Compositions and methods for reducing the chromatin content of biological products

Abstract

A method for removing chromatin from a cell culture harvest, comprising the steps of: - providing a cell culture harvest containing a desired biological product selected from parvovirus or adeno-associated virus, - incubating the cell culture harvest in an aqueous medium having a pH in the range of 3.0 to 4.0 and an ionic strength corresponding to a concentration of NaCl in the range of 3.0 M to saturation, and - separating the desired biological product from the resulting solid.

Description

Background of the Invention

[0002] The DNA content in the final drug formulation is a major safety and regulatory concern for all biopharmaceuticals. This includes, for example, adeno-associated virus (AAV) used as a delivery vector for therapeutic DNA plasmids in the field of gene therapy. The safety concern is not the therapeutic DNA within the AAV capsid, but rather the DNA left behind by the host cells used to produce the AAV. Removing this host-derived DNA has been considered an obstacle to the development of the field because it has proven to be more difficult to remove than expected. This difficulty may in part stem from the possibility that host-derived DNA binds to the outer surface of the capsid.

[0003] The therapeutic DNA payload within the AAV capsid is commonly referred to as vector DNA. Some of this DNA may also be outside the capsid due to incomplete insertion or subsequent damage to the initially filled capsids. This DNA is not considered a major safety concern, but it is an issue because it has the potential to increase the apparent number of DNA-filled capsids measured by various DNA tests. Some sample preparation methods can limit the impact of vector DNA outside the capsid, but it would be better if it were completely absent.

[0004] Treatment of the AAV harvest with nucleases to cleave host-derived DNA is a common feature of the AAV purification step. The limitation of this method is that host DNA is protected from enzymatic cleavage by strong binding to histones. A significant amount of host DNA remains undigested and is retained in the AAV preparation. Some purification steps also use anion exchange chromatography to increase the overall reduction of host DNA. The basic principle is that DNA should bind more strongly to the anion exchanger than AAV, such that AAV can be eluted while the DNA remains bound to the anion exchanger. This treatment does reduce the host DNA content, but it does not completely eliminate it, and its failure is related to the same issue that limits the effectiveness of the restriction enzyme treatment: the host cell DNA in the harvest is present in tight binding with histones. In addition to physically protecting the DNA from enzymatic cleavage, the strongly bound histones also produce mixed aggregates with intermediate charge characteristics, so a subset of the DNA will still co-elute with the virus [1].

[0005] Alternatively, by lowering the pH of the harvest, a subset of the host DNA that remains bound to histones can be precipitated from the cell culture harvest. This method was described in 1994 by titrating the pH into the range of pH 2.6 to pH 5.0 using a citrate buffer at physiological salt concentration [2]. This work was carried out on mammalian cell culture harvests, but the technique has been applied to bacteria, yeast, fungi, and insect cells [3 - 5]. It has recently been applied to mammalian cell culture harvests in the same pH and salt concentration range for flocculating AAV contaminants including host DNA [6].

[0006] It is also described that the use of cationic heterocyclic compounds and soluble cationic polymers results in clarification and reduction of host cell DNA contamination [7-10]. It is also known that anionic fatty acids and heterocyclic anions can reduce DNA levels [11-16]. A common feature of these methods is that they use salt concentrations less than 0.5 M (500 mM), for example in the range of about 15 - 300 mM salt, most commonly 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]. It is well known that it has a high affinity for endotoxin

[17] .

[0007] The pKa of DNA is known to be approximately 2.6. Host DNA in cell culture harvests is first bound to histones in nucleosome arrays of various sizes. Histones are highly hydrophobic with isoelectric points in the range of about 9 to 11. Host DNA dissociates from histones in 1 M NaCl and the dissociation increases as the NaCl concentration is raised to 4 M, but is not completely dissociated even at this concentration. Histones start to form insoluble aggregates at NaCl concentrations greater than 1 M. The dissociated DNA is soluble under high salt conditions, but if the salt concentration is decreased, it re-binds to histones.

[0008] Surfaces coated with primary amine ligands are known to bind biomolecules more firmly than surfaces with quaternary amine ligands. This includes virus particles, DNA, endotoxin, and acidic proteins. They generally bind these molecules at slightly higher salt concentrations compared to quaternary amines on similar surfaces. DNA can also be eluted from primary amine ligands at slightly higher salt concentrations but still below 1 M sodium chloride. Experiments on quaternary amine surfaces and primary amine surfaces are most often carried out at neutral to weakly basic pH (pH 7.0 to 8.5). It is known that treatment of mammalian cell culture harvests with hydrophobic quaternary amine (cholestyramine) particles at neutral pH and 50 to 150 mM sodium chloride salt concentration binds some of the DNA in mammalian cell culture harvests

[18] . Subsequent removal of the particles results in a corresponding decrease in the DNA content of the harvest.

[0009]

[19] discloses a method for purifying a sample containing a desired protein, comprising the following steps: (i) providing a packed chromatographic column having positively charged porous particles, (ii) equilibrating the chromatographic column to conditions under which the desired protein in the sample is to be eluted, (iii) contacting the sample with the packed chromatographic column, wherein the volume of the sample applied to the packed chromatographic column is less than or equal to the particle spacing of the positively charged porous particles within the packed chromatographic column, (iv) eluting the desired protein from the packed chromatographic column, wherein the desired protein is in a purer state and under the conditions under which the packed chromatographic column was equilibrated; wherein the desired protein is an antibody, an antibody fragment, an antibody derivative or an antibody fusion protein. In essence,

[19] is about removing viruses and producing the desired protein, while the viruses are unwanted. This reference does not mention removing DNA while retaining the viruses.

[0010]

[20] discloses a method for enhancing the reduction of 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 does not mention removing DNA from the viruses and leaving the viruses purified from the viral DNA.

[0011] All references cited herein are incorporated herein by reference in their entirety to the extent that such incorporation is not inconsistent with the express teachings herein.

[0012] References

[0013] [1] R. Nian, P. Gagnon, Advanced chromatin extraction improves the performance and yield of immunoglobulin G monoclonal antibody capture based on cation exchange chromatography, J. Chromatogr. A 1453 (2016) 54–61.

[0014] [2] B. Lydersen, T. Brehm-Gibson, A. Murel, Acid precipitation of mammalian cell fermentation broth, Ann. N.Y. Acad. Sci. 745 (1994) 222–231.

[0015] [3] M. Westoby, J. Chrostowski, P. de Vilmorin, J.P. Smelko, J.K. Romero, Influence of solution environment on the properties of mammalian cell fermentation broth: enhanced impurity removal and clarification performance, Biotechnol. Bioeng. 108 (2011) 50–58.

[0016] [4] E. Soares, Quantification of yeast flocculation, J. Inst. Brew. 103 (1997) 93–98.

[0017] [5] J. Westman, M. Taherzadeh, C. Franzen, Inhibitor tolerance and flocculation of yeast strains suitable for second generation bioethanol production, Electronic J. Biotechnol. 15 (2012) DOI: 10.2225 / vol15-issue3-fulltext-8

[0018] [6] US 20170130208 A1.

[0019] [7] D. Salt, O. Hay, O. Thomas, M. Hoare, P. Dunhill, Selective flocculation of cellular contaminants in soluble proteins using polyethylenimine: Studies on several organisms and polymer molecular weights. Enz. Microbiol. Technol. 17 (1995) 107–113.

[0020] [8] J. Hughes, D. Ramsden, K. Symes, Flocculation of bacteria using cationic synthetic flocculants and chitosan. Biotechnol Tech. 4 (1990) 55–60.

[0021] [9] EP 0240 348.

[0022]

[10] T. McNerney, A. Thomas, A. Senczuk, K. Petty, X. Zhao, R. Piper, J. Carvalho, M. Hammond, S. Sawant, and J. Bussiere, Flocculation of Chinese hamster ovary cells with PDMAMAC: Towards a centrifugation-free harvest process for monoclonal antibodies. MAbs 7 (2015) 413–427.

[0023]

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

[0024]

[12] M. McInney, A. Parkinson, A simple non-chromatographic step for the purification of immunoglobulins from serum and ascites, J. Immunol. Met., 96 (1987) 271-278.

[0025]

[13] US 8,063,189 B2.

[0026]

[14] Y. Brodsky, C. Zhang, Y. Yigsaw, G. Vedanthum, Precipitation of Octanoic Acid to Reduce Impurities: An Alternative to Traditional Chromatography for Monoclonal Antibody Purification, Biotechnol. Bioeng. 109 (2012) 2589 - 2598.

[0027]

[15] P. Gagnon, R. Nian, Y - S. Yang, Q - Y. Yang, C - L. Lim, Non - immunological Specific Binding of Immunoglobulin G to Chromatin during Elution from Protein A Increases Host Contaminants, Aggregate Content, and Antibody Loss, J. Chromatogr. A 1408 (2015) 151–160.

[0028]

[16] R. Nian, W. Zhang, L. Tan, J. Lee, X. Bi, Y - S. Yang, H - T. Gan, P. Gagnon, Advanced Chromatin Extraction Improves the Capture Performance of Protein A Affinity Chromatography, J. Chromatogr. A 1431 (2016) 1–7.

[0029]

[17] US 9,695,216 B2.

[0030]

[18] P. Gagnon, 1996, Purification Tools for Monoclonal Antibodies, Proven Biological Systems, Tucson, 296 pages.

[0031]

[19] WO 2013 / 180647 A1.

[0032]

[20] WO 2015 / 183180 A1. Summary of the Invention

[0033] A method has been developed for reducing host DNA contamination in mammalian cell cultures that is orthogonal to and extends the capabilities of traditional DNA lyase treatments. It is also more effective than the method of citric acid flocculation. The method is primarily directed at cell culture harvests, cell lysates, or other preparations containing AAV, but can also be used to reduce DNA from other parvoviruses and other cell - culture - derived biological products.

[0034] According to the present invention, a method for removing chromatin from a cell culture harvest comprises the following steps:

[0035] — providing a cell culture harvest containing a desired biological product, said biological product being particularly selected from parvoviruses or adeno - associated viruses,

[0036] — incubating the cell culture harvest in an aqueous medium having a pH in the range of 3.0 to 4.0 and an ionic strength corresponding to a concentration of NaCl in the range of 3.0 M to saturation, and

[0037] — separating the desired biological product from the resulting solid.

[0038] In one embodiment of the method of the present invention, the desired biological product can be parvovirus or adeno-associated virus. Similarly, the method of the present invention can also be used to remove chromatin from cell cultures to produce recombinant proteins, antibodies, IgG, and IgM, or antigen-binding fragments of antibodies.

[0039] In another embodiment of the method of the present invention, the harvest can be obtained from cultures of mammalian cells, insect cells, yeast cells, or bacterial cells.

[0040] In yet another embodiment of the method of the present invention, the aqueous medium can contain sodium chloride at a concentration in the range of about 3.0 M up to saturation.

[0041] In yet another embodiment of the method of the present invention, the pH can be about 3.5 ± 0.3.

[0042] In yet another embodiment of the method of the present invention, the incubation period can be up to 24 hours, particularly in the range of 30 minutes to 240 minutes, or 45 minutes to 90 minutes, or 55 minutes to 65 minutes.

[0043] In yet another embodiment of the method of the present invention, the solid can be separated from the soluble product by sedimentation or filtration.

[0044] In another embodiment of the method of the present invention, the incubation can be carried out in the presence of a solid-phase material having a primary amine group.

[0045] In yet another embodiment of the method of the present invention, the solid phase with a primary amine can be in the form of loose particles or in the form of a chromatography device.

[0046] In yet another embodiment of the method of the present invention, the solid-phase particles can be present in a volume ratio in the range of 1% to 10%, or 2.5% to 7.5%, or 4.5% to 5.0% relative to the volume of the cell culture harvest in the aqueous medium.

[0047] In yet another embodiment of the method of the present invention, the form of the solid phase with a primary amine can be selected from the group consisting of columns filled with porous particles, columns filled with nanofibers, columns filled with monoliths, hydrogels, porous membrane filters, and depth filters.

[0048] In yet another embodiment of the method of the present invention, the primary amine surface of the solid phase may be accompanied by chemical residues of alternative features, such as secondary amines, tertiary amines, quaternary amines, hydrophobic residues, hydrogen bond residues, metal affinity residues, or combinations thereof.

[0049] In another embodiment of the method of the present invention, the method may be carried out in the presence of one or more salts, sugars, surfactants, divalent metal ions, allantoin, polyethylene glycol, flocculants, or combinations thereof.

[0050] In another embodiment of the method of the present invention, the following steps are further included:

[0051] —Reducing the ionic strength of the solution containing the desired bioproduct;

[0052] —Contacting the solution with a cation exchanger to remove more contaminants, wherein the cation exchanger binds to the contaminants;

[0053] —Further reducing the ionic strength of the solution containing the desired bioproduct;

[0054] —Contacting the solution with a cation exchanger to purify the desired bioproduct, wherein the cation exchanger binds to the desired bioproduct.

[0055] The subject matter of the present invention also lies in the use of a kit for implementing the method of the present invention, the kit comprising solid phase particles in a concentrated culture medium in a container and instructions for performing the method, preferably wherein the solid phase particles carry primary amine groups.

[0056] "Ionic strength corresponding to NaCl" is defined by comparing the conductivity of an 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 3.0 to saturation of NaCl.

[0057] "Chromatin" is the residue of the host cell chromosome mass. Its main components are nucleosomes, composed of DNA and proteins, especially histones. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Shows the effect of NaCl concentration on chromatin reduction at pH 3.5.

[0059] Figure 2 Shows the performance improvement by adding particles with primary amines.

[0060] Figure 3 Shows the effect of pH value in the presence of solid phase particles with primary amines.

[0061] Figure 4Shows the effect of pH on AAV recovery at 5M NaCl.

[0062] Figure 5 Shows the results of cation exchange chromatography.

[0063] Figure 6 Shows the anion exchange chromatography of AAV purified by DNA extraction and cation exchange chromatography.

[0064] Figure 7 Shows size exclusion chromatography performed after DNA extraction.

[0065] Figure 8 Shows the analytical anion exchange chromatography of AAV2 / 8 after extraction with amine-functionalized particles at pH 3.5 in the presence of 5.0M NaCl.

[0066] Figure 9 Shows a comparison of citrate flocculation and solid phase extraction on amine-functionalized particles under high salt conditions.

[0067] Figure 10 Shows the refinement of the pH response.

[0068] Figure 11 Shows the effect of salt concentration on cation exchange chromatography during solid phase extraction.

[0069] Figure 12 Shows the effect of different ratios of amine-functionalized particles.

[0070] Figure 13 Shows the effect of different salt species. DETAILED DESCRIPTION OF THE INVENTION

[0072] One feature of the method is that it uses unique and unusually high salt concentrations, e.g., from 3M to saturation. The saturation concentration varies for different salts. Sodium chloride (NaCl) saturates at a concentration of about 5M. The extreme salt concentrations are intended to promote dissociation of host DNA from histones, rendering the host DNA soluble. To the extent that a subset of the host-derived DNA may bind to the AAV capsid surface, the extreme conditions are intended to jointly promote dissociation and solubilization of a specific DNA subset from the capsid. The method of specifically dissociating and solubilizing host DNA from pre-existing complexes is notable because it is anomalous relative to the task of removing it. For example, in the field of flocculation, benefit is obtained by precipitating contaminants since the soluble contaminants remain in the soluble product.

[0073] Another feature of the method of the present invention is that it simultaneously employs a pH value in the range of pH 3.0 to pH 4.0, concomitant with a highly elevated salt concentration. These conditions are thought to further facilitate the dissociation of host DNA from pre-existing complexes with other species (including histones, transcription factors) and potentially from the AAV capsid. The combination of high salt concentration and low pH to reduce host DNA from any biological product is unknown in the art. However, AAV has an unexpected characteristic in its response to pH under high salt conditions, which goes even further and makes the most effective range completely unpredictable. It could reasonably be speculated that the relationship between the pH value and the performance of the method would follow a linear progression, i.e., the recovery rate would decrease with decreasing pH value. On the contrary, experimental data show that in the presence of 5.0 M NaCl, the recovery rate of AAV capsids increases with decreasing pH value, reaches a maximum at pH 3.5, and then drops sharply at pH 2.5, revealing an unexpected window of optimal performance within the narrow range of pH 3.0 to 4.0. See the data in Examples 3 and 4.

[0074] The method of using an elevated salt concentration within this narrow window is itself more effective than other known methods for extracting chromatin from AAV preparations. Optional extensions further enhance its utility.

[0075] In addition to the narrow pH window at high salt concentration, the method can optionally be defined by binding the solid phase extraction material to a primary amine surface chemistry. Under the high salt and low pH conditions of the basic method, the primary amine surface confers an unexpectedly high affinity of the solid phase for DNA. Amine-based solid phase surfaces are generally classified as anion exchangers because they are positively charged. It is widely understood in the art that anion exchangers provide optimal DNA binding characteristics at salt concentrations below 0.1 M and weak basic pH values (e.g., pH 8.0 to pH 8.5). Even within this pH range, it is well known that anion exchangers are unable to bind DNA at salt concentrations greater than approximately 0.4 to 0.6 M NaCl. Very surprisingly, we found that the primary amine solid phase used to perform this method is able to bind DNA when exposed to a combination of high salt concentrations (such as 3 M to saturation) and strong acidic pH conditions (such as pH 3.0 to pH 4.0).

[0076] The final step of the method (with or without the primary amine solid phase) is to separate the sample from the solid produced by the method, still under the conditions of 3 M to saturation salt and pH 3.0 to 4.0.

[0077] In addition to pH, salt concentration, and the presence or absence of a primary amine solid phase, particularly where it is strongly desired to reduce host DNA contamination to the lowest possible level, the sample can optionally be treated with a DNA lyase. Such enzymes, the basic methods of using them, and methods of optimizing their performance are well known in the art, including in the field of AAV purification. Treatment with a DNA lyase can be carried out before or after this method, or at any stage of a multi-step purification sequence, such as after a tangential flow filtration step or after a chromatography step.

[0078] The reaction mixture can optionally contain a sugar, such as sucrose in a concentration range of 1% to 10%. Or it can contain a sugar, such as sorbitol in a concentration range of 2% to 20%, or mannitol or xylitol in a similar concentration range. Or it can contain other sugars or combinations of sugars. The inclusion of a sugar is considered to be beneficial for the stability of the AAV capsid and can be beneficial for a higher recovery of the product. Experimental data show that sugars do not significantly alter the extent of DNA extraction. In any such case, the sugar may be present because it is in the original sample being treated, or because it is added with this treatment.

[0079] The reaction mixture can optionally contain the surfactant Pluronic F68 in a concentration range of 0.01% to 1.00%. Or it can contain a surfactant such as Tween in a similar concentration range, or the surfactant Triton, or any other non-ionic surfactant or zwitterionic surfactant or combination of surfactants. The inclusion of a surfactant is considered to help inhibit non-specific interactions that can lead to loss of virus by adhesion to the container and / or the tube wall and can be beneficial for a higher recovery of the virus. Experimental data show that Pluronic F68 may moderately and indirectly benefit the ability of this method to extract DNA by this mechanism. In any such case, the surfactant may be present because it is in the original sample being treated, or because it is added with this method.

[0080] The reaction mixture can optionally contain divalent metal cations, such as magnesium ions. Or it can contain calcium ions. Or it can contain a combination of magnesium and calcium ions. Or it can contain other metal ions or combinations of ions. The inclusion of metal ions is considered to increase the stability of the AAV capsid and can increase the recovery of the product. Experimental data show that the addition of metal ions indirectly and moderately contributes to the ability of this method to remove host DNA during extraction. In any case, the metal ions may be present because they are in the original sample being treated, or because they are added with this treatment.

[0081] The reaction mixture may optionally contain allantoin in a concentration range of 1% to 10%, or 2% to 5%, or in a wider or narrower, higher or lower range. Experimental data show that allantoin does not directly contribute to DNA extraction, but can indirectly improve the overall performance of the method by reducing the content of turbidity-forming nanoparticles that would impede filtration or chromatographic methods. Allantoin can be added before or after other solids are removed from the sample.

[0082] The reaction mixture may optionally contain a non-ionic organic polymer, such as polyethylene glycol, at a concentration of 0.5% to 5.0%.

[0083] The reaction mixture may optionally contain common flocculants of various properties. One class of such flocculants includes fatty acids containing 6 to 10 carbon atoms at a concentration of 0.1% to 1.0%. The reaction mixture may alternatively and / or additionally contain anionic flocculants in a content range of 0.01% to 0.1%, such as chitosan, acridine, or methylene blue, or polyethyleneimine (PEI), or polydiallyldimethylammonium chloride (pDADMAC), or chlorhexidine or benzalkonium chloride. In any case, such flocculants may be present because they are in the original sample being processed or because they are added with this treatment.

[0084] In a preferred embodiment, a further purification step is carried out after the method of the present invention.

[0085] It is obvious that the high concentration of salts remaining after treatment needs to be processed to make the sample conditions suitable for subsequent purification methods. The pH value of the sample may change as the salt concentration decreases. In some cases, it may be advantageous to achieve these changes by tangential flow filtration (TFF). In some cases, it may be advantageous to achieve these changes by buffer exchange chromatography. In some cases, it may be advantageous to achieve these changes by dialysis. In some cases, it may be advantageous to achieve these changes by dilution. These options, how to select them, and how to implement them have been well known in the art for decades.

[0086] In some cases, it may be useful to change the sample conditions in two steps rather than one. In an illustrative but non-limiting example, the treated sample is diluted to a concentration of about 1.0 M NaCl. In this case, the diluent can be a buffer, such as 50 mM formic acid with a pH of about 3.5. Then the sample is contacted with a solid phase that has been chemically modified to exhibit sulfonyl (SO 3 )), where the solid phase is, for example, chromatographic particles, or a chromatographic device, or a filtration device that has been pre-equilibrated to the same conditions. Under these conditions, the AAV virus is not retained, but for SO 3Contaminants with a higher affinity for the group may be retained and eliminated from the sample in this way. Such contaminants particularly include histones and other contaminants that are positively charged at the operating pH. These contaminants have been eliminated from the sample, and the sample can be diluted to a greater extent so that the virus can be retained by the chromatographic material, which includes but is not limited to SO 3 cation exchange material. Obviously, the simplest way to achieve an initial reduction in salt concentration is simple dilution as described, but it can optionally be achieved by dialysis, TFF, or buffer exchange chromatography according to personal preference.

[0087] Experimental data show that in addition to removing host-derived DNA, this method also removes most of the contaminating host-derived RNA and host-derived proteins. At a practical level, this provides the benefit of further enhancing the performance of subsequent purification steps. From a chemical perspective, it is logical to remove RNA by the method of removing DNA because they have many chemical similarities, but this does not explain how this method reduces protein contamination. One hypothesis is that histones are released from DNA precipitates under the reaction conditions and then, due to their extreme hydrophobicity, they act as nucleation centers for the secondary proliferation of other protein species.

[0088] It is obvious to those skilled in the art that the strong binding of DNA to the primary amine solid phase means that it will support similar strong binding to other highly phosphorylated contaminants, including endotoxins, lipid-enveloped viruses, and extracellular vesicles such as exosomes, microvesicles, and apoptotic bodies; or viral or vesicular debris, as well as organelles or membrane fragments from the original host cell. Since high salt concentrations and low pH values are known to be destructive to viruses and extracellular vesicles respectively, the combination of these conditions with the ability of the solid phase to bind viruses and vesicles is expected to enhance the elimination of these two types of contaminants.

[0089] The most effective variant of this method uses neutral salts, such as sodium chloride, potassium chloride, or other halide salts, all of which can be used at concentrations from 1.0 M to saturation. Higher salt concentrations generally promote more effective dissociation and dissolution of DNA from histones. Preliminary experimental data show that NaCl is the most effective. In terms of the extent to which host DNA may bind to the outer surface of the AAV capsid protein, the highest salt concentration should also promote its most effective dissociation from the outer wall of the capsid. Generally speaking, the salt concentration should be the highest concentration at which the AAV particles remain soluble. Polyvalent anion salts such as phosphates, citrates, and sulfates can have a positive effect, but they cause AAV precipitation at concentrations greater than 1 M. They also weaken the attraction of DNA to the solid phase.

[0090] This method can be implemented within an acidic pH range of pH 2.5 to pH 6.5, 2.75 to 6.0, 3.0 to 5.0, or 3.5 to 4.5, or 3.3 to 3.7, or 3.4 to 3.6, or pH 3.5, or pH 3.5 ± 0.1, or 3.5 ± 0.2, or 3.5 ± 0.3, or 3.5 ± 0.4, or 3.5 ± 0.5, or 3.5 ± 1.0. Low pH values pose a potential risk of damaging AAV particles, but published research in this field indicates that a pH value as low as 2.5 can be tolerated for at least several hours. Experimental data clearly show that implementing this method at pH values below 3.0 or above 4.0 reduces the effectiveness of the method, but it is still better than the results of known alternative sample preparation methods.

[0091] The importance of buffer capacity for good process control is well known in the art. Many resources are known for determining suitable buffers for specific pH ranges, and their use is well known to those skilled in the art. Without limiting the methods by way of example only, acetic acid with a pKa of 4.71 is a buffer suitable for controlling the pH within the range of 4.25 to 5.25 and has good buffering capacity at pH 4.5. Formic acid with a pKa of 3.77 is a buffer suitable for controlling the pH within the range of 3.25 to 4.25 and has good buffering capacity within the range of pH 3.5 to 3.75. Glycine with a pKa of 2.96 is a buffer suitable for controlling the pH within the range of 2.5 to 3.5 and has good buffering capacity at pH 3.0. Alanine with a pKa of 2.34 is a buffer suitable for controlling the pH within the range of 1.8 to 2.8 and has good buffering capacity at pH 2.0 to pH 2.5. The combination of glycine (pKa 2.96) titrated to pH 3.5 ± 0.5 and formic acid (pKa 3.77) is a particularly suitable combination that provides good buffering capacity and controls the pH within this range.

[0092] The concentration of the buffer should be sufficient to ensure that the pH value of the reaction mixture is as close as possible to the selected target pH value. Depending on the content and buffering characteristics of the sample, the concentration of the buffer can be in the range of 10 mM to 500 mM, or 20 mM to 200 mM, or 50 mM to 100 mM. Selecting an appropriate buffer concentration is a routine and simple matter well known throughout the field of biologic purification. A convenient starting concentration is approximately 100 mM because this concentration will exceed the natural buffering characteristics of most samples to be processed by this method. Higher or lower buffer concentrations will not affect the results obtained by this method as long as the target pH value is achieved.

[0093] After achieving the target high-salt and low-pH conditions, the incubation period can be 15 minutes, or 30 minutes, or 60 minutes, or 90 minutes, or 120 minutes, or longer, or shorter, or an intermediate time period. Experimental results indicate that the results obtained with a 60-minute incubation period are very similar to those obtained with a 120-minute incubation period. Longer incubation periods 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 wide variability known to exist between AAV product media, it is within the scope of the art to consider a range of incubation periods to determine the incubation time that supports optimal results for any given sample.

[0094] In one embodiment, the method is performed on a cell culture harvest, where cells secrete AAV particles into the extracellular cell culture medium. In another embodiment, the method is performed on a cell lysate, where 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 containing excess DNA.

[0095] The method can also be performed on a harvest or lysate containing non-AAV parvovirus. Or it can be performed on a non-viral product grown in cell culture, such as an antibody, including a monoclonal antibody or other protein of interest.

[0096] In some embodiments, the incubation includes a solid phase, particularly a solid phase having a primary amine.

[0097] In one form of operation, the solid phase with a primary amine is in particulate form and is present in a mixture of AAV cell culture harvest or cell lysate at high salt and low pH. In another form of operation, the solid phase with a primary amine is in the form of a device through which the AAV cell culture harvest or cell lysate at high salt and low pH passes. In another form of operation, the solid phase with a primary amine is in the form of particles present in a mixture of AAV cell culture harvest or cell lysate at high salt and low pH, and after removal of the solid phase, the supernatant passes through a solid phase with a primary amine in the form of a chromatographic device.

[0098] It is apparent that each form of operation has characteristics that can be considered relative advantages or disadvantages. Only the particulate form is the most convenient because it does not require an additional step before performing subsequent chromatography or other purification steps. The surface contact efficiency of the solid phase device is higher than that of the particles, which should translate into a shorter contact time and a lower level of DNA removal. A combination of the two forms may be more effective than using either one alone.

[0099] Any solid-phase particles with primary amine surface chemistry can be used to implement this method. The solid-phase particles can be non-porous, or they may contain pores and / or channels that allow molecules up to 1 μm in size to diffuse and / or convect in. Experimental data show that neither the size nor the porosity of the particles has a significant effect on the effectiveness of the treatment. The key feature is that they provide a solid-phase surface that presents the primary amine chemical to the reaction solution. Thus the particle size can be in the range of 5 nm to 500 nm, or 10 nm to 400 nm, or 40 nm to 300 nm, or 80 nm to 200 nm, or 100 nm to 150 nm, or some other intermediate value within the range of 5 nm to 500 nm. The range of the average pore and / or channel size can be from less than 1 nm to 10,000 nm, or 10 nm to 7,500 nm, or 100 nm to 5000 nm, or 1000 nm to 2000 nm, or any other intermediate value within the range of less than 1 nm to 10,000 nm.

[0100] The solid-phase particles can be added in a volume ratio of about 1% of the sample volume, or 2% of the sample volume, or 3% of the sample volume, or 4% of the sample volume, or 5% of the sample volume, or 10% of the sample volume, or a larger or different ratio. Those skilled in the art should understand that the amount of DNA in different AAV preparations can vary greatly. Since the whole method is specifically for DNA removal, simple routine experiments may be needed to determine the proportion of solid-phase particles that most effectively removes DNA. Using an excessive amount, such as 5%, usually can obviate the need for such experiments, but process economic considerations may make such experiments desirable as they may prove the effectiveness of a smaller amount.

[0101] The solid-phase extraction device used to perform this method can consist of columns filled with porous particles, columns filled with nanofibers, columns filled with monoliths, hydrogels, membrane filters, and depth filters in any form or configuration.

[0102] The surface of any physical form of the solid phase is chemically modified so that at least one primary amine is covalently attached to its surface. It can contain ligands composed of only a single primary amine, or a primary amine polymer, or a combination of a primary amine with a secondary amine, or a tertiary amine, or a quaternary amine, or any combination of amines. The solid phase can also carry other chemical groups, including but not limited to hydrophobic groups, hydrogen-bonding groups, metal-binding groups, and negatively charged groups. Solid phases lacking primary amines can also be present together with solid-phase particles carrying primary amines.

[0103] As good process control, the solid phase, whether in the form of loose particles or in a chromatographic device, should be equilibrated with the reaction conditions before contacting the sample.

[0104] Solids can be removed by any convenient means. Many such methods and the criteria for selecting them are well known in the art. Any such method can be used without altering its ability to achieve its stated purpose of reducing the amount of DNA in an AAV preparation.

[0105] In one embodiment, the solids produced by the method can be removed by gravitational sedimentation.

[0106] In another embodiment, sedimentation of the solids can be accelerated by centrifugation.

[0107] In another embodiment, sedimentation of the solids can be accelerated by sonication.

[0108] In another embodiment, sedimentation of the solids can be carried out while pumping the mixture upward through a cylinder, which can cause the denser precipitate to settle under gravity while the solid-free carrier liquid containing the virus flows upward out of the cylinder. In one such embodiment, the liquid can pass through a filtration device after it exits the cylinder.

[0109] In another embodiment where the solids are not removed by sedimentation, they can be removed by filtration.

[0110] In another embodiment, the filtration method for removing solids after treatment can be a membrane filtration method or a depth filtration method.

[0111] In another embodiment, a so-called bag filter or filter bag can be used to assist in removing solids, where the porous membrane is configured as a cylinder having an open end to allow the sample to enter but the other end is closed so that the liquid is forced through the pores.

[0112] The present invention gives a non-limiting example to illustrate the basic steps of performing the method. Particles with primary amines are pre-equilibrated to 100 mM formic acid, 4 M NaCl, pH 3.5. A determined volume of cell lysate or cell culture harvest is placed in a suitable container. Sufficient dry salt such as sodium chloride is added to bring the final concentration to 4 M, and the pH is adjusted to 3.5 by titrating with formic acid. The solid-phase particles may already be present when adjusting the sample conditions or may be added after the conditions are adjusted. In either case, the particles are present in a volume ratio of approximately 5% of the original sample volume. The combination is incubated and mixed at ambient temperature for 60 minutes. The solids, including the solid-phase extraction particles, are then removed by any convenient method (including but not limited to centrifugation or filtration). For emphasis, the solids must be removed while they are still in the working solution.

[0113] The present invention gives another non - limiting example to illustrate the basic steps of performing the method. The primary amine - bearing particles are pre - equilibrated to 100 mM formic acid, 5 M NaCl, pH 3.5. A determined volume of cell lysate or cell culture harvest is placed in a suitable container. Another container contains twice - concentrated reagent mixture of the same volume, which contains sodium chloride sufficient to reach a concentration of 5 M when diluted with the sample; contains formic acid with a pH of 3.5 sufficient to produce 100 mM formic acid at pH 3.5 when diluted with the sample; and contains primary - amine - bearing particles sufficient to reach a volume ratio of 5% when diluted with the sample. The sample is added to the concentrated reagent and incubated with mixing at ambient temperature for 60 minutes. Then the solids produced by exposing the sample to high salt and low pH are removed by any convenient method (including but not limited to centrifugation or filtration).

[0114] The materials and instructions for performing the method can be provided in the form of a kit. In one embodiment, the kit provides solid - phase particles in a concentrated buffer in a pre - filled container, and the end - user can add the sample to the pre - filled container and incubate. The incubated sample is processed using a provided centrifugal filter cartridge with a 0.22 - μm membrane to remove solids from the liquid containing AAV. One version of the kit contains sufficient materials for 10 purifications, 10 mL each. Alternative versions can be configured to handle smaller or larger sample volumes, and / or use materials that can perform fewer or more purifications. An extended version of the kit can include one or more chromatography devices for purification. A further extended version can include pre - prepared tubes containing cell lysis buffer.

[0115] In an alternative embodiment, the kit includes a buffer concentrate lacking particulate solid phase in a pre - filled container, and the end - user adds the sample to the pre - filled container and incubates for an appropriate period of time. The incubated sample is processed using a provided centrifugal filter cartridge with a 0.22 - μm membrane to remove solids from the liquid containing AAV. Then the liquid is passed through a provided reusable chromatography device containing solid phase to extract DNA according to the method. One version of the kit contains sufficient materials for 10 purifications, 10 mL each. Alternative versions can be configured to handle smaller or larger sample volumes, and / or use materials that can perform fewer or more purifications. An extended version of the kit can include one or more chromatography devices for purification. A further extended version can include pre - prepared tubes containing cell lysis buffer.

[0116] In another alternative embodiment that does not require chromatography, the kit includes a buffer concentrate containing a solid phase in a pre-filled container. The end user adds the sample to the pre-filled container and incubates for an appropriate period of time. The incubated sample is optionally processed using a provided centrifugal spin filter with a 0.22 μm membrane to remove solids from the liquid containing AAV. The sample is then processed using a provided centrifuge tube with a 300,000 MWCO (molecular weight cut-off) membrane, which retains high molecular weight contaminants while allowing the virus to pass through the membrane. The sample is then processed using a provided centrifuge tube with a 100,000 MWCO membrane, which retains the virus while allowing low molecular weight contaminants to pass through the membrane. This step also concentrates the virus and enables buffer exchange into the final formulation.

[0117] The present invention is further illustrated by the following non-limiting examples. Examples

[0118] Example 1

[0119] Effect of NaCl concentration on chromatin reduction at pH 3.5. Figure 1 The experimental results using different concentrations of salt from 0 M to 5 M at pH 3.5 are shown. The analytical method is size exclusion chromatography with fluorescence monitoring to detect the intrinsic fluorescence of tryptophan residues in proteins. This method improves sensitivity and enables detection of AAV capsids in the sample. Samples were prepared by titrating the pH with 200 mM formic acid at pH 3.5 and a two-fold amount of salt needed to produce the target concentration for a 1:1 volume dilution. The experimental results show that as the NaCl concentration increases, chromatin reduction increases more and more. AAV remains soluble throughout the range. At salt concentrations of 1 M and 2 M, chromatin contamination is reduced by approximately 40%, but has little effect on reducing non-chromatin protein contamination. 3 M is slightly less effective in reducing chromatin, but shows a significant improvement in reducing non-chromatin proteins. 4 M NaCl shows at least 90% chromatin reduction and even more reduction in non-chromatin proteins, making AAV soluble. The contaminant reduction effect of 5 M NaCl is slightly better, but the recovery of AAV may be slightly reduced.

[0120] Example 2

[0121] Excellent performance is exhibited by including particles with primary amines. The ability of particles with primary amines to contribute to better overall results was evaluated. One sample was equilibrated to 5.0 M NaCl, 50 mM formic acid, 5 mM MgCl 2 and 0.1% Pluronic F68, pH 3.5. Another was equilibrated to the same conditions except including 5% particles with primary amines. The two samples were incubated at room temperature for 1 hour and then solids were removed by membrane filtration. Figure 2The results were compared with untreated controls. The condition of lacking particles reduced the chromatin content by approximately 40%, and the non-chromatin proteins decreased by a very substantial amount that could not be estimated from the figure. The inclusion of these particles reduced chromatin by more than 95% and to a greater extent reduced non-chromatin proteins. The recovery rate of AAV was roughly the same with or without particles.

[0122] Example 3

[0123] Improvement of chromatin clearance rate with the change of pH value in the presence of primary amine solid-phase particles. Figure 3 Shows the effect of including primary amine particles and pH value on removing chromatin and other contaminants while keeping AAV soluble. All experiments were carried out in the presence of 5M NaCl, 5% primary amine particles, 100mM formic acid, 5mM MgCl 2 and 0.1% Pluronic F68 (surfactant). Combining with Example 1, these results emphasize that high salt concentration is the main variable, but indicate that pH value makes a great contribution to the overall effect. The virus recovery rate is the best at pH 3.5, which also supports that the chromatin and non-chromatin protein contents are the lowest at pH 2.5. However, the AAV recovery rate at pH 2.5 is the lowest. The recovery rate at pH 4.5 is almost as good as that at pH 3.5, but the reduction of chromatin is slightly worse, and the non-chromatin protein contamination is 2 - 3 times that at pH 3.5. The results at pH 5.5 and 6.5 are worse in all aspects. The higher AAV recovery rate at pH 3.5 suggests using this pH, especially for using this method. In a separate experiment comparing formic acid and glycine as buffers, the results using formic acid were significantly better. In other experiments, it was observed that 5mM MgCl 2 made a small but clear positive contribution to reducing contaminants while maintaining a high virus recovery rate. In another series of experiments, the incubation periods of 1 hour and 2 hours were compared. The results of 1h were the same as those of 2h.

[0124] Example 4

[0125] Cation exchange chromatography was carried out after DNA extraction

[0126] The insect cell lysate containing AAV was balanced to 100mM formic acid, 5.0M NaCl, 5mM MgCl 2 , 0.1% Pluronic F68, 5% primary amine particles, pH 3.5. The mixture was incubated at room temperature for 1 hour and the solids were removed by membrane filtration. 50mM formic acid (pH 3.5) was added to dilute the supernatant until the conductivity reached 40mS / cm. The cation exchange monolithic column (CIMmultus SO 3, BIA Separations were equilibrated to the same conditions and loaded. As Figure 4 shown, a concentrated peak containing greater than 90% pure AAV was obtained. Figure 4 The AAV recovery was plotted against pH, highlighting two major unexpected findings. It was previously expected that the highest recovery would be at the pH closest to the physiological environment where the virus normally survives. In fact, the lowest pH, 2.5, had the lowest recovery among all pH values. The first unexpected finding was that the second lowest recovery was observed at pH 6.5 (closest to the physiological value), and the recovery gradually increased from pH 6.5 to pH 3.5. The second unexpected finding was that the highest virus recovery was observed at pH 3.5. These findings highlight the unpredictability of the system at high salt concentrations, which was 5M NaCl in this example. The chromatogram is as Figure 5 shown, and the inset shows a silver-stained PAGE gel.

[0127] Example 5

[0128] After DNA extraction, cation exchange chromatography was performed, followed by anion exchange chromatography

[0129] The sample processed by DNA extraction and cation exchange chromatography in Example 4 was further processed by anion exchange chromatography (CIMmac AAV, BIA Separations) to illustrate DNA extraction in the complete purification process. Briefly, fractions E2 and E3 were combined (see Figure 5 ), the pH was adjusted to 9.0, and the sample was diluted to a conductivity of approximately 2.5 mS / cm. The column was equilibrated to 50 mM bis-Tris propane, pH 9.0, and then the sample was loaded onto the column. The column was washed with 5 CV of the equilibration buffer, and then eluted with a 20 CV linear gradient ending with 50 mM bis-Tris propane, 200 mM NaCl, pH 9.0. The elution curve is as Figure 6 shown. It shows a characteristic double-peak AAV curve, where the first peak corresponds to empty capsids and the second peak corresponds to capsids filled with vector DNA. Capsid proteins transmit ultraviolet light, so the internal DNA causes the absorbance of full capsids to exceed 260. Empty capsids lacking internal DNA have a more typical 260 / 280 absorbance for proteins. The absorbance ratio at 260 nm and 280 nm is considered an indicator of AAV purity, and a value of 1.30 or lower indicates an impure preparation. Note that in this case, the ratio in full capsids is approximately 1.35, indicating high purity. This is attributed to the improved purification performance of the cation exchange and anion exchange chromatography steps due to the effectiveness of the DNA extraction step.

[0130] Example 6

[0131] After DNA extraction, size exclusion chromatography was performed

[0132] Equilibrate the insect cell lysate containing AAV to 100 mM formic acid, 5.0 M NaCl, 5 mM MgCl 2 , 0.1% Pluronic F68, 5% primary amine particles, pH 3.5. Incubate the mixture at room temperature for 1 hour and remove solids by membrane filtration. Dilute the supernatant 1:1 with 3.0 M potassium phosphate, pH 7.0. And apply it to a CIMmultus OH monolithic column (BIASeparations), which has been pre-equilibrated to 1.5 M potassium phosphate, pH 7.0. After loading, wash the column with the equilibration buffer to displace unbound contaminants on the column. AAV is eluted with a decreasing linear salt gradient ending with 50 mM potassium phosphate buffer. Figure 7 Shows the results of polyacrylamide gel electrophoresis (PAGE) analysis (silver staining) of column fractions. The original processed sample is in the last well on the far right. The next well to the left shows the sample after DNA extraction. On the far left are the molecular weight markers, followed by the DNA extraction sample diluted and ready to be loaded onto the chromatography column. The other wells show the content of the fractions collected during the chromatography run. The content of the elution peak is shown in the middle of the gel, labeled OH01E5. Note that the AAV band stains very strongly, but the contaminant bands stain very weakly, indicating high purity.

[0133] Example 7

[0134] Differential membrane filtration after DNA extraction

[0135] Equilibrate the insect cell lysate containing AAV to 100 mM formic acid, 5.0 M NaCl, 5 mM MgCl 2 , 0.1% Pluronic F68, 5% primary amine particles, pH 3.5. Incubate the mixture at room temperature for 1 hour and remove solids by membrane filtration. Titrate the sample to pH 6.5 ± 0.5, then apply it to a centrifugal membrane filtration device with a 300,000 kDa molecular weight cut-off to retain residual high molecular weight chromatin, as seen in the sample of Example 4. The virus passes through the filter membrane, and they are applied to a centrifugal membrane filtration device with a 100,000 kDa molecular weight cut-off to retain the virus while allowing low molecular weight contaminants to pass through the membrane. Restore the initial sample volume and recentrifuge by adding 50 mM Hepes, 50 mM NaCl, 50 mM arginine, 1% sorbitol, 2 mM magnesium chloride, pH 7.0. This concentrates the sample by approximately 20-fold and in a new buffer. Evaluate the product by analytical anion exchange chromatography. The results are as Figure 8 shown. Compare with Figure 6 as a reference.

[0136] Example 8

[0137] Comparison of Solid-Phase Extraction with Primary Amine Particles and Citrate Flocculation under High-Salt Conditions

[0138] The citrate flocculation method described by Potter and Byrne [6] was used to treat the harvested AAV-containing material filtered from SF9 cells 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, which relied on the added citrate to provide buffering. At the same time, formic acid (100 mM), magnesium chloride (95 mM), Pluronic F68 (0.1%), and primary amine particles (5% v:v) were added to another aliquot of the filtered harvest, and then titrated to pH 3.5. Both treatments were incubated for 60 minutes, and then the solids were removed by filtration. Virtually no visible solids were observed with the citrate flocculation treatment. The results were compared in Figure 9 As shown, the contaminant curve after citrate flocculation was worse than before treatment and was significantly lower than that treated with high salt and primary amine particles.

[0139] Example 9

[0140] Refinement of pH Response

[0141] Examples 1 and 3 showed that in the absence of primary amine particles, the optimal pH was approximately 3.5 ± 0.5. A series of additional experiments were conducted to refine the estimate of the optimal pH value in the presence of primary amine particles. Filtered AAV-containing harvests were treated with 5.0 M sodium chloride, 5 mM magnesium chloride, 1% Pluronic F68, and 5% v:v primary amine particles. The samples were titrated to pH 3.3, 3.4, 3.5, 3.6, and 3.7, respectively. They were incubated for 60 minutes, the solids were removed by filtration, and the samples were analyzed by monitoring the intrinsic fluorescence through size exclusion chromatography. As Figure 10 shown, the pH value that provided the most effective reduction of contaminants without significant loss of AAV particles was 3.3, and the contamination increased at higher pH values.

[0142] Example 10

[0143] Alternative Verification of the Utility of Extreme Salt Concentrations

[0144] Example 1 shows that in the absence of primary amine - bearing particles, as the sodium chloride concentration increases, the proportion of contaminants removed increases, where 4M NaCl provides less contamination than 1M, and 5M provides significantly less contamination than 4M. A series of experiments were conducted to demonstrate that the effect of NaCl still exists in the presence of primary amine - bearing particles and was demonstrated by another form of analysis. In the new series of experiments, the effect of salt concentration during solid - phase extraction was determined by separating the treated samples on a cation - exchange column. Samples were prepared at pH 3.5 in the presence of 50 mM formic acid, 5 mM magnesium chloride, 1% Pluronic F68, 5% primary amine - bearing particles, and sodium chloride at concentrations of 1M, 4M, and 5M. The results are as Figure 11 shown. The curve corresponding to solid - phase extraction at 1M NaCl shows heavy contamination leading to the major AAV peak. The contaminant level is significantly reduced after solid - phase extraction with 4M NaCl and reaches the lowest level after solid - phase extraction with 5M NaCl. From these results, extreme salt concentrations are even more favorable in the presence of primary amine - bearing particles than in their absence (Example 1).

[0145] Example 11

[0146] Response to the proportion of primary amine - bearing particles

[0147] A series of experiments were conducted to determine the most favorable primary amine loading ratio. At pH 3.5, filtered cell - culture harvests containing AAV2 / 8 were treated with 50 mM formic acid, 5.0M sodium chloride, 5 mM magnesium chloride, 1% Pluronic F68, and primary amine - bearing particles at volume ratios of 2%, 3%, 4%, and 5%. The mixtures were incubated for 60 minutes and then the solids were removed by membrane filtration. The samples were analyzed by size - exclusion chromatography monitored by intrinsic fluorescence. As Figure 12 shown, increasing the particle ratio can measurably improve the reduction of contaminants, with a significant improvement at 5%.

[0148] Example 12

[0149] Effect of different salt species

[0150] Example 1 demonstrated that increasing the sodium chloride level improved the utility of the method in removing contaminants while retaining AAV. Other types of salts were investigated to evaluate their relative utility. All experiments were conducted using the same filtered AAV2 / 8 harvest, which was derived from SF9 cells used in other experiments. All experiments were carried out in the presence of 5 mM magnesium chloride, 1% Pluronic F68, and 5% amine-functionalized particles, using pH 3.5, 50 mM formic acid. Lithium chloride was added to a final concentration of 1.0 M in one 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 in another experiment, and potassium acetate was added to 1.5 M in another experiment. All were compared to an experimental control using 5.0 M sodium chloride. As Figure 13 shown, and Figure 13 compared with Figure 1 , the results for sodium acetate and potassium acetate were similar but superior to those of sodium chloride at similar concentrations. The reduction of chromatin and non-chromatin proteins was prominent in both cases, indicating that higher concentrations of these salts may provide utility equal to or greater than that of sodium chloride. However, the apparent recovery of AAV seemed lower. Lithium chloride and ammonium chloride appeared to provide excellent AAV recovery and better reduction of non-chromatin proteins compared to sodium chloride, but had a poorer effect on reducing chromatin proteins. Sodium glutamate also better reduced protein contamination than sodium chloride at the same concentration, but was significantly inferior to all other salts within this concentration range. In addition to suggesting further exploration of salts other than sodium chloride, these results also indicate the potential utility of using a combination of two or more salts.

Claims

1. A method for removing chromatin from a cell culture harvest, comprising the steps of: - providing a cell culture harvest containing a desired biological product selected from parvovirus or adeno-associated virus, - incubating the cell culture harvest in an aqueous medium having a pH in the range of 3.0 to 4.0 and an ionic strength corresponding to a concentration of NaCl in the range of 3.0 M to saturation, in the presence of a solid phase material bearing a primary amine group and present in a volume ratio in the range of 1% to 10% relative to the volume of the cell culture harvest in the aqueous medium; and separating the desired biological product from the resulting solid; wherein the harvest is obtained from a culture of mammalian cells, insect cells, or yeast cells.

2. The method according to claim 1, characterized in that the biological product is parvovirus or adeno-associated virus.

3. The method according to claim 1 or 2, characterized in that the pH is 3.5 ± 0.

3.

4. The method according to any one of claims 1 to 3, characterized in that the incubation period is up to 24 hours.

5. The method according to any one of claims 1 to 3, characterized in that the incubation period is in the range of 30 minutes to 240 minutes.

6. The method according to any one of claims 1 to 3, characterized in that the incubation period is in the range of 45 minutes to 90 minutes.

7. The method according to any one of claims 1 to 3, characterized in that the incubation period is in the range of 55 minutes to 65 minutes.

8. The method according to any one of claims 1 to 7, characterized in that the solid is separated from the soluble product by sedimentation or filtration.

9. The method according to any one of claims 1 to 8, characterized in that the solid phase bearing a primary amine is in the form of loose particles or in the form of a chromatography device.

10. The method according to claim 9, characterized in that the solid phase particles are present in a volume ratio in the range of 2.5% to 7.5% relative to the volume of the cell culture harvest in the aqueous medium.

11. The method according to claim 9, characterized in that the solid phase particles are present in a volume ratio in the range of 4.5% to 5.0% relative to the volume of the cell culture harvest in the aqueous medium.

12. The method according to claim 9, characterized in that the form of the solid phase bearing a primary amine is selected from the group consisting of a column packed with porous particles, a column packed with nanofibers, a column packed with monoliths, a hydrogel, a porous membrane filter, and a depth filter.

13. The method according to any one of claims 1 to 12, characterized in that the primary amine surface of the solid phase is accompanied by chemical residues with alternative features.

14. The method according to claim 13, characterized in that the chemical residues are secondary amines, tertiary amines, quaternary amines, hydrophobic residues, hydrogen bond residues, metal affinity residues, or combinations thereof.

15. The method according to any one of claims 1 to 14, characterized in that The method is carried out in the presence of one or more of salts, sugars, surfactants, divalent metal ions, allantoin, polyethylene glycol, flocculants, or combinations thereof.

16. The method according to any one of claims 1 to 15, further comprises the following steps: — reducing the ionic strength of a solution containing the desired biologic; — contacting the solution with a cation exchanger to remove further contaminants, wherein the cation exchanger binds to the contaminants; — further reducing the ionic strength of a solution containing the desired biologic; — contacting the solution with a cation exchanger to purify the desired biologic, wherein the cation exchanger binds to the desired biologic.

17. Use of a kit for performing the method according to any one of claims 1 to 16, the kit comprising solid phase particles in a concentrated culture medium in a container and instructions for performing the method.

18. The use according to claim 17, characterized in that the solid phase particles carry primary amine groups.

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