Compositions and methods for reducing the chromatin content of biological preparations - Patents.com

Abstract

A method for removing chromatin from a cell culture harvest, comprising: providing a cell culture harvest containing a desired biological material selected from the group consisting of parvovirus and adeno-associated virus; incubating the cell culture harvest in an aqueous medium having an ionic strength equivalent to a concentration of NaCl in the range of 3.0 M to saturation and a pH value in the range of 3.0 to 4.0; and separating the desired biological material from the solids produced.

Description

[Technical field]

[0001] The amount of DNA in the final drug product is a primary safety and regulatory concern for all biopharmaceuticals. This includes, for example, adeno-associated viruses (AAVs) used as delivery vehicles for therapeutic DNA plasmids in the field of gene therapy. The safety concern is not the therapeutic DNA in the AAV capsid, but rather the residual DNA from the host cells used to produce the AAVs. Removing this host-derived DNA has proven more difficult than expected and has been recognized as an obstacle to progress in the field. The challenge may stem in part from host-derived DNA bound to the outer surface of the capsid.

[0002] The therapeutic DNA payload in the AAV capsid is commonly referred to as vector DNA. Some of this DNA may also be present outside the capsid due to incomplete insertion or subsequent damage to the initially filled capsid. Although this DNA is not considered to be a top safety concern, it is problematic due to its potential to inflate the apparent number of DNA filled capsids as measured by various DNA assays. Several sample preparation methods are available to reduce the effect of excess capsid vector DNA, but it would be preferable if the excess capsid vector DNA were simply not present.

[0003] Treatment of the AAV harvest with nuclease enzymes to degrade host-derived DNA is a common feature of AAV purification procedures. A limitation of this approach is that host DNA is protected from enzymatic degradation by its strong association with histone proteins. A significant amount of host DNA remains undigested in the AAV preparation. Some purification procedures employ additional anion exchange chromatography to improve the overall reduction of host DNA. The principle is that DNA binds more strongly to the anion exchanger than AAV, allowing AAV to be eluted while DNA remains on the anion exchanger. Such treatments reduce but do not completely eliminate the host DNA content, and the inability to do so is related to the same problem that limits the effectiveness of enzymatic treatments: the presence of host cell DNA in the harvest in strong association with histone proteins. In addition to physically protecting DNA from enzymatic degradation, the strongly associated histones create hybrid aggregates with charge-neutral properties, and some of the DNA still co-elutes with the virus [1].

[0004] Alternatively, some of the host DNA still associated with histones can be precipitated from cell culture harvests by lowering the pH of the harvest. This method was described in 1994 and involves titrating the pH to within the range of pH 2.6 to pH 5.0 using a citrate buffer with physiological salt concentrations [2]. Although this work was performed on mammalian cell culture harvests, the technique was subsequently applied to bacterial, yeast, fungal, and insect cells [3-5]. More recently, it has been applied to mammalian cell culture harvests within the same range of pH and salt concentrations for the soft aggregation of AAV contaminants, including host DNA [6].

[0005] Harvest clarification and reduction of host cell DNA contamination using cationic heterocyclic compounds and soluble cationic polymers have also been described [7-10]. Anionic fatty acids and heterocyclic anions are also known to reduce DNA levels [11-16]. A common feature of these techniques is the employment of salt concentrations less than 0.5 M (500 mM), such as in the range of about 15-300 mM salt, and most often in the range of 50-100 mM salt. Allantoin has been used alone and in combination with many of these reagents and conditions [1, 15, 16]. Allantoin is known to have a high affinity for endotoxins

[17] .

[0006] DNA has a pK of about 2.6 a It is known that host DNA in cell culture harvests initially associates with histone proteins in nucleosomal arrays of various sizes. Histone proteins are extremely hydrophobic, with isoelectric points ranging from about 9 to 11. Host DNA partially dissociates from histone proteins in 1M NaCl, and progressively dissociates as NaCl concentrations increase up to 4M, but does not dissociate completely even at that concentration. Histone proteins begin to form insoluble aggregates at NaCl concentrations above 1M. Dissociated DNA is soluble under high salt conditions, but reassociates with histone proteins when salt concentrations are reduced.

[0007] Surfaces coated with primary amine ligands are known to bind biomolecules more strongly than surfaces with quaternary amine ligands. Biomolecules include virus particles, DNA, endotoxins, and acidic proteins. Primary amines usually bind these molecules better than quaternary amines on similar surfaces at moderately high salt concentrations. DNA also elutes from primary amine ligands at moderately high, but less than 1M, salt concentrations of sodium chloride. Experiments with quaternary and primary amine surfaces are mostly performed at neutral to slightly alkaline pH (pH 7.0-8.5). Treatment of mammalian cell culture harvests with hydrophobic quaternary amine (cholestyramine) particles at neutral pH and salt concentrations of 50-150 mM sodium chloride is known to bind some DNA in the mammalian cell culture harvest

[18] . Subsequent particle removal results in a corresponding reduction in DNA content in the harvest.

[0008]

[19] discloses a method for purifying a sample containing a desired protein, the method comprising: (i) providing a packed chromatographic column having positively charged porous particles; (ii) equilibrating the column to conditions under which the desired protein in the sample elutes; (iii) contacting the sample with the packed chromatographic column such that the sample volume applied to the packed chromatographic column is equal to or less than the interparticle voids of the positively charged porous particles in the packed chromatographic column; and (iv) eluting the desired protein from the packed chromatographic column, the desired protein being in a purer state and under the conditions under which the packed chromatographic column was equilibrated, the desired protein being an antibody, an antibody fragment, an antibody derivative, or an antibody fusion protein. Essentially,

[19] is concerned with removing viruses and obtaining the desired protein, where viruses are unnecessary. The reference is silent about removing DNA while preserving the viruses.

[0009]

[20] discloses a method to enhance the reduction of both virus and viral DNA levels in a protein preparation by contacting the protein preparation with a surface having primary amines, secondary amines, or both. However, this reference is silent about removing DNA from the virus, leaving the virus purified from viral DNA.

[0010] All references cited herein are incorporated by reference unless they contradict an explicit teaching of this specification.

[0011] References [1]R. Nian, P. Gagnon, Advance chromatin extraction enhances performance and productivity of cation exchange chromatography-based capture of Immunoglobulin G monoclonal antibodies, J. Chromatogr. A 1453 (2016) 54-61. [2]B. Lydersen, T. Brehm-Gibson, A. Murel, Acid precipitation of mammalian cell fermentation broth, Ann. NY Acad. Sci. 745 (1994) 222-231. [3] M. Westoby, J. Chrostowski, P. de Vilmorin, JP Smelko, JK Romero, Effects of solution environment on mammalian cell fermentation broth properties: enhanced impurity removal and clarification performance, Biotechnol. Bioeng. 108 (2011) 50-58. [4] E. Soares, Quantification of yeast flocculation, J. Inst. Brew. 103 (1997) 93-98. [5] J. Westman, M. Taherzadeh, C. Franzen, Inhibitor tolerance and flocculation of a yeast strain suitable for second generation bioethanol production, Electronic J. Biotechnol. 15 (2012) DOI: 10.2225 / vol15-issue3-fulltext-8 [6] U.S. Patent Application Publication No. 2017 / 0130208(A1) [7] D. Salt, O. Hay, O. Thomas, M. Hoare, P. Dunhill, Selective flocculation of cellular contaminants from soluble proteins using polyethyleneimine: a study of several organisms and polymer molecular weights. Enz. Microbiol. Technol. 17 (1995) 107-113. [8] J. Hughes, D. Ramsden, K. Symes, The flocculation of bacteria using cationic synthetic flocculants and chitosan. Biotechnol Tech. 4 (1990) 55-60. [9] European Patent No. 0240348

[10] T. McNerney, A. Thomas, A.Senczuk, K. Petty, X. Zhao, R. Piper, J. Carvalho, M. Hammond, S. Sawant, and J. Bussiere, PDADMAC flocculation of Chinese hamster ovary cells: Enabling a centrifuge-less harvest process for monoclonal antibodies. MAbs 7 (2015) 413-427.

[11] A. Chantuin, R. Curnish, The precipitation of plasma proteins by short-chain fatty acids Arch. Biochem. Biophys. 89 (1960) 218-220.

[12] M. McInney, A. Parkinson, A simple, nonchromatographic procedure to purify immunoglobulins from serum and ascites fluid, J. Immunol. Met., 96 (1987) 271-278.

[13] U.S. Patent No. 8,063,189 (B2)

[14] Y. Brodsky, C. Zhang, Y. Yigsaw, G. Vedanthum, Caprylic acid precipitation for impurity reduction: an alternative to conventional chromatography for monoclonal antibody purification, Biotechnol. Bioeng. 109 (2012) 2589-2598.

[15] P. Gagnon, R. Nian, YS. Yang, QY. Yang, CL. Lim, Non-immunospecific association of immunoglobulin G with chromatin during elution from protein A inflates host contaminants, aggregate content, and antibody loss, J. Chromatogr. A 1408 (2015) 151-160.

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

[17] U.S. Patent No. 9695216 (B2)

[18] P. Gagnon, 1996, Purification Tools for Monoclonal Antibodies, Validated Biosystems, Tucson, 296 pp.

[19] International Publication No. 2013 / 180647(A1) Brochure

[20] International Publication No. 2015 / 183180(A1) Brochure Summary of the Invention

[0012] A method has been developed to reduce host DNA contamination in mammalian cell cultures that is orthogonal to and expands the possibilities of conventional levels of DNAse treatment. It is also more effective than the citrate flocculation method. Although the method is primarily directed at AAV-containing cell culture harvests, cell lysates, or other AAV-containing preparations, it may also be applied to reduce DNA from other parvoviruses and other biological materials produced by cell culture.

[0013] According to the present invention, a method for removing chromatin from a cell culture harvest comprises providing a cell culture harvest containing a desired biological material, in particular selected from the group consisting of parvoviruses and adeno-associated viruses, incubating said cell culture harvest in an aqueous medium having an ionic strength equivalent to a NaCl concentration in the range of 3.0 M to saturation and a pH value in the range of 3.0 to 4.0, and separating said desired biological material from the solid produced.

[0014] In one embodiment of the method of the invention, the desired biological material may be a parvovirus or an adeno-associated virus.Similarly, the method of the invention can also be used to remove chromatin from cell cultures for the production of recombinant proteins, antibodies, IgG, IgM, or antigen-binding fragment forms of antibodies.

[0015] In other embodiments of the methods of the present invention, the harvest may be obtained from a culture of mammalian cells, insect cells, yeast cells, or bacterial cells.

[0016] In yet another embodiment of the method of the present invention, the aqueous medium may contain sodium chloride at a concentration ranging from about 3.0 M up to saturation.

[0017] In yet other embodiments of the methods of the present invention, the pH may be about 3.5±0.3.

[0018] In a further embodiment of the method of the invention, the incubation time may be up to 24 hours, in particular within the range of 30 minutes to 240 minutes, 45 minutes to 90 minutes, or 55 minutes to 65 minutes.

[0019] In yet a further embodiment of the process of the present invention, the solids may be separated from the soluble products by settling or filtration.

[0020] In another embodiment of the method of the invention, the incubation may be carried out in the presence of a solid phase material bearing primary amine groups.

[0021] In yet another embodiment of the method of the present invention, the solid phase having the primary amines can be in the form of discrete particles or in the form of a chromatographic device.

[0022] In yet other embodiments of the method of the present invention, the solid phase particles may be present in a volume fraction in the range of 1% to 10%, 2.5% to 7.5%, or 4.5% to 5.0% relative to the volume of the cell culture harvest in the aqueous medium.

[0023] In further embodiments of the methods of the present invention, the solid phase having a primary amine may be in a form selected from the group consisting of a column packed with porous particles, a column packed with nanofibers, a column packed with monolithic materials, a hydrogel, a porous membrane filter, and a depth filter.

[0024] In yet further embodiments of the method of the present invention, the primary amine surface of the solid phase may be provided with chemical moieties of other properties, such as secondary amines, tertiary amines, quaternary amines, hydrophobic moieties, hydrogen bonding moieties, metal affinity moieties, or combinations thereof.

[0025] In other embodiments of the methods of the present invention, the methods may be carried out in the presence of one or more salts, sugars, surfactants, divalent metal ions, allantoin, polyethylene glycol, flocculating agents, or combinations thereof.

[0026] In another embodiment of the method of the invention, the method further comprises reducing the ionic strength of the solution containing the desired biological product, contacting the solution with a cation exchanger, which binds the contaminants, thereby further removing the contaminants, further reducing the ionic strength of the solution containing the desired biological product, and contacting the solution with a cation exchanger, which binds the desired biological product, thereby purifying the desired biological product.

[0027] The subject of the invention is also the use of a kit for carrying out the method of the invention, said kit comprising solid phase particles in a concentrated medium in a container and instructions for carrying out said method, preferably said solid phase particles having primary amine groups.

[0028] "Ionic strength equivalent to NaCl" is defined by comparing the conductivity of a NaCl solution in pure water at the same temperature as the aqueous medium containing the cell culture harvest. In a preferred embodiment, the aqueous buffer contains NaCl between 3.0 and saturation.

[0029] "Chromatin" is the packed remnant of a host cell's chromosomes. Its main content is nucleosomes, which consist of DNA and proteins, especially histones. [Brief description of the drawings]

[0030] [Figure 1] FIG. 1 shows the effect of NaCl concentration on chromatin reduction at pH 3.5. [Diagram 2] FIG. 2 shows the improvement in performance by including particles with primary amines. [Diagram 3] FIG. 3 shows the effect of pH in the presence of solid phase particles bearing primary amines. [Figure 4] FIG. 4 shows the effect of pH on AAV recovery at 5M NaCl. [Diagram 5] FIG. 5 shows the results of cation exchange chromatography. [Figure 6] FIG. 6 shows DNA extraction and anion exchange chromatography of AAV purified by cation exchange chromatography. [Figure 7] FIG. 7 shows selective exclusion chromatography after DNA extraction. [Figure 8] FIG. 8 shows analytical anion exchange chromatography of AAV2 / 8 after extraction with particles bearing primary amines in the presence of 5.0 M NaCl at pH 3.5. [Figure 9]FIG. 9 shows a comparison of soft flocculation with citric acid and solid-phase extraction with particles containing primary amines under high salt concentrations. [Figure 10] FIG. 10 shows fine tuning of the pH response. [Figure 11] FIG. 11 shows the effect of salt concentration during solid phase extraction on cation exchange chromatography. [Figure 12] FIG. 12 shows the effect of particles having various proportions of primary amines. [Figure 13] FIG. 13 shows the effect of different salt types. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] One feature of the method is the use of characteristic and extremely high salt concentrations, such as 3M to saturation. Saturation concentrations vary by salt species. Sodium chloride (NaCl) is saturated at a concentration of about 5M. The extreme salt concentrations are intended to promote dissociation of host DNA from histone proteins, thereby rendering the host DNA soluble. To the extent that some of the host-derived DNA can bind to the AAV capsid surface, the extreme conditions are intended to co-promote dissociation and solubilization of a specific subpopulation of DNA from the capsid. The approach of deliberate dissociation and solubilization of host DNA from existing associations is noteworthy because it is counterintuitive to the challenge of removing host DNA. In the field of soft flocculation, for example, soluble contaminants can be profitably precipitated, since they remain in the soluble product.

[0032] Another feature of the method of the invention is the simultaneous use of pH values ​​in the range of pH 3.0 to pH 4.0 in conjunction with very elevated salt concentrations. These conditions are believed to further favor dissociation of host DNA from pre-existing associations with histones, transcription factors, and potentially other species including AAV capsids. The combination of high salt and low pH for the reduction of host DNA from biological materials is not known in the art. However, there are unexpected features of the AAV response to pH at high salt concentrations that take it a step further and make the most effective range completely unpredictable. It would be reasonable to assume that the relationship between pH and method performance would follow a linear progression with recovery decreasing with pH. Instead, experimental data shows that in the presence of 5.0 M NaCl, AAV capsid recovery improves with decreasing pH, reaching a maximum at pH 3.5, then plummeting at pH 2.5, showing an unexpected region of best performance within the narrow range of pH 3.0 to 4.0. See data in Examples 3 and 4.

[0033] This approach of applying elevated salt concentrations over a narrow range is by itself more effective than other known methods of extracting chromatin from AAV preparations, and any extensions would further enhance its utility.

[0034] The method may be optionally defined by the incorporation of solid phase extraction materials with primary amine surface chemistry, in addition to a narrow pH window at high salt concentration. At the high salt and low pH conditions of the basic method, the primary amine surface confers an unexpectedly high affinity for DNA to the solid phase. Amine-based solid phase surfaces are typically positively charged and are therefore classified as anion exchangers. It is widely understood in the art that anion exchangers provide the best DNA binding properties at salt concentrations below 0.1 M and slightly alkaline pH values, such as pH 8.0 to pH 8.5. Even within this pH range, it is widely recognized that anion exchangers are unable to bind DNA at salt concentrations above about 0.4 to 0.6 M NaCl. Much to our surprise, we have found that the primary amine solid phase used to carry the method is capable of binding DNA when exposed to a combination of high salt concentrations, such as 3 M to saturation, and strongly acidic pH conditions, such as pH 3.0 to pH 4.0.

[0035] Regardless of whether or not a primary amine solid phase is included, the final step of the method is separation of the sample from the solids produced by the method, while still under conditions of 3M to saturated salt and pH 3.0 to 4.0.

[0036] In addition to pH, salt concentration, and the presence or absence of a primary amine solid phase, the sample may optionally be treated with DNase, especially if there is a strong desire to reduce host DNA contamination to the lowest possible level. Such enzymes, basic methods of using enzymes, and methods of optimizing enzyme performance are widely known in the art, including in the field of AAV purification. Treatment with DNase may occur before or after the method, or at any stage of a multi-step purification sequence, for example after a tangential flow filtration step, or after a chromatography step.

[0037] The reaction mixture may optionally include a sugar such as sucrose at a concentration ranging from 1% to 10%. Alternatively, the reaction mixture may include a sugar such as sorbitol over a range of concentrations from 2% to 20%, or may include a sugar such as mannitol or xylitol over a similar range of concentrations. Alternatively, the reaction mixture may include other sugars or combinations of sugars. The inclusion of sugars is believed to favor the stability of the AAV capsid and may favor higher recovery of the product. Experimental data indicates that sugars do not significantly alter the degree of DNA extraction. In any such case, sugars may be present as they may have been included in the original sample being processed or may have been added with the process.

[0038] The reaction mixture may optionally contain the surfactant Pluronic F68 at concentrations ranging from 0.01% to 1.00%. Alternatively, the reaction mixture may contain the surfactant Tween, the surfactant Triton, other non-ionic or zwitterionic surfactants, or combinations of surfactants over a similar concentration range. The inclusion of a surfactant is believed to help suppress non-specific interactions that may lead to virus loss due to adhesion to the container and / or tube walls, and may favor higher virus recovery. Experimental data suggests that Pluronic F68 contributes modestly and indirectly, likely through this mechanism, to the ability of the method to extract DNA. In any such case, a surfactant may be present, as it may have been included in the original sample being processed or may have been added with the method.

[0039] The reaction mixture may optionally include a divalent metal cation, such as magnesium ion. Alternatively, the reaction mixture may include calcium ion. Alternatively, the reaction mixture may include a combination of magnesium ion and calcium ion. Alternatively, the reaction mixture may include other metal ions or combinations of ions. The inclusion of metal ions is believed to improve the stability of the AAV capsid and may improve product recovery. Experimental data suggests that the addition of metal ions indirectly and modestly contributes to the ability of the extraction method to remove host DNA. In any case, metal ions may be present because they may have been included in the original sample being processed or may have been added with the process.

[0040] The reaction mixture may optionally contain allantoin at concentrations ranging from 1% to 10% or 2% to 5%, or within broader or narrower, higher or lower ranges. Experimental data indicate that allantoin does not directly contribute to DNA extraction, but may indirectly enhance the overall performance of the method by reducing the content of turbidity-forming nanoparticles that may interfere with filtration or chromatographic methods. Allantoin may be added before or after other solids are removed from the sample.

[0041] The reaction mixture may optionally contain a non-ionic organic polymer, such as polyethylene glycol, at a concentration of 0.5% to 5.0%.

[0042] The reaction mixture may optionally contain common flocculants of various characteristics. One class of such flocculants are fatty acids having 6 to 10 carbon atoms at concentrations ranging from 0.1% to 1.0%. Alternatively and / or in addition, the reaction mixture may contain anionic flocculants such as chitosan, ethacridine, methylene blue, polyethyleneimine (PEI), polydiallyldimethylammonium chloride (pDADMAC), chlorhexidine, or benzalkonium chloride in amounts ranging from 0.01% to 0.1%. In any case, such flocculants may be present as they may have been included in the original sample being processed or may have been added with the process.

[0043] In a preferred embodiment, the method of the invention is followed by a further purification step.

[0044] It is clear that the high salt concentrations remaining after treatment require treatment to condition the sample suitable for the subsequent purification method. The pH of the sample may change with the reduction of the salt concentration. In some circumstances it may make sense to achieve these changes by tangential flow filtration (TFF). In some circumstances it may make sense to achieve these changes by buffer exchange chromatography. In some circumstances it may make sense to achieve these changes by dialysis. In some circumstances it may make sense to achieve these changes by dilution. How to choose between these options and how to do so has been well known in the art for decades.

[0045] In some cases, it may be beneficial to change the sample conditions in two steps rather than a single step. In one non-limiting example, the treated sample is diluted to a concentration of about 1.0 M NaCl. The diluent in this case may be a buffer such as 50 mM formic acid at a pH of about 3.5. The sample is then contacted with a solid phase, such as chromatographic particles, that has been chemically modified to exhibit sulfo (SO3) groups, or a chromatographic or filtration device that has been pre-equilibrated to the same conditions. Under such conditions, the AAV virus is not retained, but contaminants that have a higher affinity for the SO3 groups are retained and can be eliminated from the sample by this means. Such contaminants include, among others, histone proteins and other contaminants that are positively charged at the operating pH. Once these contaminants have been eliminated from the sample, the sample may be diluted significantly to allow the virus to be retained by chromatographic materials, including but not limited to SO3 cation exchange materials. Obviously, the easiest way to achieve an initial reduction in salt concentration is by simple dilution as described, but this may be optionally achieved by dialysis, TFF, or buffer exchange chromatography, depending on personal preference.

[0046] Experimental data suggest that in addition to removing host-derived DNA, the method also removes a large proportion of contaminating host-derived RNA and host-derived proteins. In practical terms, this has the benefit of further improving the performance of subsequent purification steps. Since DNA and RNA have many chemical similarities, it makes sense from a chemical point of view that a method that removes DNA should also remove RNA, but it does not explain how the method reduces protein contamination. One hypothesis is that histone proteins released from DNA under reaction conditions precipitate and then, due to their extreme hydrophobicity, act as nucleation centers for the secondary attachment of other protein species.

[0047] It will be clear to those skilled in the art that the strong binding of DNA to a primary amine solid phase implies that it will similarly support strong binding of other highly phosphorylated contaminants including endotoxins, lipid enveloped viruses, and extracellular vesicles such as exosomes, microvesicles, and apoptotic bodies, or virus or vesicle debris, along with organelles or membrane fragments from the original host cell. Because high salt concentrations and low pH are known to have adverse effects on viruses and extracellular vesicles individually, the combination of these conditions with the ability of the solid phase to bind viruses and vesicles is expected to enhance the elimination of both contaminant classes.

[0048] The most effective variant of the method uses neutral salts such as sodium chloride, potassium chloride, or other halide salts, all of which may be used at concentrations between 1.0 M and saturation. Higher concentrations of salt will generally promote more effective dissociation and solubilization of DNA from histones. Preliminary experimental data indicate that NaCl is most effective. To the extent that host DNA may be bound to the outer surface of the AAV capsid proteins, the highest salt concentration should also promote the most effective dissociation from the outer capsid wall. As a general rule, the salt concentration should be the highest while the AAV particles remain soluble. Salts of multivalent cations such as phosphate, citrate, and sulfate can give good results, but cause precipitation of AAV at concentrations above 1 M. They also weaken the attraction of DNA to the solid phase.

[0049] The method can be performed at acidic pH values ​​in the ranges of pH 2.5 - pH 6.5, 2.75 - 6.0, 3.0 - 5.0, 3.5 - 4.5, 3.3 - 3.7, 3.4 - 3.6, pH 3.5, pH 3.5 ± 0.1, 3.5 ± 0.2, 3.5 ± 0.3, 3.5 ± 0.4, 3.5 ± 0.5, or 3.5 ± 1.0. Although lower pH values ​​create a potential risk of damage to the AAV particles, published studies in the field suggest that values ​​down to pH 2.5 are tolerated for at least several hours. Experimental data clearly show that performing the method at pH values ​​below 3.0 or above 4.0 reduces the effectiveness of the method, but still provides better results than known alternatives in sample preparation.

[0050] The importance of buffer capacity for good process control is widely known in the art. Many resources are known and their use is well known to those skilled in the art for identifying appropriate buffers for specific pH ranges. Some non-limiting examples include buffers with a pK of 4.71. a Acetic acid, which has a pK of 3.77, is a suitable buffer for pH control over the range of 4.25 to 5.25 and has excellent buffering capacity at pH 4.5. a Formic acid, with a pK of 2.96, is a suitable buffer for pH control over the range of 3.25 to 4.25 and has excellent buffering capacity at pH 3.5 to 3.75. a Glycine, which has a pK of 2.34, is a suitable buffer for pH control over the range of 2.5 to 3.5 and has excellent buffering capacity at pH 3.0. a Alanine, which has a pK of 1.8 to 2.8, is a suitable buffer for pH control over the range of 1.8 to 2.8 and has excellent buffering capacity at pH 2.0 to 2.5. Glycine (pK 1.0) titrated to pH 3.5 ± 0.5 a 2.96) and formic acid (pK a 3.77) is a particularly suitable combination providing excellent buffering capacity and pH control within this range.

[0051] The concentration of the buffer should be sufficient to ensure that the pH of the reaction mixture is as close as possible to the selected target pH. Depending on the content and buffering properties of the sample, the buffer concentration may be in the range of 10 mM to 500 mM, 20 mM to 200 mM, or 50 mM to 100 mM. Selecting an appropriate buffer concentration is a well-known and well-formulated straightforward matter in the art of purifying biological materials. A convenient starting concentration is about 100 mM, since this concentration overwhelms the inherent buffering properties of many samples processed by the method. Higher or lower buffer concentrations will not affect the results obtained by the method, so long as the target pH is achieved.

[0052] The incubation time after achieving the target high salt and low pH conditions may be 15 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, longer, shorter, or intermediate. Experimental results show that a 60 minute incubation time achieves very similar results as a 120 minute incubation. Longer incubation times may promote more complete dissociation of DNA from histones and / or more complete binding of DNA to the solid phase. Shorter intervals may be more convenient. Given the known wide variation between AAV production media, it is within the norm of the art to consider a range of incubation times to identify the incubation time that supports the best results for any sample.

[0053] In one embodiment, the method is performed on a cell culture harvest in which the cells secrete AAV particles into the extracellular cell culture medium. In another embodiment, the method is performed on a cell lysate in which the AAV-containing cells have been treated to release AAV particles into the surrounding medium. In another embodiment, the method is performed on a partially purified preparation that contains an excess of DNA.

[0054] The method may also be performed on harvests or lysates containing non-AAV parvoviruses, or on non-viral products grown in cell culture, such as monoclonal antibodies or antibodies containing other proteins of interest.

[0055] In some embodiments, the incubation involves a solid phase, particularly a solid phase having a primary amine.

[0056] In one operating format, the solid phase having a primary amine is in the form of particles that are present in a mixture with the AAV cell culture harvest or cell lysate at high salt concentration and low pH. In another operating format, the solid phase having a primary amine is in the form of a device through which the AAV cell culture harvest or cell lysate is passed at high salt concentration and low pH. In another operating format, the solid phase having a primary amine is in the form of particles that are present in a mixture with the AAV cell culture harvest or cell lysate at high salt concentration and low pH, and after removal of the solid phase, the supernatant is passed over the solid phase having a primary amine in the form of a chromatography device.

[0057] It is clear that each of the operational formats exhibits characteristics that may be considered relative strengths or weaknesses. The particle-only format offers the greatest convenience since it does not require additional steps before performing subsequent chromatography or other purification steps. The solid-phase device offers a higher surface contact efficiency than the particles, which should translate into shorter contact times and lower levels of DNA removal. The combination of the two formats may be more effective than either one alone.

[0058] Any solid phase particle having a primary amine surface chemistry may be used to carry out the method. The solid phase particle may be non-porous or may contain pores and / or channels that allow diffusive and / or convective ingress of molecules up to 1 μm in size. Experimental data indicates that neither the size nor the porosity of the particle contributes substantially to the efficacy of the process. The important feature is to provide the reaction solution with a solid phase surface that exhibits primary amine chemistry. Consequently, the size of the particle may range from 5 nm to 500 nm, 10 nm to 400 nm, 40 nm to 300 nm, 80 nm to 200 nm, or 100 nm to 150 nm, or some other interval within the range of 5 nm to 500 nm. The average size of the pores and / or channels may range from 1 nm to 10,000 nm, 10 nm to 7,500 nm, 100 nm to 5,000 nm, or less than 1,000 nm to 2,000 nm, or some other interval less than 1 nm to 10,000 nm.

[0059] The solid phase particles may be added at a volumetric rate of about 1% of the sample volume, about 2% of the sample volume, about 3% of the sample volume, about 4% of the sample volume, about 5% of the sample volume, or about 10% of the sample volume, or at a greater or different rate. Those skilled in the art will appreciate that the amount of DNA in various AAV preparations may vary significantly. Since the entire method is specifically directed to removing DNA, simple stylized experiments may be necessary to determine the percentage of solid phase particles that most effectively removes DNA. The use of excess amounts, such as 5%, usually reserves the need to perform such experiments, but process economics may make such experiments desirable, as they may reveal the effectiveness of smaller amounts.

[0060] The solid phase extraction devices used to carry out the methods may be comprised of columns packed with porous particles, columns packed with nanofibers, columns packed with monolithic materials, hydrogels, membrane filters, and depth filters in any format or form.

[0061] The surface of the solid phase in any physical format is chemically modified to have at least one primary amine covalently attached to its surface. It may include only a single primary amine, a polymer of primary amines, a combination of primary amines with secondary amines, tertiary amines, or quaternary amines, or a ligand composed of any combination of amines. The solid phase may additionally have other chemical moieties, including but not limited to hydrophobic moieties, hydrogen bonding moieties, metal binding moieties, and negatively charged moieties. Solid phases lacking primary amines may also be present along with solid phase particles having primary amines.

[0062] As a good process control, the solid phase, whether in the form of discrete particles or a chromatographic device, should be equilibrated to the reaction conditions before contacting the sample.

[0063] Removal of solids may be accomplished by any convenient means. Many such methods and the criteria for selecting them are widely known in the art. Any such means may be used without altering the ability of the method to achieve the express purpose of reducing the amount of DNA in the AAV preparation.

[0064] In one embodiment, solids produced by the process may be removed by gravity settling.

[0065] In other embodiments, settling of solids may be accelerated by centrifugation.

[0066] In other embodiments, the settling of solids may be accelerated by sonication.

[0067] In other embodiments, settling of solids may be achieved while pumping the mixture upwardly through the cylinder, allowing the denser precipitate to settle by gravity while the carrier liquid containing the virus and depleted of solids rises and is discharged from the cylinder, In one such embodiment, after being discharged from the cylinder, the liquid may be passed through a filtration device.

[0068] In other embodiments where the solids are not removed by settling, the solids may be removed by filtration.

[0069] In other embodiments, the filtration method used to remove solids after treatment may be membrane filtration or depth filtration.

[0070] In other embodiments, removal of solids may be facilitated by the use of so-called bag or sock filters in which a porous membrane is configured as a cylinder with an open end that contains the sample and the other end closed, allowing liquid to be forced through the pores.

[0071] A non-limiting example is provided to illustrate the basic steps in carrying out the method. Particles bearing primary amines are pre-equilibrated with 100 mM formic acid, 4 M NaCl, pH 3.5. A defined volume of cell lysate or cell culture harvest is placed in an appropriate container. A dry salt such as sodium chloride is added in an amount sufficient to give a final concentration of 4 M, and the pH is adjusted to 3.5 by titration with formic acid. The solid phase particles may be already present when the sample is conditioned or may be added after the conditions are conditioned. In either case, the particles are present at a volume fraction of about 5% of the original sample volume. The combination may be mixed and incubated at ambient temperature for 60 minutes. The solids, including the solid phase extraction particles, are then removed by any convenient means, including but not limited to centrifugation or filtration. It is emphasized that the solids must be removed while they are still inherent in the working solution.

[0072] Other non-limiting examples are given to illustrate the basic steps in carrying out the method. Particles bearing primary amines are pre-equilibrated with 100 mM formic acid, 5 M NaCl, pH 3.5. A defined volume of cell lysate or cell culture harvest is placed in an appropriate container. Another container is filled with the same volume of twice the concentrated reagent mixture, containing enough sodium chloride to give a concentration of 5 M when diluted with the sample, enough formic acid, pH 3.5 to give 100 mM formic acid, pH 3.5 when diluted with the sample, and enough particles bearing primary amines to give a volume fraction of 5% when diluted with the sample. The sample is added to the concentrated reagent and may be mixed and incubated at ambient temperature for 60 minutes. Solids produced by exposing the sample to high salt and low pH are then removed by any convenient method, including but not limited to centrifugation or filtration.

[0073] Materials and instructions for carrying out the method may be provided in the form of a kit. In one embodiment, the kit provides solid phase particles in a concentrated buffer in a prefilled container to which the end user will add and incubate the sample. The incubated sample will be processed in a provided centrifugal spin cartridge with a 0.22 μm membrane to remove solids from the AAV-containing liquid. One version of the kit may contain enough material to perform 10 purifications of 10 mL each. Alternative versions may be configured to work with smaller or larger sample volumes and / or enough material to perform fewer or more purifications. An expanded version of the kit may include one or more chromatography devices to perform the purification. A further expanded version may include a pre-prepared tube containing cell lysis buffer.

[0074] In an alternative embodiment, the kit may include a buffer concentrate without a particulate solid phase in a pre-filled container to which the end user will add the sample and incubate for an appropriate time. The incubated sample will be processed in a provided centrifugal spin cartridge with a 0.22 μm membrane to remove solids from the AAV-containing liquid. The liquid will then be passed through a provided reusable chromatography device that contains a solid phase for extracting DNA by the method. One version of the kit may include enough material to perform 10 purifications of 10 mL each. Alternative versions may be configured to work with smaller or larger sample volumes and / or enough material to perform fewer or more purifications. An expanded version of the kit may include one or more chromatography devices to perform the purification. A further expanded version may include a prepared tube containing cell lysis buffer.

[0075] In a further alternative embodiment that does not require chromatography, the kit may include a buffer concentrate with a solid phase in a prefilled container to which the end user will add the sample and incubate for an appropriate time. The incubated sample will be optionally processed in a provided centrifugal spin cartridge with a 0.22 μm membrane to remove solids from the AAV-containing liquid. The sample will then be processed into a provided centrifugal cartridge with a 300,000 MWCO (molecular weight cut-off) membrane that allows the virus to pass through the membrane while retaining high molecular weight contaminants. The sample will then be processed into a provided centrifugal cartridge with a 100,000 MWCO membrane that allows the virus to pass through the membrane while retaining low molecular weight contaminants. This step will also concentrate the virus and allow buffer exchange into the final formulation.

[0076] Aspects of the present disclosure include the following. <Section 1> providing a cell culture harvest containing a desired biological product selected from the group consisting of parvovirus and adeno-associated virus; incubating the cell culture harvest in an aqueous medium having an ionic strength corresponding to a concentration of NaCl ranging from 3.0M to saturation and a pH value ranging from 3.0 to 4.0; separating said desired biological material from the produced solids. <Section 2> The method according to <Item 1>, wherein the harvested product is obtained from the culture of mammalian cells, insect cells, yeast cells, or bacterial cells. <Section 3> The method according to <Item 1> or <Item 2>, wherein the aqueous medium contains sodium chloride at a concentration within the range of 3.0 M to saturation. <Section 4> The method according to any one of <Item 1> to <Item 3>, wherein the pH is 3.5±0.3. <Section 5> The method according to any one of <Item 1> to <Item 4>, wherein the incubation time is up to 24 hours, preferably within the range of 30 to 240 minutes, 45 to 90 minutes, or 55 to 65 minutes. <Section 6> The method according to any one of <Item 1> to <Item 5>, wherein the solid is separated from the soluble product by settling or filtration. <Section 7> The method according to any one of <Item 1> to <Item 6>, wherein the incubation is carried out in the presence of a solid phase material having a primary amine group. <Section 8> The method according to <Item 7>, wherein the solid phase having the primary amine is in the form of discrete particles or in the form of a chromatography device. <Section 9> The method according to <Item 7> or <Item 8>, wherein the solid phase particles are present at a volume fraction within a range of 1% to 10%, 2.5% to 7.5%, or 4.5% to 5.0% relative to the volume of the cell culture harvest in the aqueous medium. <Section 10> The method according to <Item 7> or <Item 8>, wherein the solid phase having a primary amine is in a form selected from the group consisting of a column packed with porous particles, a column packed with nanofibers, a column packed with a monolithic material, a hydrogel, a porous membrane filter, and a depth filter. <Section 11> The method according to any one of <Item 1> to <Item 10>, wherein the surface of the primary amine of the solid phase is accompanied by a chemical moiety of alternative properties, such as a secondary amine, a tertiary amine, a quaternary amine, a hydrophobic moiety, a hydrogen-bonding moiety, a metal affinity moiety, or a combination thereof. <Section 12> The method according to any one of <Item 1> to <Item 11>, wherein the method is carried out in the presence of one or more salts, sugars, surfactants, divalent metal ions, allantoin, polyethylene glycol, flocculants, or combinations thereof. <Section 13> reducing the ionic strength of the solution containing the desired biological material; Contacting the solution with a cation exchanger that binds contaminants to further remove the contaminants. To do, further reducing the ionic strength of the solution containing the desired biological material; The method according to any one of <Item 1> to <Item 12>, further comprising contacting the solution with a cation exchanger that binds to the desired biological substance, thereby purifying the desired biological substance. <Section 14> Item 14. Use of a kit comprising solid-phase particles in a concentrated medium in a container and instructions for carrying out the method, preferably the solid-phase particles having primary amine groups, for carrying out the method according to any one of items 1 to 13. The invention is further illustrated by the following non-limiting examples. EXAMPLES

[0077] Example 1 Effect of NaCl concentration on chromatin reduction at pH 3.5. Figure 1 shows the results of an experiment at pH 3.5 with different salt concentrations ranging from 0M to 5M. The analytical method was size-exclusion chromatography with fluorescence monitoring to detect the intrinsic fluorescence of tryptophan residues in the protein. This method amplifies the sensitivity and allows the detection of AAV capsids in the sample. Samples were adjusted by titrating the pH by diluting 1:1 by volume with 200 mM formic acid, pH 3.5, and twice the amount of salt required to reach the target concentration. The experimental results showed that chromatin reduction improved as the NaCl concentration increased. AAV remained soluble throughout the entire range. Salt concentrations of 1M and 2M reduced chromatin contamination by perhaps 40%, but had little effect on reducing contamination by non-chromatin proteins. 3M was only slightly effective in reducing chromatin, but showed a significant improvement in reducing non-chromatin proteins. 4 M NaCl showed at least 90% reduction of chromatin and greater reduction of non-chromatin proteins while AAV remained soluble. The effect of 5 M NaCl was slightly better for reducing contaminants but may have slightly reduced AAV recovery.

[0078] Example 2 Superior performance with the inclusion of particles with primary amines. The ability of particles with primary amines to contribute to better overall results was evaluated. One sample was equilibrated with 5.0 M NaCl, 50 mM formic acid, 5 mM MgCl2, and 0.1% Pluronic F68, pH 3.5. The other sample was equilibrated under the same conditions except for the inclusion of particles with 5% primary amines. Both samples were incubated at room temperature for 1 hour, after which the solids were removed by membrane filtration. The results are compared to the untreated control in Figure 2. In conditions lacking particles, the chromatin content was reduced by approximately 40%, and non-chromatin proteins were reduced to a significant extent that could not be assessed from the figure. With the inclusion of particles, chromatin was reduced by more than 95%, and non-chromatin proteins were reduced to a much greater extent. AAV recovery was nearly the same with and without particles.

[0079] Example 3 Improved chromatin removal as a function of pH in the presence of solid-phase particles with primary amines. Figure 3 shows the effect of inclusion of primary amine particles and pH on the removal of chromatin and other contaminants while AAV remains soluble. All experiments were performed at 5 M NaCl and in the presence of 5% particles with primary amines, 100 mM formic acid, 5 mM MgCl2, and 0.1% Pluronic F68 (surfactant). In combination with Example 1, these results highlight that high salt concentration is the main variable, but show that pH makes a large contribution to overall efficacy. The best virus recovery was observed at pH 3.5, which also supports the lowest chromatin and non-chromatin protein content at pH 2.5. However, pH 2.5 results in the lowest AAV recovery. Recovery at pH 4.5 is almost as good as pH 3.5, but chromatin reduction is moderately poorer and non-chromatin protein contamination is 2-3 times higher than at pH 3.5. Results at pH 5.5 and 6.5 are inferior in all respects. The higher AAV recovery at pH 3.5 makes this pH especially recommended for carrying out the method. In another experiment comparing formic acid and glycine as buffers, the results with formic acid were clearly superior. In another experiment, it was observed that 5 mM MgCl2 made a small but clearly positive contribution to reducing contaminants while maintaining high virus recovery. In another series of experiments, incubation times of 1 and 2 hours were compared. 1 hour gave the same results as 2 hours.

[0080] Example 4 Cation exchange chromatography after DNA extraction AAV-containing insect cell lysate was equilibrated with 100 mM formic acid, 5.0 M NaCl, 5 mM MgCl2, 0.1% Pluronic F68, and particles with 5% primary amines, pH 3.5. The mixture was incubated at room temperature for 1 h and solids were removed by membrane filtration. The supernatant was diluted by adding 50 mM formic acid, pH 3.5 until the conductivity reached 40 mS / cm. A cation exchange monolith (CIMmultus SO3, BIA Separations) was equilibrated under the same conditions and loaded with the sample. As shown in Figure 4, a concentrated peak was obtained containing more than 90% pure AAV. Figure 4 plots AAV recovery as a function of pH and highlights two main unexpected findings. It was expected that the recovery would be highest at a pH closest to the physiological environment in which the virus normally resides. In fact, the lowest pH, 2.5, resulted in the lowest recovery at all pH values. The first unexpected finding is that the second lowest recovery was observed at pH 6.5, the closest physiological value, with an increase in recovery from pH 6.5 to pH 3.5. The second unexpected finding is that the highest virus recovery was observed at pH 3.5. These findings highlight the unpredictable nature of the system at high salt concentrations, in this case 5 M NaCl. The chromatograms are shown in Figure 5, along with an inset image showing the silver-stained PAGE gel.

[0081] Example 5 Cation exchange chromatography followed by anion exchange chromatography after DNA extraction To show DNA extraction in the context of the complete purification process, the sample processed by DNA extraction and cation exchange chromatography in Example 4 was further processed by anion exchange chromatography (CIMmac AAV, BIA Separations). Briefly, fractions E2 and E3 (see FIG. 5) were pooled, the pH was adjusted to 9.0, and the sample was diluted to a conductivity of about 2.5 mS / cm. The column was equilibrated with 50 mM Bis-Tris-Propane, pH 9.0, and the sample was loaded onto the column. The column was washed with 5 column volumes of equilibration buffer and then eluted with a linear gradient of 20 column volumes to a final concentration of 50 mM Bis-Tris-Propane, 200 mM NaCl, pH 9.0. The elution profile is shown in FIG. 6. A characteristic two-peak AAV profile is shown, the first peak corresponding to empty capsids and the second peak corresponding to capsids filled with vector DNA. Because capsid proteins are transparent to UV light, the internal DNA contributes to the excess absorbance at 260 nm of intact capsids. Empty capsids lacking internal DNA result in a protein-specific 260 nm / 280 nm absorbance. The absorbance ratio at 260 nm and 280 nm is considered an indicator of AAV purity, with values ​​below 1.30 indicating an impure preparation. Note that in this case, the ratio for intact capsids is approximately 1.35, indicating high purity. This is due to the effect of the DNA extraction step, which enhances the performance of both the cation and anion exchange chromatography steps of purification.

[0082] Example 6 Selective exclusion chromatography after DNA extraction AAV-containing insect cell lysate was equilibrated with 100 mM formic acid, 5.0 M NaCl, 5 mM MgCl2, and particles with 5% primary amines, pH 3.5. The mixture was incubated at room temperature for 1 hour and solids were removed by membrane filtration. The supernatant was diluted 1:1 with 3.0 M potassium phosphate, pH 7.0 and applied to a CIMmultus OH monolith (BIA Separations) pre-equilibrated with 1.5 M potassium phosphate, pH 7.0. After loading the sample, the column was washed with equilibration buffer to transfer unbound contaminants from the column. AAV was eluted with a linear salt gradient going down to a final 50 mM potassium phosphate buffer. Figure 7 shows the results of polyacrylamide gel electrophoresis (PAGE) analysis (silver stained) of the column fractions. The original processed sample is in the last well on the far right. The next well on the left shows the sample after DNA extraction. On the far left are molecular weight markers, followed by the DNA extracted sample that has been diluted and is ready to be loaded onto the column. The other wells show the contents of the fractions collected during the chromatography run. The content of the elution peak is shown in the center of the gel, labeled OH 01 E5. Note that the AAV band stains very strongly, while the staining of contaminants is noticeably weaker, indicating high purity.

[0083] Example 7 Differential membrane filtration after DNA extraction AAV-containing insect cell lysate was equilibrated with 100 mM formic acid, 5.0 M NaCl, 5 mM MgCl2, 0.1% Pluronic F68, and 5% primary amine particles, pH 3.5. The mixture was incubated at room temperature for 1 hour, and solids were removed by membrane filtration. The sample was titrated to a pH of 6.5±0.5 and then applied to a centrifugal membrane filter with a molecular weight cut-off of 300,000 kDa to retain the remaining high molecular weight chromatin, as seen in the sample of Example 4. The virus passed through the filter membrane and was then applied to a centrifugal membrane filter with a molecular weight cut-off of 100,000 kDa to retain the virus while allowing low molecular weight contaminants to pass through the membrane. The original sample volume was restored by adding 50 mM Hepes, 50 mM NaCl, 50 mM arginine, 1% sorbitol, 2 mM magnesium chloride, pH 7.0, and centrifuged again. This left the sample approximately 20-fold concentrated in new buffer. The product was evaluated by analytical anion exchange chromatography. The results are shown in Figure 8. For reference, compare with Figure 6.

[0084] Example 8 Comparison of citric acid flocculation and solid-phase extraction with particles containing primary amines under high salt concentrations Filtered AAV-containing harvest from SF9 cells was treated with the citrate flocculation method described by Potter and Byrne [6] to highlight the relative performance of the present invention. Citric acid was added to a final concentration of 20 mM, and the pH of the preparation was titrated to pH 3.5, relying on citrate salt added to provide buffering. In parallel, another aliquot of the filtered harvest was added with formic acid (100 mM), magnesium chloride (95 mM), Pluronic F68 (0.1%), and particles with primary amines (5% v:v), and then titrated to a pH of 3.5. Both treatments were incubated for 60 min before removing solids by filtration. Almost no visible solids were observed with the citrate flocculation treatment. The results are compared in Figure 9. As can be seen, the contaminant profile after citrate flocculation was worse than before treatment, and was dramatically worse than the treatment with high salt concentration and particles with primary amines.

[0085] Example 9 Fine-tuning pH response Examples 1 and 3 revealed that the optimal pH was about 3.5±0.5 in the absence of particles with primary amines. An additional series of experiments was performed to fine-tune the estimate of the pH optimum in the presence of particles with primary amines. Filtered AAV-containing harvest was treated with 5.0 M sodium chloride, 5 mM magnesium chloride, 1% Pluronic F68, and 5% v:v particles with primary amines. The samples were titrated to pH 3.3, 3.4, 3.5, 3.6, and 3.7, respectively. They were incubated for 60 minutes, solids were removed by filtration, and the samples were analyzed by size exclusion chromatography with monitoring by intrinsic fluorescence. As shown in FIG. 10, the pH that resulted in the most effective reduction of contaminants without obvious loss of AAV particles was pH 3.3, and higher pH values ​​increased contamination.

[0086] Example 10 Demonstration of alternatives to the use of extreme salt concentrations Example 1 showed that in the absence of particles with primary amines, the increase in the proportion of contaminants with increasing sodium chloride concentration was eliminated, with 4M NaCl resulting in lower contamination than 1M and 5M resulting in significantly lower contamination than 4M. To demonstrate that the effect of NaCl remains in the presence of particles with primary amines, a series of experiments was performed to demonstrate this with an alternative analytical format. In a new series of experiments, the effect of salt concentration during solid phase extraction was determined by fractionating the treated samples on a cation exchanger. Samples were prepared at pH 3.5 in the presence of 50 mM formic acid, 5 mM magnesium chloride, 1% Pluronic F68, 5% particles with primary amines, and sodium chloride at concentrations of 1M, 4M, and 5M. The results are shown in Figure 11. The profile corresponding to solid phase extraction with 1M NaCl shows heavy contamination leading to the main AAV peak. Contaminant levels are strongly reduced after solid phase extraction with 4M NaCl, with the lowest levels being obtained after solid phase extraction with 5M NaCl. These results show that extreme salt concentrations are much more beneficial in the presence of particles with primary amines than in their absence (Example 1).

[0087] Example 11 Proportional response to particles with primary amines A series of experiments were performed to determine the most favorable ratio of particles with primary amines. Filtered AAV2 / 8-containing cell culture harvest was treated with 50 mM formic acid, 5.0 M sodium chloride, 5 mM magnesium chloride, 1% Pluronic F68, and particles with primary amines at volume fractions of 2%, 3%, 4%, and 5%, pH 3.5. The mixture was incubated for 60 minutes, after which the solids were removed by membrane filtration. Samples were analyzed by size exclusion chromatography monitored by intrinsic fluorescence. As shown in Figure 12, increasing the ratio of particles resulted in clearly better contaminant reduction, with a significant improvement at 5%.

[0088] Example 12 Effects of different salt types Example 1 showed that increasing the level of sodium chloride improved the utility of the method in terms of removing contaminants while preserving AAV. Additional salt types were investigated to evaluate their relative utility. All experiments were performed with the same filtered AAV2 / 8 harvest from SF9 cells used in the other experiments. All experiments were performed with 50 mM formic acid, pH 3.5, in the presence of 5 mM magnesium chloride, 1% Pluronic F68, and particles with 5% primary amines. In one experiment, lithium chloride was added to a final concentration of 1.0 M. In another experiment, ammonium chloride was added to a final concentration of 1.0 M. In another experiment, sodium glutamate was added to a final concentration of 1.0 M. In another experiment, sodium acetate was added to a final concentration of 1.0 M, and in another experiment, potassium acetate was added to a final concentration of 1.5 M. All were compared to a 5.0 M sodium chloride control. As shown in FIG. 13, and comparing FIG. 13 with FIG. 1, sodium acetate and potassium acetate gave similar but better results than sodium chloride at similar concentrations. Reduction of chromatin and non-chromatin proteins was both significantly better, suggesting that higher concentrations of these salts may show utility over sodium chloride. However, the apparent recovery of AAV appeared to be lower. Lithium chloride and ammonium chloride appeared to show significantly better recovery of AAV and better reduction of non-chromatin proteins than sodium chloride, but poorer reduction of chromatin proteins. Monosodium glutamate also gave better reduction of contaminating proteins than sodium chloride at the same concentration, but was clearly inferior to all other salts in this concentration range. Beyond suggesting further investigation of salts other than sodium chloride, these results suggest the potential utility of combinations of two or more salts.

Claims

1. Providing a cell culture harvest containing the adeno-associated virus; incubating said cell culture harvest in an aqueous medium having a pH value in the range of 3.0 to 4.0 with a NaCl ionic strength in the range of 3.0 M to saturation; and and separating said adeno-associated virus from the produced solids.

2. 10. The method of claim 1, wherein the cell culture harvest is obtained from a culture of mammalian cells, insect cells, yeast cells, or bacterial cells.

3. 3. The method of claim 1 or claim 2, wherein the pH value is 3.5±0.

3.

4. The method according to any one of claims 1 to 3, wherein the incubation time is up to 24 hours.

5. The method according to any one of claims 1 to 3, wherein the incubation time is in the range of from 30 minutes to 240 minutes, or from 45 minutes to 90 minutes, or from 55 minutes to 65 minutes.

6. The process according to any one of claims 1 to 5, wherein the solids are separated from the soluble product by settling or filtration.

7. The method according to any one of claims 1 to 6, wherein the incubation is carried out in the presence of a solid phase carrying primary amine groups.

8. 8. The method of claim 7, wherein the solid phase having primary amine groups is a solid phase particle in the form of discrete particles or a chromatographic device.

9. 9. The method of claim 8, wherein the solid phase particles are present in a volume fraction in the range of 1% to 10%, 2.5% to 7.5%, or 4.5% to 5.0% relative to the volume of the cell culture harvest in the aqueous medium.

10. 9. The method of claim 7 or claim 8, wherein the solid phase having primary amine groups is in a form selected from the group consisting of a column packed with porous particles, a column packed with nanofibers, a column packed with monolithic materials, a hydrogel, a porous membrane filter, and a depth filter.

11. 9. The method according to claim 7 or claim 8, wherein the surface of the primary amine groups of the solid phase is accompanied by chemical moieties of alternative properties selected from the group consisting of secondary amines, tertiary amines, quaternary amines, hydrophobic moieties, hydrogen bonding moieties, metal affinity moieties, and combinations thereof.

12. 12. The method of any one of claims 1 to 11, wherein the method is carried out in the presence of one or more salts, sugars, surfactants, divalent metal ions, allantoin, polyethylene glycol, flocculants, or combinations thereof.

13. Reducing the ionic strength of a solution containing the adeno-associated virus; contacting the solution with a cation exchanger that binds contaminants to further remove the contaminants; further reducing the ionic strength of the solution containing the adeno-associated virus; The method of any one of claims 1 to 12, further comprising contacting the solution with a cation exchanger that binds the adeno-associated virus to purify the adeno-associated virus.

14. 10. Use of a kit comprising the solid phase particles having primary amine groups in the concentrated aqueous medium in a container and instructions for carrying out the method according to claim 9 for carrying out the method.

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