Method for heat inactivation of adenovirus

Abstract

A method for heat inactivating adenovirus is provided. The present disclosure generally relates to a method for protecting the genomic integrity and / or biological activity of AAV viral particles in a sample containing both AAV particles and helper viral particles during heat inactivation. The method includes heating a sample containing helper viral particles, AAV particles, and a buffer to a temperature greater than or equal to 45° C. The buffer contains a kosmotropic salt at a concentration of 10 mM or greater and / or a divalent or trivalent cation at a concentration of 10 mM or greater.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority and benefit to U.S. Provisional Patent Application No. 62 / 314,116, filed on March 28, 2016, entitled "Methods of Heat Inactivation of Adenoviruses", the entire disclosure of which is incorporated herein by reference.

Background Art

[0002] Adeno - associated virus (AAV) is a non - pathogenic replication - defective parvovirus. AAV vectors have many unique features that make them attractive as vectors for gene therapy. In particular, AAV vectors can deliver therapeutic genes to both dividing and non - dividing cells, and these genes can persist for long periods without integration into the genome of the target cells. However, to produce AAV vectors, helper virus functions must sometimes be provided in the form of an actively infectious virus such as adenovirus (AV). During the AAV vector purification process to isolate therapeutic AAV vectors, helper virus particles must be inactivated. However, common methods of helper virus inactivation, such as heat inactivation, also result in the disruption or degradation of the AAV vector genome and can reduce the quality and quantity of AAV vectors that can be recovered from the inactivation process. This is particularly true for larger AAV vector genomes. Therefore, there is a need in the art to develop methods to protect the integrity of AAV vectors during the heat inactivation of helper viruses.

Summary of the Invention

Means for Solving the Problems

[0003] The present disclosure generally relates to methods for protecting the integrity and / or biological activity of the genome of AAV viral particles in a sample containing both AAV particles and helper virus particles during heat inactivation. These methods generally enable the selective inactivation of helper virus particles by having a greater effect on helper virus particles than on AAV particles. The method includes heating a sample containing helper virus particles, AAV particles, and a buffer to a temperature above or equal to 45°C. The buffer includes a cosmotrophic salt at a concentration of 10 mM or more and / or a divalent or trivalent cation at a concentration of 10 mM or more.

[0004] The method includes heating the sample to a temperature above or equal to 45°C, above or equal to 46°C, above or equal to 47°C, above or equal to 48°C, above or equal to 49°C, above or equal to 50°C, or above or equal to 51°C. In certain embodiments, the sample is heated to a temperature between 45°C and 65°C, between 45°C and 60°C, between 45°C and 55°C, between 47°C and 53°C, between 48°C and 51°C, or between 48°C and 50°C. For example, in certain embodiments, the sample is heated between 48°C and 50°C, or to 48°C or 49°C.

[0005] The period during which the sample is heated can vary. For example, in certain embodiments, the sample is heated to the target temperature for a period between 1 minute and 6 hours, such as between 10 and 180 minutes, between 20 and 180 minutes, between 20 and 60 minutes, or between 20 and 40 minutes. Generally, higher temperatures or other conditions that promote the inactivation of helper virus particles are used with relatively short heating times, and vice versa.

[0006] In some methods, a buffer containing a divalent or trivalent metal cation at a concentration of 10 mM or greater is used. Exemplary cations include the following metals: Mg, Ca, Mn, Ni, Zn, Co, Sr, Cu, Cr, Fe, and Sc, which form cations such as Mg 2+ , Ca 2+ , Mn 2+ , Ni 2+ , Zn 2+ , Co 2+ , Sr 2+ , Cu 2+ , Cr 2+ and Sc 3+ . In certain embodiments, the buffer contains Mg 2+ and / or Ca 2+ at a concentration of 10 mM or greater.

[0007] In a subset of these buffers containing a divalent or trivalent metal cation at a concentration of 10 mM or greater, the concentration is greater than 15 mM. For example, the cation concentration can be greater than 20 mM, greater than 50 mM, greater than 100 mM, or greater than 200 mM. In another subset of buffers containing a divalent or trivalent metal cation at a concentration of 10 mM or greater, the concentration is from 10 mM to 500 mM. For example, the cation concentration can be from 20 mM to 400 mM, from 30 mM to 300 mM, from 50 mM to 250 mM, from 70 mM to 200 mM, or concentrations of 50 mM, 100 mM, 150 mM, or 200 mM.

[0008] In some methods, a buffer containing a cosmotropic salt at a concentration of 10 mM or greater is used. Exemplary cosmotropic salts include ammonium sulfate, ammonium acetate, sodium citrate, sodium acetate, sodium sulfate, potassium phosphate, and cesium chloride, either individually or in any combination. When present, the cosmotropic salt is typically used at a concentration greater than 10 mM, between 0.1 M and 1 M; between 0.2 M and 0.8 M; between 0.3 M and 0.7 M; between 0.4 M and 0.6 M; or 0.5 M, etc.

[0009] In some methods, the buffer used contains a chaotropic salt. In a subset of buffers containing a chaotropic salt, the chaotropic salt is a salt of urea or a salt of guanidine. In some methods, the buffer used contains a polyol. In a subset of buffers containing a polyol, the polyol is glycerol, propylene glycol, or 1,6 - hexanediol.

[0010] In some methods, the buffer maintains the pH over a temperature range from 4°C to 70°C. A subset of the buffers maintains the pH between 3.0 and 10.0 at a temperature between 4°C and 70°C. A further subset of these buffers maintains the pH between 7.0 and 9.0 at a temperature between 4°C and 70°C.

[0011] In some methods, the buffer is a Tris buffer, a phosphate buffer, a trizoleamine buffer, or a bis - tris propane buffer. In a subset of Tris and bis - tris propane buffers, the buffer contains additional components including HEPES, citrate, NaCl, and Pluronic F68. In a subset of bis - tris propane buffers, the buffer contains 40 mM bis - tris propane, 20 mM HEPES, 20 mM citrate, 200 mM NaCl, and 0.001% (w / v) Pluronic F68.

[0012] The methods disclosed herein are useful for preserving the integrity of the genome of any AAV particle. In some methods, the AAV particle contains a genome of approximately 4.7 kb of DNA, DNA greater than 4.7 kb, DNA greater than 5.0 kb, or approximately 5.1 kb of DNA. Alternatively, the AAV particle can contain a genome of less than 4.0 kb of DNA, or approximately 3.0 kb of DNA. The methods disclosed herein can be used with AAV particles containing a genome that is single - stranded or substantially self - complementary.

[0013] Some methods of inactivating the present invention achieve a logarithmic reduction of more than 5.0 of the active helper virus. A subset of these methods achieves a logarithmic reduction of more than 6.0, or more than 6.3, of the helper virus. In some methods, the helper virus is an adenovirus, a herpesvirus, or a baculovirus. In a subset of methods where the helper virus is an adenovirus, the adenovirus is Ad5. In embodiments of the present invention, for example, the following items are provided. (Item 1) A method for inactivating a helper virus in a sample containing helper virus particles, adeno-associated virus particles, and a buffer, comprising: heating the sample to a temperature above or equal to 45°C wherein the buffer contains a divalent or trivalent cation at a concentration of 10 mM or more, or a cosmotrophic salt at a concentration of 10 mM or more. (Item 2) The method according to item 1, wherein the sample is heated to a temperature between 45°C and 65°C. (Item 3) The method according to item 1, wherein the sample is heated to a temperature between 45°C and 60°C. (Item 4) The method according to item 1, wherein the sample is heated to a temperature between 45°C and 55°C. (Item 5) The method according to item 1, wherein the sample is heated to a temperature between 47°C and 53°C. (Item 6) The method according to item 1, wherein the sample is heated to 48°C. (Item 7) The method according to item 1, wherein the sample is heated to 49°C. (Item 8) The method according to item 1, wherein the sample is maintained at the temperature for a period between 1 minute and 6 hours. (Item 9) The method according to item 1, wherein the sample is maintained at the temperature for a period between 10 minutes and 180 minutes. (Item 10) The method according to item 1, wherein the sample is maintained at the temperature for a period between 20 minutes and 180 minutes. (Item 11) The method according to item 1, wherein the sample is maintained at the temperature for a period between 20 minutes and 60 minutes. (Item 12) The method according to item 1, wherein the sample is maintained at the temperature for a period between 20 minutes and 40 minutes. (Item 13) The method according to item 1, which results in a logarithmic decrease of helper virus of 6.3 or more. (Item 14) The method according to item 1, wherein the adeno-associated virus particles contain a DNA genome of more than 4.7 kb. (Item 15) The method according to item 1, wherein the adeno-associated virus particles contain a DNA genome of more than 5.1 kb. (Item 16) The method according to item 1, wherein the adeno-associated virus particles contain a DNA genome of approximately 4.7 kb. (Item 17) The method according to item 1, wherein the adeno-associated virus particles contain a DNA genome of less than 4.0 kb. (Item 18) The method according to item 1, wherein the adeno-associated virus particles contain a DNA genome of approximately 3.0 kb. (Item 19) The method according to item 1, wherein the adeno-associated virus particles contain a genome that is substantially self-complementary. (Item 20) The method according to item 1, wherein the buffer further contains a chaotropic salt. (Item 21) The method according to item 20, wherein the chaotropic salt is a salt of urea. (Item 22) The method according to item 20, wherein the chaotropic salt is a salt of guanidine. (Item 23) The method according to item 1, wherein the buffer solution further contains a polyol selected from the group consisting of glycerol, propylene glycol, and 1,6 - hexanediol. (Item 24) The method according to item 1, wherein the buffer solution maintains a pH between 3.0 and 10.0 at a temperature between 4°C and 70°C. (Item 25) The method according to item 1, wherein the buffer solution maintains a pH between 7.0 and 9.0 at a temperature between 4°C and 70°C. (Item 26) The method according to item 1, wherein the buffer solution further contains 40 mM bis - tris propane, 20 mM HEPES, 20 mM citrate, 200 mM NaCl, and 0.001% (w / v) Pluronic F68. (Item 27) The method according to item 24, wherein the buffer solution is a tris buffer solution. (Item 28) The method according to item 24, wherein the buffer solution is a phosphate buffer solution. (Item 29) The method according to item 24, wherein the buffer solution is a triazoleamine buffer solution. (Item 30) The method according to item 1, wherein the helper virus is an adenovirus. (Item 31) The method according to item 28, wherein the adenovirus is Ad5. (Item 32) The method according to any one of items 1 to 31, wherein the concentration of the divalent or trivalent cation exceeds 15 mM. (Item 33) The method according to any one of items 1 to 31, wherein the concentration of the divalent or trivalent cation exceeds 20 mM. (Item 34) The method according to any one of items 1 to 31, wherein the concentration of the divalent or trivalent cation exceeds 50 mM. (Item 35) The method according to any one of items 1 to 31, wherein the concentration of the divalent or trivalent cation exceeds 100 mM. (Item 36) The method according to any one of items 1 to 31, wherein the concentration of the divalent or trivalent cation exceeds 200 mM. (Item 37) The method according to any one of items 1 to 31, wherein the concentration of the divalent or trivalent cation is from 10 mM to 500 mM. (Item 38) The method according to item 37, wherein the concentration of the divalent or trivalent cation is from 20 mM to 400 mM. (Item 39) The method according to item 37, wherein the concentration of the divalent or trivalent cation is from 30 mM to 300 mM. (Item 40) The method according to item 37, wherein the concentration of the divalent or trivalent cation is from 50 mM to 250 mM. (Item 41) The method according to item 37, wherein the concentration of the divalent or trivalent cation is from 70 mM to 200 mM. (Item 42) The method according to any one of items 1 to 31, wherein the buffer solution contains a divalent or trivalent cation of a metal selected from the group consisting of Mg, Ca, Mn, Ni, Zn, Co, Sr, Cu, Cr, Fe, and Sc at a concentration of 10 mM or more. (Item 43) The method according to any one of items 1 to 31, wherein the cation is a divalent cation. (Item 44) The cation is Mg 2+ , Ca 2+ , Mn 2+ , Ni 2+ , Zn 2+ , Co 2+ , Sr 2+ , Cu 2+ and Cr 2+ The method according to item 43, which is selected from the group consisting of. (Item 45) The cation is Ca 2+ The method according to item 43. (Item 46) The method according to item 43, wherein the cation is Mg 2+ . (Item 47) The method according to any one of items 1 to 31, wherein the cation is a trivalent cation. (Item 48) The method according to item 47, wherein the cation is Sc 3+ . (Item 49) The method according to any one of items 1 to 31, wherein the buffer solution contains a cosmotropic salt selected from the group consisting of ammonium sulfate, ammonium acetate, sodium citrate, sodium acetate, sodium sulfate, potassium phosphate, and cesium chloride at a concentration of 10 mM or more. (Item 50) The method according to item 49, wherein the buffer solution contains ammonium sulfate at a concentration of 10 mM or more. (Item 51) The method according to item 49 or 50, wherein the concentration of the cosmotropic salt is 0.1 M to 1 M. (Item 52) The method according to item 49 or 50, wherein the concentration of the cosmotropic salt is 0.2 M to 0.8 M. (Item 53) The method according to item 49 or 50, wherein the concentration of the cosmotropic salt is 0.3 M to 0.7 M. (Item 54) The method according to item 49 or 50, wherein the concentration of the cosmotropic salt is 0.4 M to 0.6 M. (Item 55) The method according to item 49 or 50, wherein the concentration of the cosmotropic salt is 0.5 M.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0025] A method for inactivating helper viruses such as AV in a sample containing helper virus particles and AAV particles, which results in increased recovery of AAV particles having an intact genome and / or biological activity, is described herein. The method includes heating a sample containing helper virus particles, AAV particles, and a buffer at a temperature and for a time sufficient to inactivate the helper virus particles. Unexpectedly, it has been shown that the addition of divalent or trivalent metal ions or cosmotropic salts to the buffer according to the present invention results in increased recovery of AAV particles containing an intact genome. Adeno-associated virus

[0026] AAV is a small non-enveloped icosahedral virus of the Dependoparvovirus genus in the Parvovirus family. AAV has a single-stranded linear DNA genome of approximately 4.7 kb. AAV includes a number of serologically distinguishable types, including not only serotypes AAV-1 to AAV-12, but also more than 100 serotypes from non-human primates. See, for example, Srivastava, J. Cell Biochem., Vol. 105(1):17-24 (2008); Gao et al., J. Virol., Vol. 78(12), pp. 6381-6388 (2004). Any AAV type can be used in the methods of the present invention. AAV can infect both dividing and quiescent cells of several tissue types by different AAV serotypes that exhibit different tissue tropisms. AAV replicates non-autonomously and has a life cycle with a latent and a productive phase. During the latent phase, after a cell is infected with AAV, AAV is site-specifically integrated into the host genome as a provirus. The productive phase does not occur unless the cell is also infected with a helper virus (e.g., AV or herpes simplex virus), which enables the replication of AAV and results in the production of both AAV particles and helper virus particles. The production of this mixed population of AAV particles and helper virus particles poses a significant problem when AAV is used as a therapeutic vector because the contaminating helper virus must be removed or inactivated due to its potential pathogenicity and / or immunogenicity.

[0027] The wild-type AAV genome contains two 145-nucleotide inverted terminal repeats (ITRs) that contain signal sequences directing AAV replication, capsid formation, and integration of the genome. In addition to the ITRs, three AAV promoters, p5, p19, and p40, drive the expression of two open reading frames encoding the rep and cap genes. The two rep promoters, in combination with differential splicing of a single AAV intron, result in the production of four rep proteins (Rep78, Rep68, Rep52, and Rep40) from the rep gene. The rep proteins are involved in genome replication. The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3, which are splice variants of the cap gene. These proteins form the capsid of the AAV particle.

[0028] Since the cis-acting signals for replication, capsid formation, and integration are contained within the ITRs, some or all of the 4.3 kb internal genome can be replaced with foreign DNA, such as an expression cassette for a foreign protein of interest. In this case, the rep and cap proteins are provided in trans, for example, on a plasmid. To produce an AAV vector, a cell line that permits AAV replication must express the rep and cap genes, an expression cassette adjacent to the ITRs, and helper functions provided by a helper virus, such as the AV genes E1a, E1b55K, E2a, E4orf6, and VA (Weitzman et al., Adeno-associated virus biology. Adeno-Associated Virus:Methods and Protocols, pp. 1-23, 2011). Production of the AAV vector can also result in the production of helper virus particles, which must be removed or inactivated prior to use of the AAV vector. A number of cell types, including HEK293 cells, COS cells, HeLa cells, Vero cells, as well as insect cells, are suitable for the production of AAV vectors (see, for example, U.S. Patent No. 6,156,303, U.S. Patent No. 5,387,484, U.S. Patent No. 5,741,683, U.S. Patent No. 5,691,176, U.S. Patent No. 5,688,676, U.S. Patent No. 8,163,543, US20020081721, WO00 / 47757, WO00 / 24916, and WO96 / 17947). AAV vectors are typically produced in these cell types by one plasmid containing an expression cassette adjacent to the ITRs and one or more additional plasmids that provide additional AAV and helper virus genes.

[0029] AAV vectors of any serotype can be used in the present invention. Similarly, it is contemplated that any AAV type can be used, and one of ordinary skill in the art can identify the AAV type and AAV type suitable for the production of the desired AAV vector. AAV particles and AAV particles can be minimally purified, for example, by affinity chromatography, iodixonal gradient, or CsCl gradient. Samples containing more purified AAV particles and AAV particles can also be used in the method of the present invention, similar to samples with low purification degrees.

[0030] The genome of wild-type AAV is single-stranded DNA and is 4.7 kb. An AAV vector can have a single-stranded genome larger than or smaller than 4.7 kb, including a genome as large as 5.2 kb or as small as about 3.0 kb. Furthermore, the vector genome can be substantially self-complementary such that the genome is substantially double-stranded within the virus. As shown here, AAV vectors containing large genomes are more sensitive to thermal degradation. Therefore, AAV vectors containing large genomes are preferred for use in the method of the present invention. However, one of ordinary skill in the art understands that increasing the stability of all types of AAV vectors during heat inactivation is valuable because it allows for increasingly stringent inactivation conditions. Therefore, AAV vectors containing genomes of all types are suitable for use in the method of the present invention. Helper virus

[0031] As discussed above, AAV requires co - infection with a helper virus to enter the infectious stage of its life cycle. Helper viruses include adenovirus (AV) and herpes simplex virus (HSV), and there are systems that use baculovirus to produce AAV in insect cells. It has also been proposed that papillomavirus may provide helper functions for AAV. See Hermonat et al., Molecular Therapy 9, pp. S289 - S290 (2004). Helper viruses include any virus that can create an allowing AAV replication. Any helper virus can be used in the present invention, provided that it exhibits lower thermal stability than AAV. AV is a non - enveloped nuclear DNA virus containing a double - stranded DNA genome of approximately 36 kb. AV can rescue latent AAV proviruses in cells by providing the E1a gene, E1b55K gene, E2a gene, E4orf6 gene, and VA gene, which enable AAV replication and capsid formation.

[0032] HSV is a family of viruses with a relatively large double - stranded linear DNA genome encapsulated in an icosahedral capsid surrounded by a lipid bilayer envelope. HSV is infectious and highly contagious. The following HSV - 1 replication proteins: helicase / primase complex (UL5, UL8, and UL52) and the DNA - binding protein ICP8 encoded by the UL29 gene, along with other proteins that enhance helper functions, have been identified as necessary for AAV replication.

[0033] Other helper viruses, such as baculovirus, can be used in conjunction with the present invention, provided that they can assist AAV replication either naturally or in a modified form and exhibit a lower level of thermal stability than AAV. Production of AAV vectors

[0034] AAV vectors can be produced in mammalian or insect cells by a number of methods known in the art. Any production method is suitable for producing starting materials for the present invention provided that the result of the production method is a sample containing both AAV and helper virus. The manufacturing process includes providing cells with a plasmid containing an AAV vector genome having not only the functions of the AAV rep gene and cap gene but also additional helper functions. The additional helper functions can be provided, for example, by AV infection, by a plasmid carrying all of the necessary AV helper function genes, or by other viruses such as HSV or baculovirus. Suitable AAV production methods for use with the methods of the present invention include those disclosed in Clark et al., Human Gene Therapy, Vol. 6: pp. 1329-1341 (1995); Martin et al., Human Gene Therapy Methods, Vol. 24: pp. 253-269 (2013); Thorne et al., Human Gene Therapy, Vol. 20: pp. 707-714 (2009); Fraser Wright, Human Gene Therapy, Vol. 20: pp. 698-706 (2009); Virag et al., Human Gene Therapy, Vol. 20: pp. 807-817 (2009).

[0035] The AAV product is collected from the cell lysate or from the cell culture medium. The primary purification step includes affinity chromatography and ion exchange chromatography to remove cell contaminants. The purified sample is filtered and stored at -60°C or below. Cell lysis

[0023] AAV particles can be obtained from infected cells by lysing the cells. Lysis of AAV-infected cells can be achieved by chemically or enzymatically treating the cells to release infectious virus particles. These methods include the use of nucleases such as benzonase or DNase, proteases such as trypsin, or detergents or surfactants. Physical disruption such as homogenization or grinding, or pressurization with a microfluidizer pressure cell, or freeze-thaw cycles can also be used. Alternatively, the supernatant can be harvested from AAV-infected cells without the need for cell lysis. Purification of virus particles

[0036] Prior to the inactivation method of the present invention, it may be necessary to purify a sample containing AAV particles and helper virus particles, for example, to remove cell debris resulting from cell lysis. Methods for minimal purification of helper virus particles and AAV particles are known in the art, and any suitable method can be used to prepare a sample containing both AAV particles and helper virus particles for use in the methods of the present invention. Two exemplary purification methods are density gradient purification based on cesium chloride (CsCl) and iodixanol. Both methods are described in Strobel et al., Human Gene Therapy Methods, Vol. 26(4):147-157 (2015). Minimal purification can also be achieved, for example, using affinity chromatography using AVB Sepharose affinity resin (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The method for AAV purification using AVB Sepharose affinity resin is described, for example, in Wang et al., Mol Ther Methods Clin Dev., Vol. 2:15040 (2015). Heat inactivation

[0037] The heat inactivation technique is based on the different thermal stabilities of AAV particles and helper virus particles. For example, AAV particles can be heated to a high temperature of 56 °C and still remain intact, while AV particles are inactivated. Conway et al., Gene Therapy 6, pp. 986-993, 1999, describe the differential heat inactivation of HSV in samples containing AAV. Heat inactivation can be achieved by any known methodology. In the examples described below, heat inactivation was achieved using a thermocycler to rapidly heat and cool sample volumes of 300 μL or less. This system was chosen because it relies primarily on conductive heat transfer and serves as a viable model for both continuous flow systems and larger batch systems that employ active mixing. Examples of continuous flow systems include the passage of a sample through a continuous flow heat exchanger such as the DHX™ Single-Use Heat Exchanger for Bio-therapeutic Manufacturing (Thermo Fisher Scientific, Millersberg, PA). Such systems enable the operator to control the heat inactivation process by controlling the flow rate of the sample through the heat exchanger, and as a result, the duration of the heating process and the temperature of the heat exchanger, and as a result, the temperature of the heat inactivation.

[0038] Alternatively, heat inactivation can be achieved using batch systems of various sizes. For example, heat inactivation can be achieved on a 1L scale by placing a sample containing AAV in a 1L PETG bottle and placing the bottle in a water bath set to the desired inactivation temperature for a desired period with mixing, e.g., the sample can be heated to 47°C for 20 minutes. On a larger scale, heat inactivation can be achieved by placing a sample containing AAV in a 5L bioprocessing bag on a temperature-controlled rocking platform set to the desired inactivation temperature for a desired period. For example, the rocking platform can be set at a mixing angle of 12°, a rocking speed of 30 RPM, and 49°C for 40 minutes.

[0039] Heat inactivation can be performed at any temperature because the stability between AAV particles and helper virus particles is sufficiently different such that while the helper virus particles are substantially inactivated, active AAV particles remain. In the present invention, a sample containing AAV particles and helper virus particles is heated to a temperature above or equal to 45°C, generally a temperature between 45°C and 55°C, and most frequently a temperature of 49°C ± 2°C is used. Generally, the sample is held at the temperature for a period from 1 minute to 60 minutes, and 10 - 40 minutes is most frequently used. However, as shown in Figure 1, heat inactivation is almost independent of time, so the duration of the heating step is a function of the time required to bring the entire sample to that temperature. For example, a 300μL sample size can be brought to a uniform temperature within seconds, while a multi-liter tank may take many minutes to reach a uniform temperature. Further, one of ordinary skill in the art will understand that higher temperatures may be required to achieve a greater level of AV reduction. Measurement of inactivation efficacy

[0040] Once heat inactivation is achieved, it may be necessary or desirable to determine the efficiency of inactivation. The potency of an inactivation protocol is determined by an assay that detects the presence of a replicable helper virus, such as a plaque assay. Plaque assays for helper viruses, including those for AV, HSV, baculovirus, etc., are well known to those skilled in the art. The adenovirus plaque assay can be performed using any suitable cell type, such as HeLa cells or HEK293 cells. Standard plaque assay protocols are described, for example, in Current Protocols in Human Genetics, 2003. Alternative assays for measuring adenovirus titers include those that enable the identification of infected cells in culture by detecting viral proteins, such as hexon protein, using immunocytochemical staining. Such assays include the QuickTiter™ Adenovirus Titer Immunoassay Kit (Cell Biolabs, San Diego, CA). Generally, the efficiency of inactivation is reported as the log reduction in virus (LRV). Quantification of AAV Particles

[0041] Quantification of AAV particles is complicated by the fact that AAV infection does not result in a cytopathic effect in vitro and thus plaque assays cannot be used to determine infectivity titers. However, AAV particles can be quantified using several methods, including quantitative polymerase chain reaction (qPCR) (Clark et al., Hum. Gene Ther., 10:1031-1039 (1999)) or dot blot hybridization (Samulski et al., J. Virol., 63:3822-3828 (1989)), or by the optical density of highly purified vector preparations (Sommer et al., Mol. Ther., 7:122-128 (2003)). DNase-resistant particles (DRP) are quantified using a thermocycler (e.g., iCycler iQ It can be quantified by real-time quantitative polymerase chain reaction (qPCR) (DRP-qPCR) in a 96-well block format thermocycler (Bio-Rad, Hercules, CA). Samples containing AAV particles are incubated at 37 °C for 60 minutes in the presence of DNase I (100 U / ml; Promega, Madison, WI), followed by proteinase K (Invitrogen, Carlsbad, CA) digestion (10 U / ml) at 50 °C for 60 minutes and then denaturation at 95 °C for 30 minutes. The primer-probe set used should be specific for the non-native portion of the AAV vector genome, such as the poly(A) sequence of the protein of interest. The PCR products can be amplified using any suitable set of cycling parameters based on the length and composition of the primers, probes, and amplified sequences. Alternative protocols are disclosed, for example, in Lock et al., Human Gene Therapy Methods, Volume 25(2):115-125 (2014).

[0042] The infectivity of AAV particles can be determined, for example, using the TCID 50 (50% tissue culture infective dose) assay described in Zhen et al., Human Gene Therapy, Volume 15:709-715 (2004). In this assay, AAV vector particles are serially diluted and used to co-infect a Rep / Cap-expressing cell line with AV particles in a 96-well plate. At 48 hours post-infection, total intracellular DNA is extracted from the infected wells and control wells. Then, qPCR is used with a transgene-specific probe and primers to measure the replication of the AAV vector. The TCID 50 infectivity (TCID 50 / ml) is calculated by the Kaerber equation using the ratio of AAV-positive wells in a 10-fold serial dilution. Background buffer

[0043] The method of the present invention involves the use of a background buffer. The background buffer can maintain a stable pH over a wide temperature range. For example, when the temperature of the buffer changes between 4°C and 70°C, it can maintain a pH in the range of 3.0 to 10.0. It can also maintain a pH between 7.0 and 9.0 when the temperature of the buffer changes between 4°C and 70°C. Exemplary background buffers include glycine, citrate, succinate, acetate, MES (2-(N-morpholino)ethanesulfonic acid), bis-tris (bis-(2-hydroxyethyl)-imino-tris-(hydroxymethyl)-methane), phosphate, pipes (1,4-piperazinediethanesulfonic acid), mopso (3-morpholino-2-hydroxypropanesulfonic acid), BTP (1,3-bis(tris(hydroxymethyl)methylamino)propane), MOPS (3-morpholinopropane-1-sulfonic acid), TES 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TEA (tris base, acetic acid, and EDTA), tris, tricine, bicine, lactate, formate, and systems buffered by MMA (2-methylpropanedioic acid). One exemplary background buffer contains 40 mM bis-trispropane, 20 mM HEPES, 20 mM citrate, 200 mM NaCl, and 0.001% (w / v) Pluronic F68 and has a pH of 8.0 at room temperature. Divalent and trivalent metal ions

[0044] A subset of the buffers used in the present invention further includes divalent or trivalent metal cations, such as Mg 2+ , Ca 2+ , Mn 2+ , Ni 2+ , Zn 2+ , Co 2+ , Sr 2+ , Cu 2+ , Cr 2+ and Sc 3+It contains. The salts of the above cations can also be adopted. When adopted in the present invention, the divalent or trivalent cations are present at a total concentration exceeding 10 mM. As shown in the following examples, all buffers containing divalent or trivalent cations exceeding 10 mM tested provided significant protection compared to samples heated in the control buffer. The highest level of protection was observed in buffers containing 50 mM to 200 mM of MgCl 2 It was observed in the buffer containing. For highly soluble metal ions such as Ca or Mg cations, increasing the cation concentration beyond the amount necessary to achieve maximum protection of AAV has no negative effect on the protection of AAV and results in equivalent results, which is understood by those skilled in the art. The buffer of the present invention may also contain chaotropic salts, including salts of urea or guanidine. The buffer can also contain polyols, and preferred polyols are glycerol, propylene glycol, and 1,6-hexanediol. Cosmotropic salts

[0045] A subset of the buffers used in the present invention contains cosmotropic salts. Cosmotropic salts are cosolvents that contribute to the stability and structure of water-water interactions. Cosmotropic salts also stabilize proteins, membranes, and hydrophobic aggregates in solution. In one embodiment of the present invention, the buffer contains one or more cosmotropic salts, particularly strong cosmotropic salts such as ammonium sulfate, ammonium acetate, sodium citrate, sodium acetate, sodium sulfate, potassium phosphate, and cesium chloride. Cosmotropic salts are useful in the present invention at concentrations exceeding 10 mM, for example, at concentrations of 0.1 M to 1 M, 0.2 M to 0.8 M, 0.3 M to 0.7 M, 0.4 M to 0.6 M, or 0.5 M.

Examples

[0046] The following examples are not intended to limit the scope of the present invention in any way and are provided to illustrate aspects of the disclosed methods. Many other embodiments of the present invention will be apparent to those skilled in the art. (Example 1)

[0047] Ad5 AV particles were produced using standard techniques and minimally purified by CsCl gradient. The Ad5 particles were dialyzed into a background buffer (40 mM bis-tris propane, 20 mM HEPES, 20 mM citrate, 200 mM NaCl, 0.001% (w / v) Pluronic F68, pH 8.0). Heating was performed using a thermocycler to immediately heat the sample to the desired temperature, hold at the desired temperature, and then immediately cool the sample. Samples of Ad5 particles in buffer were contained in polypropylene tubes and had a volume of 300 μL or less. Except for the Ad5 AV sample that was only heated to 51°C and held at that temperature for 10 minutes, the Ad5 AV samples were heated to 45°C, 47°C, 49°C, or 51°C for a duration of either 10 minutes, 15 minutes, or 40 minutes. Subsequently, following the heat treatment, Ad5 infectivity was measured using the QuickTiter™ Adenovirus Titer Immunoassay Kit (Cell Biolabs, San Diego, CA) according to the manufacturer's instructions.

[0048] The thermal inactivation data of Ad5 AV are shown in Figure 1 as the viral log reduction (LRV) of the Ad5 virus, and it is desirable to exceed 6 LRV. As can be seen in Figure 1, the inactivation of the Ad5 material is highly temperature-dependent, and the time at temperature has little effect on inactivation. Based on these data, it is desirable to perform the inactivation step at 47°C or higher. (Example 2)

[0049] AAV vectors with different serotypes and containing different genomic types and sizes were tested for the effect of the AV thermal inactivation protocol on the stability of the AAV genome. Two serotypes of AAV8 and hu37 with three different genomes were tested. The following AAV vectors: (A) hu37 capsid containing a single-stranded construct (4.7 kb) (B) hu37 capsid containing an extra-large single-stranded construct (5.1 kb) (C) An AAV8 capsid containing a small single-stranded construct (4.1 kb) (D) An AAV8 capsid containing a self-complementary construct (4.6 kb) (A), (C), and (D) were tested. AAV vectors (A), (C), and (D) were produced in HEK293 cells, and particle (B) was produced in HeLa cells. All vectors were minimally purified by affinity chromatography or iodixanol gradient using standard techniques. The vectors were dialyzed into a background buffer (40 mM bis-tris propane, 20 mM HEPES, 20 mM citrate, 200 mM NaCl, 0.001% (w / v) Pluronic F68, pH 8.0). Heating was performed using a thermocycler to immediately heat the vector samples to the desired temperature, hold at the desired temperature, and then immediately cool the samples. The vector samples were contained in polypropylene tubes and had a volume of 300 μL or less. The vector samples were exposed to the following heat inactivation conditions: vectors (A), (B), and (C) were heated to 45 °C, 47 °C, or 49 °C, and vector (B) was heated to 45 °C, 47 °C, 49 °C, 51 °C, or 53 °C. After heating for 10, 20, or 40 minutes, all vectors were tested. Samples after heat treatment were analyzed for genomic stability by DRP-qPCR assay.

[0050] The data from this experiment are shown in Figure 2. The genomic degradation of the AAV products after the inactivation protocol was determined by the DRP-qPCR assay and expressed as the qPCR yield (%). The qPCR yield (%) was determined by comparing to the starting sample that was not exposed to the temperature increase and was kept at 4°C. As can be seen in the figure, for serotype AAV8 containing single-stranded or self-complementary constructs less than 4.7 kb in length (Figure 2C and Figure 2D, respectively), the temperature increase and the time at that temperature did not significantly degrade the AAV material. In contrast, the hu37 serotype containing the single-stranded construct (4.7 kb) was more sensitive to heat and heating time compared to a similarly sized construct produced in AAV8 (Figure 2A vs. Figure 2C). Finally, for the hu37 serotype, the extra-large construct (5.1 kb) had a significant negative impact on the stability to heat compared to the construct of normal genomic size (4.7 kb) (Figure 2B vs. Figure 2A). These results show that different serotypes have different thermal stability profiles, but more importantly, that the construct size has a significant impact on the stability of the capsid to heat. (Example 3)

[0051] Two methods for determining the integrity of the AAV vector genome were compared. Samples of the hu37 vector containing the extra-large genome (5.1 kb) were heated at 22°C, 45°C, 47°C, 49°C, 51°C, or 53°C for 20 minutes, and the recovery rate was measured by the DRP-qPCR assay or by the TCID 50 infectivity assay. Control samples were kept frozen until the integrity of the genome was measured.

[0052] Figure 3 shows DRP-qPCR vs. TCID 50Represents the comparison of data collected using the infectivity assay. As can be seen in the figure, the recovery rates resulting from the two techniques produced qualitatively similar results, producing the same trend of decreasing capsid quality with increasing temperature. These data thus strengthen the use of the DRP-qPCR assay as a model to perform preliminary investigations into reducing capsid degradation when exposed to elevated temperatures. (Example 4)

[0053] The effect of the cell production system on heat sensitivity was tested by comparing the thermal stability of AAV vectors produced using different cell types. Vectors of serotype hu37 containing the extra-large genome (5.1 kb) were produced using either HeLa cells or HEK293 cells. Vector samples were heated to 45 °C, 47 °C, 49 °C, 51 °C, or 53 °C for 10, 20, or 40 minutes. The integrity of the genome was measured by DRP-qPCR. As shown in Figure 4, not all data points were repeated for both vector types. However, the results from the two experiments were qualitatively similar. These data suggest that the cell line production system used to create the AAV material had no significant effect on the resulting thermal stability of the AAV. In addition, these data support the overall finding that the hu37 serotype containing the extra-large construct material is sensitive to the heating conditions required for Ad5 inactivation (as shown in Figure 1). (Example 5)

[0054] An AAV vector sample of serotype hu37 containing a large genome was produced as described in Example 2. The sample was dialyzed either in (A) a standard background buffer (40 mM bis-tris propane, 20 mM HEPES, 20 mM citrate, 200 mM NaCl, 0.001% (w / v) Pluronic F68, pH 8.0) or (B) a standard background buffer containing 0.5 M ammonium sulfate. The sample was heated as follows: 10 and 40 minutes at 45 °C, 20 minutes at 47 °C, 10 and 40 minutes at 49 °C, and 20 minutes at 51 °C. Genome integrity was measured by DRP-qPCR.

[0055] As shown in Figure 5, addition of 0.5 M ammonium sulfate to the background buffer improved the thermal stability of the AAV vector (Figure 5A vs. Figure 5B). These data indicate that addition of a strong cosmotropic salt to the buffer formulation used during the heat inactivation step can protect AAV vectors against the effects of thermal degradation during heating.

[0056] These data demonstrate that AAV can be sensitive to the conditions required for heat inactivation of Ad5 virus. The stability of AAV appears to be serotype-dependent, and the use of large constructs (defined as large constructs with a length of 4.7 kb or greater) can have a significantly detrimental effect on the stability of AAV materials. The data presented in this report also suggest that addition of cosmotropic salts can have a beneficial effect of increasing the stability of AAV materials when exposed to elevated temperatures. (Example 6)

[0057] An AAV vector sample of serotype hu37 containing a large genome was produced as described in Example 2. (A) A standard background buffer (40 mM bis-tris propane, 20 mM HEPES, 20 mM citrate, 200 mM NaCl, 0.001% (w / v) Pluronic F68, pH 8.0), (B) 0.1 mM MgCl 2 , (C) 10 mM MgCl 2, (D) 25 mM MgCl 2 , (E) 50 mM MgCl 2 , (F) 100 mM MgCl 2 , or (G) 200 mM MgCl 2 The sample was dialyzed in any of the standard background buffers containing it. The sample was heated to the temperature shown in Table 1 and for the duration shown in Table 1. The integrity of the genome of the AAV particles was measured by DRP-qPCR. The results of the experiment are presented in Table 2. By comparing with the starting sample that was not exposed to the temperature increase and was kept at 4 °C, the residual titer was determined and reported as a percentage. The coefficient of variation (%CV) was also reported.

[0058] Figure 6 shows the recovery rate of the AAV vector in the buffer containing MgCl from 0 to 100 mM 2 over 40 minutes at 47 °C. Samples in buffers containing less than 25 mM MgCl 2 had significantly lower recovery rates at all time points.

[0059] Figure 7 shows the recovery rate of the AAV vector in the buffer containing 100 mM or 200 mM MgCl 2 over 180 minutes at 49 °C. Figure 8 shows the recovery rate of the AAV vector in the buffer containing 100 mM or 200 mM MgCl 2 over 180 minutes at 51 °C. Figure 9 shows the recovery rate of the AAV vector in the buffer containing 100 mM or 200 mM MgCl 2 over 180 minutes at 53 °C. Overall, these data suggest that the buffer containing 200 mM MgCl 2 provides slightly better protection against degradation during long-term heating. These differences are shown in Figures 10 and 11, where a slightly increased loss of the vector is shown with increasing duration and temperature for the buffer containing 100 mM MgCl 2 versus the buffer containing 200 mM MgCl 2 .

Table 1

Table 2-1

Table 2-2

Table 2-3

[0060] In addition to what is shown and described herein, various modifications of the invention and many further embodiments thereof will become apparent to those skilled in the art from the entire contents of this document, including references to scientific and patent documents cited herein. The subject matter of this specification contains important information, exemplification and guidance that can be adapted to the practice of the invention in its various embodiments and their equivalents.

Claims

1. A method for inactivating helper virus in a sample containing helper virus particles, adeno-associated virus particles, and a buffer, comprising: heating the sample to a temperature above or equal to 45°C for a period of 1 minute to 6 hours; wherein the buffer contains ammonium sulfate at a concentration of 0.1 M to 1 M.

2. The method according to claim 1, wherein the sample is heated to a temperature between 45°C and 55°C.

3. The method according to claim 1, wherein the sample is maintained at the temperature for a period of 10 minutes to 40 minutes.

4. The method according to claim 1, wherein the buffer further contains a chaotropic salt selected from the group consisting of salts of urea and salts of guanidine.

5. The method according to claim 1, wherein the buffer further contains a polyol selected from the group consisting of glycerol, propylene glycol, and 1,6 - hexanediol.

6. The method according to claim 1, wherein the buffer maintains the pH of the sample between 3.0 and 10.0, or between 7.0 and 9.0, at a temperature between 4°C and 70°C.

7. The method according to claim 1, wherein the buffer further contains 40 mM bis - tris propane, 20 mM HEPES, 20 mM citrate, 200 mM NaCl, and 0.001% (w / v) Pluronic® F68.

8. The method according to claim 6, wherein the buffer is a tris buffer; a phosphate buffer; or a triazoleamine buffer.

9. The method according to claim 1, wherein the helper virus is an adenovirus.

10. The method according to claim 9, wherein the adenovirus is Ad5.

11. The method according to claim 1, wherein the concentration of the ammonium sulfate is 0.5 M to 0.6 M.

12. The method according to claim 1, wherein the buffer further contains a divalent or trivalent cation at a concentration between 25 mM and 500 mM, and the divalent or trivalent cation is selected from the group consisting of Mg, Ca, Mn, Ni, Zn, Co, Sr, Cu, Cr, Fe, and Sc.

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