Improving purification of adeno-associated virus to more effectively remove contaminating DNA

Abstract

A method for reducing the content of contaminating DNA in a preparation containing AAV capsids and contaminating DNA, comprising the steps of: a) extracting the DNA with a solid phase having a positive charge on its surface, contacting the solid phase with the preparation at a pH of 7.0 ± 1.0 and a salt concentration of 10 mM to 200 mM, to obtain a first fraction, b) diafiltering the first fraction by first tangential flow filtration to obtain a second fraction, c) treating the second fraction with a DNAse, d) diafiltering the DNAse-treated second fraction obtained by step c) by second tangential flow filtration, e) filtering into a buffer having a pH of 7.0 ± 1.0 and a salt concentration of 10 mM to 20 mM to produce a third fraction, and optionally f) concentrating the third fraction by tangential flow filtration prior to performing a supplementary chromatography.

Description

[0001] A method of reducing the amount of contaminating DNA in a preparation containing AAV capsids and contaminating DNA is disclosed.

[0002] BACKGROUND

[0003] Adeno-associated virus (AAV) has become a common delivery vehicle for therapeutic DNA in the field of human gene therapy. The virus is produced by cell culture, most commonly using mammalian or insect host cells. The cell culture is manipulated so that the desired therapeutic DNA plasmid is inside the AAV capsid. The cell culture is harvested and the DNA-containing AAV capsids are purified using standard chromatographic methods (e.g., affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, etc.).

[0004] As with all human injectable therapeutics, to ensure safety, contaminating DNA needs to be reduced to very low levels. It has proven to be more challenging than expected. Recently developed analytical techniques suggest that the difficulty can be partially due to residual DNA bound on the outside of the AAV capsid. This can not be surprising since it is well known that AAV capsids bind tightly to cationic exchangers. The strong binding to cationic exchangers suggests that the outside of the AAV capsid has a strong positive charge. DNA is negatively charged and binds tightly to positively charged surfaces. Regardless of the mechanism, these findings highlight the difficulty of removing DNA.

[0005] Currently, it is common practice in the industry to treat AAV harvests by simply adding DNAse. Unfortunately, the DNA reduction by this method is limited and does not solve the problem of reducing contaminating DNA to very low levels. It provides a secondary benefit of improving the filterability of the harvest from hopeless to possible, but filter clogging remains a regular problem, requires excessive filter media, and results in loss of AAV capsids. It also prevents processing methods like tangential flow filtration from being able to concentrate the harvest to a large extent. Concentration factors as high as 2 are sometimes possible, but only that.

[0006] WO 02 / 12455 Al discloses a method for large scale purification of recombinant AAV (rAAV) virus produced in the absence of infectious adenovirus. Preferably, the rAAV is produced in a host cell line by triple transfection using a helper function vector, an AAV vector, and an AAV helper vector. The method includes preparing a lysate from the host cell line and passing the lysate through various combinations of ion exchange chromatography media and / or affinity chromatography media. The affinity chromatography media is an AAV receptor or an antibody having binding affinity for AAV, such as heparin sulfate. Various cationic and anionic exchange media are contemplated. In certain embodiments, optional purification steps can be included, such as filtering the lysate through one or more filters, or treating the lysate with a nuclease.

[0007] WO 2010 / 148143 A1 discloses a method for purifying recombinant adeno-associated virus (rAAV) vectors, which can be used for gene transfer, particularly for gene therapy or vaccination. The recombinant AAV vectors are substantially free of impurities from the process, including production components such as cellular nucleic acids, cellular proteins, helper viruses, and culture medium components.

[0008] WO 03 / 097797A2 discloses a method for purifying viral particles, particularly recombinant adenovirus vector particles. This method relies on various combinations of cell lysis, detergent-based host cell contaminant precipitation detached from the virus, deep filtration or centrifugation, ultrafiltration, nuclease digestion, and chromatography to reliably and economically produce highly purified products. This method results in contaminating DNA levels consistently below detectable levels.

[0009] WO 2019 / 006390 A1 discloses a method for purifying, producing, and manufacturing recombinant adeno-associated virus (rAAV) vector particles, comprising at least two column chromatography steps. The column chromatography steps include, for example, cation exchange chromatography, anion exchange chromatography, size exclusion chromatography, and / or AAV affinity chromatography, performed individually or in combination and in any order.

[0010] All references cited in this article are incorporated by way of citation, to a degree that does not contradict the explicit teachings of this article. Invention Summary

[0011] A novel method has been developed that provides a surprising improvement in the purification of AAV, particularly in reducing DNA contamination. Cell cultures or lysates are exposed to one or more positively charged surfaces to bind DNA independent of AAV particles. Initial DNA reduction is achieved with treatment of positively charged surfaces, and tangential flow filtration (TFF) is promoted, resulting in a concentration factor of 10-20 or higher. Given that DNase treatment of cell harvests or lysates cannot achieve this improvement in filterability, it is surprising that solid-phase extraction with positively charged surfaces can accomplish this.

[0012] Brief Description of the Invention

[0013] According to the method of the present invention, in a formulation containing AAV capsid and contaminating DNA, the content of contaminating DNA is reduced, the method comprising the following steps:

[0014] a) DNA is extracted using a solid phase with a positively charged surface. The solid phase is contacted with a formulation at a pH of 7.0 ± 1.0 and a salt concentration of 10 mM to 200 mM to obtain a first component.

[0015] b) Percolate the first component by a first tangential flow percolation to obtain the second component.

[0016] c) Treat the second component with DNase.

[0017] d) Percolate the second DNase-treated fraction obtained in step c) through a second tangential flow filtration to a buffer solution with a pH of 7.0 ± 1.0 and a salt concentration of 10 mM to 20 mM to obtain the third fraction, and optionally...

[0018] e) Concentrate the third component by tangential flow filtration prior to supplemental chromatography.

[0019] In one embodiment of the method of the present invention, the solid phase may be in the form of loose bulk particles or a flow device.

[0020] In another embodiment of the method of the present invention, the tangential flow filtration can be performed using a membrane selected from the group consisting of: hollow fiber membranes, including single hollow fiber membranes or bundled membranes; or flat membranes, including single-layer membranes or membranes stacked or rolled into cylinders.

[0021] In another embodiment of the method of the present invention, the first filtration may be performed in a buffer solution prepared to facilitate the digestion of DNA by DNases.

[0022] In another embodiment of the method of the present invention, the tangential flow filter membrane has a porosity in the range of molecular weight cutoff of 10 kDa to 300 kDa, or 30 kDa to 300 kDa, or 60 kDa to 300 kDa, or 100 kDa to 300 kDa, or 200 kDa to 300 kDa, or 250 kDa to 300 kDa, preferably with a molecular weight cutoff of 100 kDa or above.

[0023] In another embodiment of the method of the present invention, the preparation containing AAV capsid and contaminating DNA may contain AAV particles selected from the group consisting of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or other serotypes, including recombinant serotypes having more than one serotype characteristic.

[0024] In another embodiment of the method of the present invention, the component obtainable after step d) or step e) may be further processed according to the following options (i) to (iii):

[0025] Option (i) Cation exchange chromatography,

[0026] Option (ii) Affinity chromatography, or

[0027] Option (iii) is an anion exchange chromatography.

[0028] In another embodiment of the second aspect of the method of the present invention, in purification step (i), the component of step d) or e) is loaded onto a cation exchange chromatography material with a pH of 4 to 6, particularly a pH of about 5, and then:

[0029] - Reequilibrate the cation exchange chromatography material to a pH of approximately 3.5 ± 0.5.

[0030] - Elution using a salt gradient

[0031] Then it is processed by anion exchange chromatography. This process produces, for example, clinical-quality AAV.

[0032] In another embodiment of the method of the present invention, in purification step (ii), the component of step d) or e) is loaded onto an affinity chromatography material and subjected to anion exchange chromatography after elution. This process produces, for example, purified AAV.

[0033] In another embodiment of the method of the present invention, in purification step (iii), the component from step d) or e) is subjected to anion exchange chromatography. This process produces, for example, research-grade AAV.

[0034] Brief description of the attached figures

[0035] Figure 1 An overview of the method of the present invention is shown.

[0036] Figure 2 The cation exchange chromatogram is shown, demonstrating the application of the method of the present invention.

[0037] Figure 3 The analytical reduction SDS-polyacrylamide gel electrophoresis (PAGE) results are shown, comparing the standard conditions with those of the method of the present invention.

[0038] Figure 4 Affinity chromatography was demonstrated to capture AAV from cell lysates.

[0039] Figure 5 It shows, as Figure 4 The affinity chromatograms shown are analyzed by SDS-PAGE of the components. Detailed Implementation

[0040] Using AAV as an example of the entity to be purified, various aspects of the present invention will now be described in more detail. Similarly, other entities can be purified in a similar manner, as will be readily apparent to those skilled in the art who read and understand this disclosure.

[0041] Those skilled in the art will recognize that DNA can be removed from the treatment solution by adding a positively charged soluble polymer instead of a positively charged solid material. From a clinical safety perspective, this can be unstable and may alter the biological behavior of the purified capsid in vitro, leading to misinterpretations of results and potentially causing results not representative of AAV but caused by the positively charged polymer. They may also have a protective effect against certain DNAs from DNase cleavage. DNA is known to bind actively to the exterior of the AAV capsid. If a positively charged polymer is added to a formulation containing such an AAV capsid, the positively charged polymer may form a coating on the associated DNA on the exterior of the capsid. Tight binding between DNA and positively charged proteins is known to interfere with the action of DNases. Positively charged polymers can do this to a greater extent. This extent is such that, after purification, the positively charged polymer remains bound to the AAV capsid, and they can subsequently be leached into in vitro samples or gradually released into living organisms, including laboratory animals or humans. Positively charged polymers are all chemical irritants that induce inflammation. Given that purified AAV capsids will ultimately be administered to already ill individuals, this inadvertently poses an unnecessary and potentially serious risk to them from taking known classes of inflammatory agents. This highlights the inventiveness of the present invention, as it provides a method for extracting DNA using a solid-phase material that can be completely removed from the AAV capsid formulation after treatment.

[0042] A buffer solution is prepared for the TFF concentration step to facilitate DNA digestion by a selected DNase in subsequent processing steps. Adding the selected DNase and incubating it for the appropriate duration differs significantly from conventional DNA lysis methods in two ways: 1) Due to the achieved TFF concentration, the volume is smaller and less enzyme is required. 2) Removing inhibitors and exchanging the buffer for the optimal lysis conditions of the selected DNase allows for a more efficient reduction compared to the standard practice of simply adding it to the original cell culture or lysate.

[0043] The porosity of TFF membranes benefits from the porosity at which the coating is retained to the greatest extent possible. Porosity grades vary depending on the manufacturer and membrane material, but generally, membranes with a molecular weight cutoff of 250 kDa to 300 kDa are ideal. Lower MWCO values ​​support lower contaminant removal efficiency, but still significantly reduce the contaminant content of the treated formulation. In general, TFF membranes with the following MWCO grades can be used: 10 kDa to 300 kDa, 30 kDa to 300 kDa, 60 kDa to 300 kDa, 100 kDa to 300 kDa, 200 kDa to 300 kDa, or 250 kDa to 300 kDa, but are preferably higher than 100 kDa.

[0044] TFF membranes in various physical forms and sizes are commercially available to meet diverse needs in research and development, scaling up, and manufacturing. These include flat membranes, either individually or in stacks or rolls; and hollow fibers, either single strands or bundles.

[0045] Tangential flow filtration (TFF) technology is typically performed in any one or any combination of three principle modes, as in the method of this invention. The first of these modes is concentration, which aims to remove excess fluid to make subsequent processing operations more efficient. The second mode is percolation. In percolation, the original fluid, salts, sugars, and buffer components are filtered out, and fresh fluid free of these components is introduced. This has the effect of "buffer exchange" with the sample's liquid, with the aim of preparing the sample for subsequent processing. The term percolation is a combination of the words "dialysis" and "filtration." The third mode is size-based purification. Concentration and percolation can be performed using tangential flow filtration membranes of any porosity to retain the desired product, including membranes with a molecular weight cutoff of 10 kDa or less. Using TFF as a purification tool represents an extension where macroporous membranes are specifically used to remove biomolecular contaminants smaller than the target product. The condition is that the porosity must be small enough to retain the target product; membranes with the largest pores that meet this requirement offer the benefit of removing the widest range of contaminating biomolecules. In the current context, because the AAV capsid is quite large on the scale of biological products, membranes with very large pores, such as those with a molecular weight cutoff of up to 300 kDa, can eliminate most proteins and other biomolecules. The smaller those contaminating biomolecules are, the more efficiently they can be eliminated. This makes the technology particularly suitable for removing DNases after DNA cleavage, as well as for removing histones released from host cell DNA by cleaving previously associated DNA.

[0046] Many DNases are commercially available, and each requires its own specific conditions to achieve optimal results. Preliminary experimental data suggest that so-called salt-tolerant DNases may provide better results. Some DNases also cleave RNA, which may be useful in terms of the degree to which RNA stabilizes large heteropolymers of DNA. If a DNase lacks this ability, an RNase can be added to it. However, the ability of the cleavage formulation to digest RNA is not essential for the method of the present invention.

[0047] After lysis, the sample is treated again with TFF to remove the lysin, DNA fragments, and host histones released by lysing the host DNA.

[0048] A particularly unexpected experimental finding was that even with fully optimized lysis conditions, DNA lysis alone was insufficient to completely solve the problem. Experimental data indicated that residual histones interfered with chromatography, thereby impairing its ability to remove contaminants, including DNA. Removing histones prior to chromatography enhanced the removal of all contaminant categories and further improved the removal of contaminating DNA.

[0049] The second TFF step uses a buffer solution that roughly represents physiological conditions, such as a pH between 6.5 and 7.5 and a sodium chloride concentration of 25-200 mM. Maintaining roughly physiological conditions is considered a valuable contributing factor to the enhancements provided by the method of this invention.

[0050] Samples prepared using TFF typically employ buffers with chromatographic binding conditions close to those described below. While physiological conditions are generally suitable for AAV capture via affinity chromatography, the method of this invention reduces the total contaminant load to such a low level that alternative methods, such as capture via cation exchange chromatography or anion exchange chromatography, can produce high-purity AAV without relying on affinity chromatography. This is important because commercially available affinity chromatography media require low flow rates, which increases processing time, and they are not resistant to washing with 1.0 M NaOH. Ion exchange chromatography media can be used for capture, which support high flow rates, higher product binding capacity, and longer sterilization times with 1.0 M NaOH.

[0051] In one implementation, the preceding method steps are used to prepare a sample for loading onto an affinity chromatography column.

[0052] In another embodiment, the aforementioned method steps are used to prepare the sample for loading into a positively charged chromatographic column, such as an anion exchanger. In this case, the sample is diluted after the second TFF step to ensure its conductivity is low enough to guarantee high-capacity binding of AAV to the column. Its pH can also be adjusted to a higher value for better differentiation between empty and intact capsids. Magnesium and / or calcium salts can be added to modify the selectivity of the empty-intact separation. This method may be suitable for producing research-grade intact capsids.

[0053] In another embodiment, the aforementioned method steps are used to prepare a sample for loading onto a cation exchanger. Typical preparation of the sample for binding to the cation exchanger includes titrating the sample to pH 3.5. This alters the titration state of the amino acid residues on the capsid protein surface, resulting in a net positive charge on the capsid surface. Their positive surface charge causes them to bind to the negatively charged surface of the cation exchanger. However, it also has adverse effects. By making the capsid surface positively charged, it induces strong DNA binding, or if DNA is already bound to the capsid, the low pH stabilizes this binding. Either way, a low binding pH promotes DNA binding to the capsid exterior.

[0054] Another aspect of this problem makes it even more problematic. If DNA binds to the capsid exterior, then excess histones in the system are highly likely to bind to the DNA associated with the capsid exterior. This highlights the value of the second TFF step, which can remove not only DNA fragments but also histones. In this case, it is important to emphasize that extremely low pH (such as pH 3.5) can also amplify the positive charge on histones and other DNA-binding proteins (such as transcription factors and even ubiquitin).

[0055] The method of this invention addresses another characteristic of these potentials, which also produce surprising effects. In the preparation of the cation exchanger, the sample is equilibrated to a milder pH of 5.0 ± 0.25 instead of pH 3.5. The cation exchanger is equilibrated to the same conditions, and the sample is loaded. After sample binding, the column is reequilibrated to pH 3.5, and AAV is eluted with an increased salt gradient. Experimental data show that this method significantly reduces contamination of the AAV components eluted from the cation exchanger. In particular, it removes small contaminants within the size range of DNA-binding proteins. This indicates that histones and other DNA-binding proteins still present in the sample after the previous steps of the method of this invention are bound to the cation exchanger when the sample is applied to the column, and they remain bound there when the AAV is eluted. This is consistent with the known behavior of histones, which do not elute from cation exchangers in NaCl but require elution with guanidine or NaOH. Regardless of the mechanism employed, the quality of the eluted AAV is significantly improved.

[0056] Following the initial cation exchange chromatography step, AAV is applied to a positively charged chromatographic apparatus (e.g., an anion exchanger) to further extract DNA and separate empty capsids from intact capsids, referring to capsids containing the desired therapeutic DNA plasmid. A similar treatment can be performed on the AAV capsids after the initial affinity chromatography step.

[0057] Cation exchange chromatography media are widely available on the market, with a variety of different ligands and different physical forms. A key defining characteristic of cation exchangers is that they are negatively charged. Biological species with a sufficiently positive charge bind to them and can then be eluted, most commonly by increasing the salt concentration. However, all cation exchangers are multimodal, meaning that electrostatic interactions represent only one of the chemical mechanisms that facilitate the binding of biomolecules. The two most common cation exchange ligands are carboxyl alkane groups, such as carboxymethyl, carboxyethyl, and carboxypropyl; and sulfonyl alkane groups, such as sulfonylmethyl, sulfonylethyl, and sulfonylpropyl. Carboxyl alkane cation exchange groups contain a carbonyl oxygen atom with two free lone pairs of electrons and a carboxyl oxygen atom with three free lone pairs of electrons. These lone pairs of electrons act as hydrogen acceptors, giving these ligands the potential to participate in hydrogen bonding. Sulfonyl alkane cation exchange groups contain three oxygen atoms, two of which are uncharged but carry two lone pairs of free electrons. The third carries a negative charge and has three lone pairs of electrons, making the ligand a hydrogen donor and enabling it to participate in hydrogen bonding. Cation exchangers sometimes utilize alkane-phosphatidic acid residues. Depending on the pH, they have two negatively charged oxygen atoms, each with three lone pairs of electrons, and one uncharged oxygen atom with two lone pairs of electrons, or they have one negatively charged oxygen atom with three lone pairs of electrons and two uncharged oxygen atoms, each with two lone pairs. Furthermore, phosphatidic acid-based cation exchangers have the ability to participate in metal-coordinated bonding. Dicarboxyl, tricarboxyl, and polycarboxyl cation exchange ligands also have the ability to bind metals. All of the above ligands also exhibit varying degrees of hydrophobicity, depending on whether the bound alkane contains one, two, three, or more carbon residues. Some cation exchange ligands also include a hydrophobic ring. Therefore, for the purposes of this invention, cation exchangers are defined as negatively charged chromatographic solid phases, regardless of any other chemical reactivity they may exhibit. Cation exchangers are commercially available from numerous suppliers worldwide.

[0058] The same general logic applies to anion exchange chromatographic media. For the purposes of this invention, they are defined as positively charged chromatographic solid phases, although they may exhibit any additional chemical reactivity. The charged nitrogen atom in weak anion exchangers has a single pair of free electrons, making it a hydrogen acceptor. A hydrogen atom directly bonded to the nitrogen group can act as a hydrogen donor. The charged nitrogen atom in strong anion exchangers lacks a lone pair of free electrons. The alkane group associated with the nitrogen atom in strong anion exchangers and most weak anion exchangers imparts hydrophobicity. Some employ other hydrophobic structures. Some anion exchangers use polymer-based ligands that produce combinations of other chemical reactivity. All anion exchangers are multimodal. Anion exchangers are commercially available from numerous suppliers worldwide.

[0059] Ligands used in affinity chromatography consist of peptides and proteins, typically representing antibodies or antibody substructures, such as the F(ab)'2 region or Fab region, or single-chain light chain derivatives (scFV) or other recombinant structures, all of which mimic the specificity of antibodies. Similar to ion exchangers, numerous commercial variants are available from various suppliers worldwide.

[0060] The solid-phase DNA extraction process begins when AAV-containing cell culture harvests or lysates are brought into contact with a positively charged surface under approximately physiological conditions. The sample is brought into contact with the positively charged surface for approximately 1 minute to 4 hours.

[0061] In one embodiment, when the positively charged surface is in particulate form, the volume ratio of the particles to the sample can be 0.1% to 10%, 1% to 5%, 2% to 5%, or higher, lower, or intermediate.

[0062] In one embodiment, when the positively charged surface is in particulate form, the contact time can be 10 to 240 minutes, or 20 to 120 minutes, or 30 to 60 minutes, or longer, shorter, or intermediate times.

[0063] In one or more embodiments, when the positively charged surface is a flow device, the contact time can be from 1 minute to 60 minutes, or from 2 minutes to 30 minutes, or from 5 minutes to 15 minutes, or longer, shorter, or intermediate times.

[0064] In some cases, the positively charged particles may be mixed with particles of other chemical properties. Experimental data show that a mixture of negatively charged and positively charged particles removes more pollutants than positively charged particles alone.

[0065] DNase treatment can be performed for 15 minutes to 24 hours, or 30 minutes to 16 hours, or 1 hour to 8 hours, or longer, shorter, or intermediate times. Since achieving the highest possible degree of DNA cleavage is a specific goal, a longer interval (e.g., 16 to 24 hours) is preferred.

[0066] DNase treatment is preferably performed in the same TFF unit used for concentrating and equilibrating the DNase treatment buffer. The transmembrane pressure is set to zero, and the sample is recirculated in the system after the addition of DNase to ensure thorough mixing. After digestion, the TFF buffer is replaced with a buffer at approximately pH 5 and a salt concentration of 20 mM to 100 mM. The transmembrane pressure is set to a positive value, and the TFF is restarted. Most enzymes are eliminated along with the DNA fragments, especially histones.

[0067] The term pH approximately 5 should be understood to mean a pH value within the range of 4.0 to 6.0, or 4.5 to 5.5, or 4.75 to 5.25. Those skilled in the art will understand that the lower the pH, the higher the concentration of salts that may be present in the sample, and that AAV can still be captured by the cation exchanger. The sample may also be diluted at this point to improve the binding capacity of the cation exchanger. Generally, the visual clarity of the equilibrated sample will be an indicator of suitability for the conditions. The presence of turbidity or white filaments or particles indicates that the pH is too low.

[0068] When AAV particles are loaded onto a cation exchanger, the cation exchanger is reequilibrated to a pH of approximately 3.5 ± 0.5, for example, using a buffer containing formic acid, or acetic acid, or glycine, or some combination of these substances, at a concentration of 20 mM to 50 mM. This buffer may contain 20 mM to 200 mM NaCl to prevent binding to contaminants weaker than the AAV capsid. After reequilibration, the column is eluted with a salt gradient to 1.0 M NaCl or higher. Following elution, the column is washed with 1.0 M NaOH (optionally including 1–3 M NaCl) and 20 mM to 50 mM EDTA.

[0069] Many commercially available DNases are available for practicing the methods of this invention. Examples include endonucleases (Protean), deoxyribonucleases (Worthington), Salton enzymes (Blirt), San-HQ and M-San HQ (ArcticZymes), totipotent nucleases (“DENARASE”, C-Lecta), Turbo nucleases (Accelagen), Kaneka endonucleases (Kaneka), DNases (New England Biolabs), and Kryptonase (BIA isolate). Each of these enzymes has its unique requirements for providing the most efficient DNA digestion. Some enzymes are more salt-tolerant than others. Some are more efficient at lower temperatures than others. Some enzymes are capable of cleaving both RNA and DNA. If desired, RNases can be added to enzymes lacking this capability.

[0070] Positively charged particles are commercially available in various forms from multiple suppliers. Particle-based anion exchange chromatography media provide the majority of candidate media, where the positive charge may originate from various forms and combinations of amine derivatives on the particle surface. These forms and combinations include primary amine groups, single residues, and polymers, such as polyallylamine and chitosan; secondary amine groups, single residues, and polymers; tertiary amine groups, single residues, and polymers, such as DEAE and DEAE dextran; quaternary amine groups, single residues, and polymers, such as Q, QA, and Q or QA dextran, or cholestyramine; and individual ligands and polymers containing primary, secondary, and tertiary amine groups. Individual ligands containing such combinations include immobilized ethylenediamine containing primary and secondary amine groups, and tris(2-aminoethyl)amine, whose immobilized form contains primary, secondary, and tertiary amine groups. Polymers in mixed amine forms include polyethyleneimine containing varying proportions of primary, secondary, and tertiary amines, depending on the degree of branching within the polymer. The physical matrix of the particles can be a polymer, silica-based, metal oxide-based, or a combination thereof. The particles can be of any size, for example, from 0.1 μm to 200 μm. The particles can be non-porous or have a porosity ranging from 0.1 nm to 10 μm. The particles can be spherical, ellipsoidal, non-spherical, irregular, filamentous, or have mixed characteristics.

[0071] The positively charged particles can be mixed with other particles exhibiting different chemical reactivity. Preliminary experimental data indicate that, in certain cases (the electronegative particles themselves), the combination of electronegative (ethylenediamine) particles and electronegative (SO3) particles removes more protein contaminants. As mentioned above, electronegative particles can also be multimodal, where specific types of positively charged particles can interact with contaminants not only through electrostatic interactions but also potentially through additional reactivity such as hydrogen bonding, hydrophobicity, or metal affinity.

[0072] Positively charged flow devices are widely available on the market. One increasingly popular form includes so-called depth filters. Other potential forms include simple membrane forms, also known as membrane adsorption units. Others include so-called hydrogels, particle-filled columns, fiber-filled columns, and devices comprising positively charged surfaces of various physical forms. The surface chemistry of such flow devices can include any surface chemistry described for the particles.

[0073] Example

[0074] Example 1

[0075] Purification of AAV using the method of the present invention

[0076] Starting with 300 mL of cell lysate containing AAV serotype 8, 5 mL of positively charged particles were added. These particles were aspherical polymethacrylate particles, ranging in size from 20 μm to 40 μm, with an average pore size of 2 μm, and their surface was coated with ethylenediamine. The ethylenediamine immobilized on the solid surface was positively charged due to each residue carrying both primary and secondary amines. The formulation was mixed for 30 minutes. The particles were removed by centrifugation at 10,000 × g for 10 minutes and filtration through a 0.45 μm PES membrane.

[0077] Using a membrane with a molecular weight cutoff of 300 kDa, the particle-free supernatant was concentrated 14-fold by tangential flow filtration. Consistent with the concentration, the supernatant was buffer-exchanged to 50 mM Tris, 0.5 M NaCl, 5 mM magnesium chloride, 1% sucrose, 0.1% poloxamer, pH 8.0.

[0078] Add salt-tolerant DNase to a final concentration of 50 units / mL and incubate the mixture at room temperature for 16 hours. Perform tangential flow filtration again, still in the same mechanical unit with the same original membrane, adding 20 mM HEPES, 30 mM sodium chloride, 1% sucrose, 0.1% poloxamer, pH 7.0.

[0079] The optically clear supernatant containing AAV was titrated to pH 5.25 by gradually adding 2M acetic acid. The strong cation exchanger (SO3) was equilibrated to 20 mM MES, 30 mM sodium chloride, 1% sucrose, 0.1% poloxamer, pH 5.25. The equilibrated sample was loaded onto the equilibrated column and then washed with equilibration buffer to remove unbound contaminants.

[0080] The cation exchange column was then reequilibrated to 50 mM formic acid, 30 mM sodium chloride, 1% sucrose, 0.1% poloxamer, pH 3.5. After reequilibration, the column was eluted with a linear gradient of sodium chloride, followed by washing with 1 M sodium hydroxide and 2 M sodium chloride. The chromatogram is shown below. Figure 2 As shown. The AAV peak was evaluated using SDS-PAGE. Figure 3 ).

[0081] Example 2

[0082] Purifying AAV using some steps of the method of this invention

[0083] The sample of the same material described in Example 1 was treated in the same manner as in the second TFF step. It was then titrated to pH 3.5 by gradually adding 2M formic acid.

[0084] Equilibrate the same cation exchanger to 50 mM formic acid, 0.2 M sodium chloride, 1% sucrose, 0.1% poloxamer, pH 3.5. Load the sample, wash the column with equilibration buffer, and then elute with the same linear gradient configuration as described in Example 1. Wash the column with 1 M NaOH and 2 M sodium chloride.

[0085] Figure 3 The degree of purification achieved in the two examples was compared. The first group of three sample channels illustrates the results from Example 2, where the cation exchange step was performed at pH 3.5. The second group of three sample channels illustrates the results from Example 1, where the cation exchanger and sample were initially equilibrated to pH 5.25, then the sample was loaded and washed at that pH, the column was reequilibrated to pH 3.5, and eluted at that pH. The results show that the equilibrated sample at pH 5.25 contained less contaminant, allowing the cation exchanger to produce a cleaner AAV component. The sample equilibrated to pH 3.5 contained a higher contaminant load, and the cation exchange produced a less pure component.

[0086] Example 3

[0087] Experimental controls using affinity chromatography were run to demonstrate the potential benefits of applying the method of this invention to other AAV capture methods. The AAV affinity column was equilibrated with 50 mM phosphate, 100 mM NaCl, pH 7.2. The sample was equilibrated to the same pH, filtered, and then loaded onto the column. The column was washed with equilibration buffer and then eluted with a gradient to 50 mM glycine at pH 3.0. After elution, the column was washed with 25 mM sodium hydroxide.

[0088] Chromatogram as shown Figure 4 As shown. Figure 2 chromatograms and Figure 4 Comparison of the chromatograms in the studies showed that affinity chromatography has a similar problem to cation exchange chromatography, namely, the problem of excessive contaminant binding.

[0089] The results of SDS-PAGE analysis are as follows: Figure 5 As shown. Figure 3 PAGE gel in Figure 5 Comparison of the chromatograms shows that the method of the present invention, with cation exchange chromatography, provides better purification compared to affinity chromatography that does not benefit from the method of the present invention.

[0090] Affinity chromatography is generally considered to provide the best capture purification among all chromatography methods. The method of the present invention enables conventional purification methods such as cation exchange chromatography to achieve significantly superior purification capabilities, highlighting the purification potential of the method of the present invention. It also notes that it will significantly improve the purification performance of affinity chromatography as a capture method, and in some cases, it will be able to purify at least research-grade AAV using anion exchange chromatography by following initial steps as described in Example 2.

[0091] Example 4

[0092] This method is adaptable to initial capture by affinity chromatography.

[0093] Perform the steps of the present invention up to the second TFF step, but omitting the subsequent steps of lowering the pH and applying the sample to the cation exchanger. Instead, apply the sample directly to the affinity chromatography column. Then, wash and elute the column as if without the improvements of the present invention.

[0094] Example 5

[0095] This method is adaptable to initial capture by anion exchange chromatography.

[0096] Perform the steps of the invention up to the second TFF step, but omitting the subsequent steps of lowering the pH and applying the sample to the cation exchanger. Instead, dilute the sample to a sufficiently low conductivity (salt concentration) to allow the AAV to bind with the anion exchanger equilibrated to the same conditions. Then, wash and elute the column as if without the improvements of the invention.

[0097] Example 6

[0098] Replace the positively charged particles with the inner surface of the positively charged flow device.

[0099] Any of the above embodiments can be practiced by replacing positively charged particles with a large, positively charged surface. Such surfaces are found in many commercial filter media, such as depth filters.

Claims

1. A method for reducing the content of contaminating DNA in a formulation containing AAV capsid and contaminating DNA, comprising the following steps: a) DNA is extracted using a solid phase with a positively charged surface, and the solid phase is contacted with a formulation at a pH of 7.0 ± 1.0 and a salt concentration of 10 mM to 200 mM to obtain a first component. b) Percolate the first component through a first tangential flow filter to obtain the second component. c) Treat the second component with DNase. d) The second component, which has been treated with DNase and obtained in step c), is percolated through a second tangential flow filtration into a buffer solution with a pH of 7.0 ± 1.0 and a salt concentration of 10 mM to 20 mM to obtain the third component.

2. The method as described in claim 1, characterized in that, The method includes the following steps: e) Concentrate the third component by tangential flow filtration prior to supplemental chromatography.

3. The method as described in claim 1 or 2, characterized in that, The solid phase is in the form of loose bulk particles or a flow device.

4. The method as described in claim 1 or 2, characterized in that, The tangential flow filtration is performed using a membrane selected from the group consisting of: hollow fiber membranes, including single hollow fiber membranes or bundled membranes; or flat membranes, including single-layer membranes or membranes stacked or rolled into a cylinder.

5. The method as described in claim 1 or 2, characterized in that, The first tangential flow filtration is performed in a buffer solution prepared to facilitate DNA digestion by DNases.

6. The method as described in claim 4, characterized in that, The tangential flow filter membrane has a porosity in the range of molecular weight cutoff of 10 kDa to 300 kDa, or 30 kDa to 300 kDa, or 60 kDa to 300 kDa, or 100 kDa to 300 kDa, or 200 kDa to 300 kDa, or 250 kDa to 300 kDa.

7. The method as described in claim 6, characterized in that, The tangential flow filter membrane has a porosity, and the molecular weight cutoff of the porosity is 100 kDa or above.

8. The method as described in claim 1 or 2, characterized in that, The formulation containing AAV capsid and contaminating DNA contains AAV particles selected from the group consisting of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11.

9. The method as described in claim 1 or 2, characterized in that, The formulation containing AAV capsid and contaminating DNA contains AAV particles that are recombinant serotypes with characteristics of one or more serotypes.

10. The method as described in claim 1 or 2, characterized in that, After step d) or step e), at least one of the following purification steps is performed: (i) Cation exchange chromatography step or, (ii) Affinity chromatography step or, (iii) Anion exchange chromatography steps.

11. The method as described in claim 10, characterized in that, In purification step (i), the fraction from step d) or e) is loaded onto cation exchange chromatography material with a pH of 4 to 6, and then: - Reequilibrate the cation exchange chromatography material to a pH of 3.5 ± 0.

5. - Elution using a salt gradient, and Then, anion exchange chromatography is performed.

12. The method as described in claim 10, characterized in that, In purification step (ii), the fraction from step d) or e) is loaded onto affinity chromatography material and subjected to anion exchange chromatography after elution.

13. The method as described in claim 10, characterized in that, In purification step (iii), the fraction obtained in step d) or e) is subjected to anion exchange chromatography.

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