the sample is processed after metal affinity by biological affinity chromatography. In one such embodiment, the affinity chromatography ligand is a biological ligand specific for one or more serotypes of AAV. In another such embodiment, the affinity ligand is a biological ligand specific for an antibody. In one such embodiment, the affinity ligand is protein A or a variant thereof.

[0090] In a related embodiment, the sample is processed by cation exchange chromatography after the metal affinity step, then processed by anion exchange chromatography. In one such embodiment, the cation exchanger is used to capture AAV. In another embodiment, the cation exchanger is used to capture an antibody

[0091] In another related embodiment, the sample is processed by hydrophobic interaction chromatography after metal affinity then processed by anion exchange chromatography.

[0092] In another related embodiment, the sample is processed by an anionic immobilized metal affinity chromatography after the anionic metal affinity DNA-removal step, then processed by anion exchange chromatography. In one such embodiment, this variation is applied to biomolecules that naturally bear histidine clusters or which have been produced from recombinant gene constructs that cause them to bear an artificial histidine cluster, tail, or tag. In one such embodiment, IgG, which naturally bears a histidine cluster in its hinge region of the desired product. In one such embodiment, the anionic metal affinity ligand for the DNA removal step bears ferric iron and the anionic metal affinity ligand for the subsequent purification step bears nickel. This step binds IgG. The IgG may be eluted by competition with imidazole, reduction of pH, or a combination of both. The eluted IgG is then polished with anion exchange chromatography. In one such embodiment, the anion exchanger is a multimodal anion exchanger. In a 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 variant of the above embodiments, the two metal affinity steps are performed with a pair of columns plumbed together, where the first in the sequence is an IDA column loaded with ferric iron and the second is IDA loaded with zinc. In one such embodiment, filtered cell culture harvest containing IgG monoclonal antibodies is passed through both columns, where the first removes DNA and the second captures IgG. The columns are washed, then a buffer is applied to elute IgG from the second column while DNA remains bound to the first until it is later removed with NaOH. In another variation of this approach, the product of interest, instead of being an antibody, is a His-tagged protein, or a His-tagged exosome, or a His-tagged virus particle.

[0093] In another related embodiment, the sample is processed by tangential flow filtration after metal affinity and before anion exchange chromatography. Since virus particles and extracellular vesicles represent large complex assemblages, commonly ranging in size from 20 nm to more than 200 nm, many such embodiments will benefit from integrated processing by tangential flow filtration with the largest pores that retain the product of interest. In some such embodiments, this will involve TFF membranes with pore size cutoffs in the range of 200 kDA to 700 kDA, and in some cases very large pore size ratings such as 1 MDa. Such filters permit the elimination of smaller contaminants by their passage through the pores of the membranes. In one such embodiment, metal affinity particles or polymers loaded with magnesium are mixed with a cell culture harvest of lysate at alkaline pH to bind the DNA. Solids are then removed by centrifugation and/or membrane filtration, and the clarified supernatant is concentrated and/or diafiltered to concentrate and/or buffer exchange the sample in preparation for anion exchange chromatography. In a related embodiment where the desired product is an IgG antibody, the pore size cutoff of the membrane may be 30-50 kDa. In one such embodiment, a subsequent anion exchange chromatography step is conducted with a multimodal anion exchanger. In a closely related embodiment where the desired product is an IgM antibody, the pore size cutoff of the membrane may be 30-100 kDa and a subsequent anion exchange chromatography step is conducted with a strong anion exchanger such as a quaternary amine anion exchanger.

[0094] In one embodiment, the method of the invention is used to process adeno-associated virus, where the anion exchanger fulfills the additional function of fractionating empty capsids from full capsids. The term full capsids is understood to refer to capsids which contain their intended payload of therapeutic plasmid DNA. The term empty capsids is understood to refer to capsids lacking the complete therapeutic DNA plasmid. 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 ascending pH gradient. In another such embodiment, the anion exchanger is mixed amino anion exchanger. In one such embodiment the anion exchanger is TREN.

[0095] In one embodiment, the method of the 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.

[0096] In one embodiment, the method of the invention is used to replace sample treatment with nuclease enzymes to reduce DNA content. In another embodiment, the method of the invention is used to augment the degree of DNA reduction achieved by treatment with nuclease enzymes. In one such embodiment, the metal affinity step of the invention is performed in advance of treatment with nuclease enzymes. In another such embodiment, the method of the invention is performed after treatment with nuclease enzymes, where it provides additional utility by binding the nuclease enzymes by their associated metal ion cofactors. In one such embodiment, the anionic metal affinity ligand is loaded with the same metal ion species used as a cofactor for the nuclease enzyme. In one such embodiment, the metal ion species is magnesium. In another such embodiment, the metal ion species is calcium. In an embodiment where the metal affinity step is performed at the same time the sample is treated with a nuclease enzyme, the metal ion used to load the affinity substrate is different from the metal ion species than the enzyme cofactor. For example, if the enzyme co-factor is magnesium, the metal affinity ligand may be loaded with ferric iron so that the metal affinity substrate will not bind the enzyme during lysis of DNA. In any of the foregoing embodiments, the metal affinity ligand may be one of many affixed covalently to a plurality of soluble polymers. In another such embodiment, the metal affinity ligand may be affixed covalently to a plurality of insoluble solid phase particles.

[0097] In one embodiment where the metal-affinity DNA-reduction step is performed with loose particles or soluble polymers bearing ligand-metal complexes, precipitates and co-precipitates may be formed. These solids may be removed before further processing of the supernatant containing the desired virus or vesicle species. In one embodiment they may be removed by membrane filtration, or centrifugation, or a combination of the two. After removal of solids, the sample may be processed by means of tangential flow filtration (TFF). In one such embodiment, TFF is performed using membranes with the largest pore size that retains the virus or vesicles of interest while but allows smaller contaminating species to be eliminated by their passage through the pores.

[0098] In one such embodiment, the pore size cutoff rating may be 100 kDa, or 300 kDa, or 500 kDa, or 700 kDa, or 1 MDa, or a larger or intermediate molecular weight cutoff (MWCO). In most or all of the foregoing embodiments, the TFF step particularly enables elimination of histone proteins liberated by lysis of the host cell DNA they were associated with. In any of the foregoing embodiments, the TFF step may also be used to concentrate the sample and/or diafilter the sample into a buffer suitable for performing a chromatography step.

[0099] In any of the foregoing embodiments, treatment of a sample by the metal affinity step may remove large aggregates and cell debris to an extent that render the sample more filterable and easier to process by TFF or chromatography. In one such embodiment, TFF may be used to concentrate the sample and diafilter it into conditions for enzymatic digestion by nuclease enzymes to further reduce DNA levels. In one embodiment, a sample treated by metal affinity may be further processed by TFF to remove histone proteins before processing the sample by anion exchange chromatography, or by an intermediate chromatography step prior to anion exchange chromatography.

[0100] In some embodiments, secondary additives may be included in product preparations suppress non-specific interactions between the desired product and processing surfaces or to stabilize the desired product. Such additives may include non-ionic or zwitterionic surfactants such as octaglucoside, poloxamer 188, Pluronic F68, CHAPS, or CHAPSO, among others. Such stabilizing compounds may instead or additionally include sugars such as sucrose, sorbitol, xylose, mannitol, or trehalose, among others. Such stabilizing compounds may instead or additionally include amino acids such as betaine, tauro-betaine, arginine, histidine, or lysine, among others. All of these agents are known in biopharmaceutical field because they tend to improve solubility and/or recovery of stable product. In some cases, they also improve fractionation of a desired product from undesired species.

[0101] It will be recognized that both steps of the method of the invention have potential to remove other phosphorylated contaminants as a byproduct of removing chromatin. Other phosphorylated contaminants potentially include RNA, endotoxins, phosphoproteins, and phospholipids.

EXAMPLES

Example 1

[0102] Advance removal of host cell DNA from a preparation of adeno-associated virus (AAV) by anionic metal affinity with magnesium A monolith bearing iminodiacetic acid (IDA) chelating residues was loaded with magnesium and equilibrated to pH 9.0. A sample of cation exchange-purified capsids was equilibrated to the same conditions and loaded onto the column. AAV capsids passed through the column unbound. Host cell DNA bound and was later removed with 1 M NaOH. Results are illustrated in FIG. 2.

Example 2

[0103] Advance Removal of Host Cell DNA from a Preparation of AAV by Anionic Metal Affinity with Magnesium

[0104] A monolith bearing IDA chelating residues was loaded with magnesium and equilibrated to pH 7.0. A sample of cation exchange-purified capsids was equilibrated to the same conditions and loaded onto the column. AAV capsids were partially bound, along with DNA (FIG. 3. Compare with FIG. 2). Despite partial binding of AAV capsids at pH 7.0, the results show their elution at about 250 mM NaCl. This means inclusion of that amount of salt in the equilibrated sample and column would have prevented their binding. FIG. 4 illustrates separation of empty and full AAV capsids coincident with removal of DNA by anion exchange chromatography with a strong (quaternary amine) anion exchanger eluted with a sodium chloride gradient. FIG. 5 illustrates separation of empty and full AAV capsids coincident with removal of DNA by anion exchange chromatography with a weak (primary amine) anion exchanger eluted with a pH gradient.

Example 3

[0105] Advance Removal of DNA from a Preparation of Extracellular Vesicles

[0106] A monolith bearing IDA chelating residues was loaded with ferric iron and equilibrated with 50 mM Hepes, 50 mM NaCl, pH 7.0. A clarified mammalian cell culture harvest was passed through the monolith. FIG. 6 illustrates an analytical size exclusion chromatography (SEC) profile of the sample before it was applied to the IDA-Fe monolith. Note the excess UV absorbance at 260 nm from about 10 minutes to about 23 minutes. This indicates the presence of nucleic acids and corresponds to the zone in which chromatin normally elutes. FIG. 7 illustrates an analytical size exclusion chromatography profile of the sample after it was applied to the IDA-Fe monolith. Note the elimination of excess absorbance at 260 nm and the general reduction of UV signal from 10-23 min. This is consistent with chromatin removal. FIG. 8 illustrates results from before and after analytical size exclusion chromatography monitored by Multi-Angle Light Scattering (MALS, LS) and by immunofluorescence (IFL). Light scatter selectively amplifies 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 before chromatography then monitoring the run with a fluorescence detector. It detects only solutes that bear the specific immunological marker to which the antibody is directed. In this experiment, the antibody was directed to CD63, which is a marker known to be characteristic of exosomes. MALS and IFL signal intensity for the “after treatment” sample were adjusted upward by a factor of 5 to compensate for 5-fold sample dilution during earlier processing by the method of the invention. Note that the ratio of IFL to MALS increased as a result of treatment. This indicates that the metal affinity step of the method selectively removes sample components lacking the exosome marker and thereby produces a more enriched exosome fraction. This suggests that a large proportion of the solutes eluting from 10-12 minutes and detected by MALS in FIG. 6 were associated with chromatin. FIG. 9 illustrates processing of a partially purified extracellular vesicle preparation that was loaded onto a strong anion exchanger (quaternary amine) equilibrated to 50 mM Hepes, 50 mM NaCl, pH 7.0, then eluted with a linear gradient to 2 M NaCl before being cleaned with 1 M NaOH. Extracellular vesicles mostly elute in less than 1 M NaCl. Chromatin mostly requires NaOH for elution.

Example 4

[0107] Advance Removal of Host DNA from a Preparation of Bacteriophage T4

[0108] A monolith bearing IDA chelating residues was loaded with ferric iron and equilibrated to pH 7.0. A filtered cell culture harvest was run through the monolith to remove DNA. FIG. 10 illustrates the elution profile. The bacteriophage flowed through the monolith. Some contaminants were bound and eluted with NaCl. DNA was later removed by 1 M NaOH. FIG. 11 illustrates polishing purification by anion exchange chromatography on a strong anion exchanger (quaternary amine) eluted with a sodium chloride gradient at pH 7. FIG. 12 illustrates polishing purification by anion exchange chromatography on a weak anion exchanger (primary amine) eluted with a sodium chloride gradient at pH 7.

Example 5

[0109] Removal of Host DNA from a Preparation Containing an IgG Monoclonal Antibody

[0110] An IDA monolith is loaded with ferric iron and excess iron is washed away with 1 M NaCl. The IDA-Fe substrate is washed with water to remove excess salt. Filtered cell culture harvest containing IgG monoclonal antibodies is passed through the monolith under roughly physiological conditions. The term physiological conditions is understood to include a pH of about 6.5 to 7.5 and a salt concentration corresponding to a conductivity of 50-200 mS/cm. The antibody flows through. Chromatin is bound. The monolith is rinsed to recover all of the antibody. The antibody is then processed by multimodal anion exchange chromatography to further reduce DNA content.

Example 6

Purification of an IgG Monoclonal Antibody

[0111] The method of Example 5 is repeated except inserting a TFF step after metal affinity removal of chromatin. TFF is performed with a membrane with a molecular weight cutoff (MWCO) of 30 kDA to retain the IgG while the content of lower molecular weight proteins and low molecular weight contaminants is reduced before the anion exchange chromatography step. In a variation of this process, metal affinity removal of DNA may be performed by treating the harvest in a bulk format with IDA-Fe particles instead of a flow-through chromatography device as described in Example 5.

Example 7

[0112] Removal of Host DNA from a Preparation Containing an IgM Monoclonal Antibody

[0113] An IDA monolith is loaded with ferric iron and excess iron is washed away with 1 M NaCl. The IDA-Fe substrate is washed with water to remove excess salt. Filtered cell culture harvest containing IgM monoclonal antibodies is passed through the monolith under roughly physiological conditions. The antibody flows through. Chromatin is bound. The monolith is rinsed to recover all of the antibody. The antibody is then processed with a strong anion exchanger eluted with a salt gradient to further reduce DNA content.

Example 8

Purification of an IgM Monoclonal Antibody

[0114] The method of Example 7 is repeated except inserting a TFF step after metal affinity removal of chromatin. TFF is performed with a membrane with a MWCO of 100 kDA to retain the IgM while the content of lower molecular weight proteins and low molecular weight contaminants is reduced before the anion exchange chromatography step. In a variation of this process, metal affinity removal of DNA may be performed by treating the harvest in a bulk format with IDA-Fe particles instead of a flow-through chromatography device as described in Example 7.

LIST OF REFERENCES

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