Method for producing whole virus particles, and method for recovering whole virus particles

Abstract

The present invention addresses the problem of providing: a method by which it becomes possible to efficiently produce whole virus particles; and a method for efficiently recovering whole virus particles. The present invention provides a method for producing whole virus particles, the method comprising: a step A in which a zonal rotor is rotated at a low speed; a step B in which the zonal rotor is operated in an ultracentrifugal mode to separate hollow virus particles from whole virus particles; and a step C in which the recovery of a fraction starts at a point in time when A260 / A280, which is the ratio of the ultraviolet absorbance of an eluate at 260 nm to the ultraviolet absorbance of the eluate at 280 nm, reaches an inflection point or a point in time when the electroconductivity of the eluate becomes 300 ms / cm or more, and the recovery of the fraction ends at a point in time when the electroconductivity of the eluate becomes 370 ms / cm or less.

Description

Method for producing whole virus particles and method for recovering whole virus particles

[0001] The present invention relates to a method for producing whole virus particles, which comprises performing ultracentrifugation. The present invention further relates to a method for recovering whole virus particles from a virus particle mixture containing hollow virus particles and whole virus particles, which comprises performing ultracentrifugation.

[0002] Gene therapy, in which a gene or cells transfected with a gene are administered into the human body, is known as a method for treating intractable diseases. One method for introducing a gene into mammalian cells for the purpose of gene therapy is to use a viral vector. A viral vector is a virus strain in which the replication and proliferation capabilities of the virus have been lost or partially lost, and the gene to be introduced for treatment is incorporated into the virus. Known viruses from which viral vectors are derived include enveloped viruses (viruses with an envelope) such as retroviruses, lentiviruses, herpes viruses, and Sendai viruses; and non-enveloped viruses (viruses without an envelope) such as adenoviruses and adeno-associated viruses (AAVs). In particular, AAVs are used in gene therapy for the treatment of various diseases because they can infect many types of cells, are non-pathogenic to humans, and their viral particles are physically stable.

[0003] Known methods for purifying viral vectors such as AAV vectors include chromatography using an ion exchange column or an affinity purification column, a method including a filtration step by tangential flow filtration (TFF), and a method combining these chromatography and TFF steps with sucrose density gradient centrifugation or cesium chloride equilibrium density gradient centrifugation.

[0004] Patent Document 1 describes a method for purifying complete adeno-associated virus (AAV) capsids from a concentrated AAV fraction containing empty AAV capsids and complete AAV capsids. The method described in Patent Document 1 includes the steps of: (i) loading a) a concentrated AAV fraction and b) at least two sugar solutions, each having a different predetermined range of sugar concentration, into a zonal rotor; (ii) operating the ultracentrifuge including the zonal rotor in batch mode to form a sugar density gradient; and (iii) obtaining fractions of the sugar density gradient to obtain an AAV fraction containing complete AAV capsids.

[0005] Patent Document 2 describes a method for efficiently separating genome-containing AAV9 vector particles and genome-deficient rAAV9 intermediates, in which a mixture containing recombinant AAV9 virus particles and AAV9 vector intermediates is subjected to high-performance liquid chromatography, the eluate is subjected to a salt gradient while monitoring ultraviolet absorbance at about 260 nm and about 280 nm, and AAV9 full capsids are recovered from the fraction eluted when the A260 / A280 ratio reaches an inflection point.

[0006] Patent Document 3 describes a method for producing whole virus particles, including a step of purifying whole virus particles from a virus particle mixture containing hollow virus particles, intermediate virus particles, and whole virus particles. The method in Patent Document 3 includes the steps of: (a) rotating a zonal rotor at a low speed and arranging, in this order from the rotor axis toward the outside, the virus particle mixture, a liquid having a lower density than the whole virus particles, and a liquid having a higher density than the liquid; (b) operating the zonal rotor in ultracentrifugation mode to separate the hollow virus particles, intermediate virus particles, and whole virus particles; and (c) fractionating and removing the contents of the zonal rotor and recovering a fraction containing whole virus particles.

[0007] JP 2020-502997 A International Publication No. WO2017 / 160360 A International Publication No. WO2022 / 220273 A

[0008] An object of the present invention is to provide a method for efficiently producing whole virus particles and a method for efficiently recovering whole virus particles.

[0009] The present inventors have found that whole virus particles can be efficiently produced or recovered by centrifuging a virus particle mixture, starting fraction collection when A260 / A280, which is the ratio of the ultraviolet absorbance at 260 nm to the ultraviolet absorbance at 280 nm of the eluate, reaches an inflection point, or when the electrical conductivity of the eluate becomes 300 ms / cm or higher, and ending fraction collection when the electrical conductivity of the eluate becomes 370 ms / cm or lower. The present invention was completed based on the above findings.

[0010] That is, the present invention provides the following: <1> A method for producing whole virus particles, comprising: step A: rotating a zonal rotor at a low speed, and arranging, from the rotor axis toward the outside, a virus particle mixture containing hollow virus particles and whole virus particles, liquid L having a lower density than the whole virus particles, and liquid H1 having a higher density than liquid L, in this order; step B: operating the zonal rotor in an ultracentrifugal mode after step A to separate the hollow virus particles from the whole virus particles; and step C: fractionating and recovering the contents of the zonal rotor after step B, in which fraction recovery is started when A260 / A280, which is the ratio of the ultraviolet absorbance at 260 nm to the ultraviolet absorbance at 280 nm of the eluate, reaches an inflection point or when the electrical conductivity of the eluate becomes 300 ms / cm or higher, and ending the fraction recovery when the electrical conductivity of the eluate becomes 370 ms / cm or lower. <2> The method for producing whole virus particles according to <1>, wherein in step C, the collection of fractions is terminated when the conductivity of the eluate is 360 mS / cm or less. <3> The method for producing whole virus particles according to <1> or <2>, wherein in step C, the collection of fractions is terminated when the conductivity of the eluate is 355 mS / cm or less. <4> The method for producing whole virus particles according to any one of <1> to <3>, wherein the virus particles are adeno-associated virus particles. <5> The method for producing whole virus particles according to any one of <1> to <4>, wherein the virus particle mixture is obtained by culturing cells capable of producing virus particles to obtain a virus particle-containing culture solution, and filtering the virus particle-containing culture solution. <6> The method for producing whole virus particles according to <5>, wherein the filtration is performed using a depth filter having a nominal pore size of 0.1 μm or more. <7> The method for producing whole virus particles according to any one of <1> to <6>, wherein the conductivity of the eluate is monitored after starting the collection of fractions in step C. <8> The method for producing whole virus particles according to any one of <1> to <7>, wherein the volume of the conductivity measurement section in step C is 1 mL or less and the measurement interval is 10 times / minute or more.<9> The method for producing whole virus particles according to any one of <1> to <8>, wherein the liquid L and the liquid H1 contain cesium chloride. <10> The density of the liquid L is 1.21 g / cm. ３ ~1.38g / cm ３ and the density of liquid H1 is 1.39 g / cm ３ <11> The method for producing whole virus particles according to <1>, which uses an automatic fluid processing system including a reservoir connected to a zonal rotor by a flow path and containing a virus particle mixture, a low-density liquid L, or a high-density liquid H1, and a detector, wherein step A of placing the virus particle mixture, the low-density liquid L, and the high-density liquid H1 in the zonal rotor is performed after the virus particle mixture, the low-density liquid L, and the high-density liquid H1 have passed through the flow path of the detector from the reservoir before being placed. <12> A method for recovering whole virus particles from a virus particle mixture containing hollow virus particles and whole virus particles, the method comprising: step A: rotating a zonal rotor at a low speed; and arranging, in this order from the rotor axis toward the outside, a virus particle mixture containing hollow virus particles and whole virus particles, liquid L having a lower density than the whole virus particles, and liquid H1 having a higher density than liquid L; after step A, step B: operating the zonal rotor in an ultracentrifugal mode to separate the hollow virus particles from the whole virus particles; and after step B, step C: eluting the contents of the zonal rotor while fractionating them to recover fractions, the recovery of fractions being started when A260 / A280, which is the ratio of the ultraviolet absorbance at 260 nm to the ultraviolet absorbance at 280 nm of the eluate, reaches an inflection point or when the electrical conductivity of the eluate becomes 300 ms / cm or more, and ending the recovery of fractions when the electrical conductivity of the eluate becomes 370 ms / cm or less.

[0011] According to the present invention, whole virus particles can be efficiently produced, and whole virus particles can be efficiently recovered.

[0012] FIG. 1 shows the relationship between conductivity and purity in Experiment 1. FIG. 2 shows the relationship between conductivity and concentration in Experiment 1. FIG. 3 shows the relationship between density and purity in Experiment 1. FIG. 4 shows the relationship between density and concentration in Experiment 1. FIG. 5 shows the relationship between conductivity and purity in Experiment 2. FIG. 6 shows the relationship between conductivity and concentration in Experiment 2. FIG. 7 shows the relationship between density and purity in Experiment 2. FIG. 8 shows the relationship between density and concentration in Experiment 2.

[0013] The present invention will be described in detail below. In this specification, a numerical range indicated using "to" means a range that includes the numerical values ​​before and after "to" as the minimum and maximum values, respectively.

[0014] The present invention relates to a method for producing whole virus particles, comprising: step A: rotating a zonal rotor at a low speed; and arranging, in this order from the rotor axis toward the outside, a virus particle mixture containing hollow virus particles and whole virus particles, liquid L having a lower density than the whole virus particles, and liquid H1 having a higher density than liquid L; step B: operating the zonal rotor in an ultracentrifugal mode after step A to separate the hollow virus particles from the whole virus particles; and step C: fractionating and recovering the contents of the zonal rotor after step B, in which fraction recovery is started when A260 / A280, which is the ratio of the ultraviolet absorbance at 260 nm to the ultraviolet absorbance at 280 nm of the eluate, reaches an inflection point or when the electrical conductivity of the eluate becomes 300 ms / cm or higher, and ends the fraction recovery when the electrical conductivity of the eluate becomes 370 ms / cm or lower.

[0015] The present invention further relates to a method for recovering whole virus particles from a virus particle mixture containing hollow virus particles and whole virus particles, the method comprising: step A: rotating a zonal rotor at a low speed; and arranging, from the rotor axis toward the outside, a virus particle mixture containing hollow virus particles and whole virus particles, liquid L having a lower density than the whole virus particles, and liquid H1 having a higher density than liquid L, in this order; after step A, step B: operating the zonal rotor in an ultracentrifugal mode to separate the hollow virus particles from the whole virus particles; and after step B, step C: eluting the contents of the zonal rotor while fractionating them to recover fractions, in which recovery of fractions is started when A260 / A280, which is the ratio of the ultraviolet absorbance at 260 nm to the ultraviolet absorbance at 280 nm of the eluate, reaches an inflection point or when the electrical conductivity of the eluate becomes 300 ms / cm or higher, and ends the recovery of fractions when the electrical conductivity of the eluate becomes 370 ms / cm or lower.

[0016] The above-described "method for producing whole virus particles" according to the present invention and the above-described "method for recovering whole virus particles from a virus particle mixture containing hollow virus particles and whole virus particles" according to the present invention are collectively referred to as the method of the present invention.

[0017] In one example of the present invention, solutions of two different concentrations (densities) are used to form a density gradient of solutes in the solutions within a zonal rotor, and whole virus particles can be separated and purified from hollow particles by the density gradient.

[0018] More specifically, one embodiment of the present invention is a method for producing whole virus particles, the method comprising the steps of purifying whole virus particles from a virus particle mixture containing hollow virus particles and whole virus particles; step A: rotating a zonal rotor at a low speed and arranging, in this order from the rotor axis toward the outside, the virus particle mixture, a liquid (liquid L) having a lower density than the whole virus particles, and a liquid (liquid H1) having a higher density than Liquid L; and step B: operating the zonal rotor in ultracentrifugation mode after step A to separate the hollow virus particles, the intermediate virus particles, and the whole virus particles. the step C of fractionating and recovering the contents of the zonal rotor after the step B, in which the collection of fractions is started when A260 / A280, which is the ratio of the ultraviolet absorbance at 260 nm to the ultraviolet absorbance at 280 nm of the eluate, reaches an inflection point or when the electrical conductivity of the eluate becomes 300 ms / cm or more, and the collection of fractions is ended when the electrical conductivity of the eluate becomes 370 ms / cm or less.

[0019] If desired, step A may be a step of rotating a zonal rotor at a low speed and arranging a liquid (liquid B) having a lower density than liquid L and liquid H1, the virus particle mixture, liquid L, and liquid H1 in this order from the rotation axis side of the rotor toward the outside, i.e., liquid B, virus particle mixture, liquid L, and liquid H1.

[0020] A whole virus particle is a virus particle in which a full-length genome (in the case of a viral vector, a full-length vector genome containing an exogenous gene of interest) is packaged and which has the ability to infect target cells. An empty virus particle is a virus particle that does not encapsulate a gene of interest. For virus particles used in gene therapy such as AAV vectors, there is a strong demand for preparing highly pure whole virus particles to ensure safety, etc., and there is an extremely high need to separate whole virus particles from empty virus particles.

[0021] Step A is a step of injecting a virus particle mixture into the zonal rotor while forming a density gradient using a solution (Liquid L) having a lower density than whole virus particles and a solution (Liquid H1) having a higher density than Liquid L. Step A can be performed, for example, by rotating the zonal rotor at a low speed (e.g., 1,000 rpm to 4,000 rpm; approximately 100 × g to 1,600 × g for approximately 30 minutes to 1 hour) and loading the virus particle mixture, Liquid L, and Liquid H1 into the zonal rotor in this order, or a liquid having a lower density than Liquid L and Liquid H (Liquid B), the virus particle mixture, Liquid L, and Liquid H1 in this order (i.e., Liquid B, virus particle mixture, Liquid L, and Liquid H1). After the low-speed rotation is completed, a linear density gradient is formed between the density of Liquid L and the density of Liquid H1 from the rotation axis side of the rotor toward the outside. The virus particles remain on the rotation axis side of the rotor.

[0022] When the virus particles are AAV vectors, the density of the complete virus particles and the empty virus particles is approximately 1.39 g / cm, although there may be some error. ３ ~Approx. 1.41g / cm ３ , about 1.3g / cm ３ ~Approx. 1.38g / cm ３ In order to separate the whole virus particles from the hollow virus particles, the density of the liquid L is, for example, 1.21 g / cm ３ ~1.38g / cm ３ , or 1.22 g / cm ３ ~1.29 g / cm ３ It may also be possible to use the following.

[0023] The density of the liquid H1 is preferably higher than the density of the whole virus particle, for example, 1.39 g / cm ３ As the density of the liquid H1 increases, the density gradient that is formed becomes steeper, and as a result, whole virus particles are concentrated and recovered, but the possibility of hollow virus particles and the like being mixed in also increases. Therefore, the density of the liquid H1 is set to, for example, 1.39 g / cm ３ ~1.45g / cm ３ It may be adjusted to.

[0024] The ratio of the volume of Liquid L and Liquid H1 (total volume of Liquid L and Liquid H1; Liquid L + Liquid H1) to the volume of the virus particle mixed solution (volume of Liquid L + Liquid H1:volume of virus particle mixed solution) injected into the zonal rotor is not particularly limited, but may be, for example, about 1:1 to 4, about 1:1 to 3, or about 1:1 to 2. Furthermore, the ratio of the volume of Liquid H1 to the volume of Liquid L (volume of Liquid H1:volume of Liquid L) injected into the zonal rotor is not particularly limited, but may be, for example, about 1:1 to 3, about 1:1 to 2, or about 1:1 to 1.5.

[0025] The total volume of the virus particle mixed solution, liquid L, and liquid H1 to be injected into the zonal rotor depends on the rotor volume of the zonal rotor used, and a person skilled in the art can appropriately select the optimal injection volume.

[0026] Step B is a step in which the zonal rotor after step A is operated in ultracentrifugation mode (high-speed rotation) to separate hollow virus particles and whole virus particles. The ultracentrifugation mode in step B is not particularly limited, but may be operated at a rotation speed of, for example, about 30,000 rpm to about 40,000 rpm (about 90,000 x g to 160,000 x g). The operation time in ultracentrifugation mode may be, for example, about 3 hours to about 10 hours, or about 4 hours to about 6 hours.

[0027] Step C is a step of fractionating and recovering the contents of the zonal rotor after step B. Those skilled in the art can easily select a method for removing the contents of the zonal rotor from the rotor. For example, the contents may be removed by introducing a liquid (liquid H2) with a higher density than liquid H1 from the radially outer side of the zonal rotor, thereby sequentially extruding the contents of the zonal rotor. The contents extruded from the rotor can be separated into whole virus particles and hollow virus particles by fractionating the contents extruded from the rotor shaft side at fixed volumes using a fraction collector or the like. The contents of the zonal rotor can be extruded from the rotor by injecting liquid H2 into the zonal rotor while rotating the rotor at a low speed (e.g., 1,000 rpm to 4,000 rpm; 100 × g to 1,600 × g). The contents extruded from the rotor may be fractionated and recovered from the rotor shaft side using a fraction collector or the like. The density of liquid H2 may be higher than that of liquid H1.

[0028] In step C, fraction collection is started when A260 / A280, which is the ratio of the ultraviolet absorbance at 260 nm to the ultraviolet absorbance at 280 nm of the eluate, reaches an inflection point or when the conductivity of the eluate becomes 300 ms / cm or higher, and fraction collection is terminated when the conductivity of the eluate becomes 370 ms / cm or lower. Preferably, in step C, fraction collection may be terminated when the conductivity of the eluate becomes 360 ms / cm or lower. More preferably, in step C, fraction collection may be terminated when the conductivity of the eluate becomes 355 ms / cm or lower.

[0029] The ultraviolet absorbance at 260 nm and the ultraviolet absorbance at 280 nm of the eluate can be measured by a conventional method using a spectrophotometer or the like. In a preferred embodiment, a spectrophotometer (e.g., a UV meter) can be installed between the zonal rotor outlet and the fraction collector, and the absorbance at UV 260 nm and UV 280 nm can be continuously measured while the solution extruded from the rotor is being collected. The conductivity of the eluate can be measured by a conventional method using a conductivity meter or the like. In a preferred embodiment, a conductivity meter can be installed between the zonal rotor outlet and the fraction collector, and the conductivity of the solution extruded from the rotor can be continuously measured. In a particularly preferred embodiment, the absorbance at UV 260 nm and UV 280 nm can be continuously measured while the solution extruded from the rotor is being collected, and the conductivity of the solution extruded from the rotor can also be continuously measured. As described above, by installing a spectrophotometer (such as a UV meter) and / or a conductivity meter between the zonal rotor outlet and the fraction collector and performing continuous measurements, it becomes possible to separate empty virus particles and whole virus particles more precisely. In step C, it is preferable to monitor the conductivity of the eluate after starting fraction collection. The conductivity can be monitored by a method known to those skilled in the art. In step C, it is preferable that the volume of the conductivity measurement section is 1 mL or less and the measurement interval is 10 times / minute or more.

[0030] The virus particle mixture, Liquid L, Liquid H1, and Liquid H2 may contain cesium chloride (CAS No. 7647-17-8), iodixanol (CAS No. 92339-11-2), iohexol (CAS No. 66108-95-0), amidotrizoic acid (CAS No. 737-31-5), metrizamide (CAS No. 31112-62-6), or the like. The density gradient formed in the zonal rotor may be a density gradient based on cesium chloride, iodixanol, iohexol, amidotrizoic acid, metrizamide, or the like, and is preferably a density gradient based on cesium chloride. Furthermore, Liquid B, the virus particle mixture, Liquid L, Liquid H1, and Liquid H2 may contain, in addition to water, a buffer solution (e.g., phosphate buffer, HEPES buffer, Tris buffer), salt, or the like.

[0031] The purity of the whole virus particles obtained after step C (proportion of whole virus particles: also called "full rate") is, for example, 90% or more, preferably 95% or more.

[0032] Viruses (particles) include, but are not limited to, wild-type viruses as well as viruses carrying foreign genes used as vectors (viral vectors). The type of virus is not particularly limited, and includes both enveloped and non-enveloped viruses.

[0033] Enveloped virus refers to a virus whose viral genome and a protein shell called capsid are covered with a membrane structure (envelope), and non-enveloped virus refers to a virus that does not have an envelope.Known enveloped virus includes DNA viruses such as herpesvirus, poxvirus, and hepadnavirus, and RNA viruses such as flavivirus, togavirus, coronavirus, orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus, and retrovirus.Known non-enveloped virus includes DNA viruses such as adenovirus, adeno-associated virus (AAV), and papillomavirus, and RNA viruses such as picornavirus, calicivirus, norovirus, and rotavirus.Preferred virus is adeno-associated virus (including adeno-associated virus vector).That is, preferred virus particle is adeno-associated virus particle.

[0034] The following will be further explained by taking as an example the case of producing whole virus particles of an adeno-associated virus vector (AAV vector). As the density gradient medium, for example, cesium chloride or the like can be used. As the liquid L, for example, about 25 wt % to 28 wt % (1.22 g / cm ３ ~1.29 g / cm ３ As the liquid H1, for example, a cesium chloride solution of about 40 wt % to 42 wt % (1.42 g / cm ３ ~1.45g / cm ３A cesium chloride solution of about 1000 rpm may be prepared and poured into a zonal rotor (e.g., P32ZT or P35ZT; both rotors have a rotor lid with a maximum diameter of 24 cm, an inner maximum diameter of 17.78 cm, and a maximum rotation radius (Rmax) of 8.89 cm, manufactured by Eppendorf Himac Technologies, etc.). The virus particle mixture, liquid L, and liquid H1 may be poured into the rotor in this order while the rotor is running at a low speed (e.g., about 3,000 rpm when using P32ZT or P35ZT). By running the rotor at a low speed, a density gradient is formed from the rotor axis toward the outside, resulting from the cesium chloride solution with a density varying from that of the virus particle mixture and liquid L to that of liquid H1 (this is step A of this embodiment).

[0035] Once the cesium chloride density gradient has been formed, the mixture may be run for approximately 4 to 5 hours at high speed (for example, approximately 30,000 to 35,000 rpm when using P32ZT or P35ZT). By running the mixture in ultracentrifugation mode at high speed, hollow virus particles, intermediate virus particles, and whole virus particles are banded in this order from the rotor axis toward the outside (the edge of the rotor) (this is step B of this embodiment).

[0036] The contents inside the rotor are liquid H2 (for example, about 42 wt % to 45 wt % (1.45 g / cm 3 )) which has a higher density than liquid H1 from the outside of the rotor. ３ ~1.50 g / cm ３ By rotating the rotor at a low speed (for example, 3,000 rpm when using P32ZT or P35ZT) while injecting a cesium chloride solution (ca.s., cesium chloride solution of about 1000 rpm), the solution can be extruded out of the rotor, starting from the solution closest to the rotor shaft. The solution extruded out of the rotor can be fractionated and collected using a fraction collector or the like to obtain a fraction containing intact virus particles (this is step C of the present embodiment).

[0037] A virus particle mixture (a mixture containing at least whole virus particles) can be prepared by culturing virus-producing cells and using the virus-producing cells or cell culture solution, and can be easily prepared by a person skilled in the art using appropriate means. Alternatively, a virus particle mixture obtained by concentrating or roughly purifying a culture solution of virus-producing cells or a lysate of the cells using TFF (Tangential Flow Filtration) or column chromatography may be used as the starting sample. Preferably, the virus particle mixture is obtained by culturing cells capable of producing virus particles to obtain a virus particle-containing culture solution, and then filtering the virus particle-containing culture solution. Filtration can be preferably performed using a depth filter with a nominal pore size of 0.1 μm or more.

[0038] A virus-producing cell is a cell that produces elements necessary for forming virus particles and has the ability to produce a virus. The virus-producing cell may be an artificially produced cell capable of producing a virus, or may be a cell that has been infected with a virus in a natural environment and has become capable of producing a virus. The virus-producing cell is preferably an artificially produced virus-producing cell, and particularly preferably the virus is a non-enveloped virus.

[0039] The methods for artificially producing virus-producing cells vary depending on the virus, and details have already been described in many reviews, etc. An outline of the production of cells that produce viral vectors is provided below.

[0040] When producing virus particles that function as vectors, virus-producing cells can be produced by introducing into any cell a plasmid in which the regions encoding viral nonstructural proteins (proteins involved in viral replication, etc.) and the regions encoding viral structural proteins (proteins such as capsid) have been deleted and the gene of interest has been inserted instead, a plasmid encoding the viral nonstructural proteins and structural proteins, or a plasmid encoding other necessary genes depending on the type of viral vector.

[0041] For example, in the case of an AAV vector, an AAV vector-producing cell can be prepared by introducing a plasmid containing a gene of interest, a plasmid containing genes encoding Rep proteins (proteins necessary for viral replication) and Cap proteins (proteins that constitute the capsid), and a plasmid containing genes encoding adenovirus-derived E1a protein, E1b protein, E2 protein, E4 protein, etc. into HEK293 cells, HEK293T cells, or the like.

[0042] Culture conditions for virus-producing cells are already known, and those skilled in the art can select appropriate conditions depending on the type of virus. For example, the conditions are not particularly limited, but include culturing the cells in a medium such as DMEM or IMDM containing necessary supplements (growth factors, amino acids, etc.) and serum at about 30 to 38°C and about 5 to 10% CO ２ The culture may be carried out at the same concentration for several days to about 20 days.

[0043] The sample containing the virus may be an extract obtained by extracting the virus from virus-producing cells, or may be a crudely purified version of the extract. For example, in the case of a virus released into a medium, the culture medium after culturing the virus-producing cells may be collected and used as a sample. In the case of a virus that accumulates intracellularly, the collected virus-producing cells may be disrupted by a freeze-thaw method, ultrasonic disruption, or the like, and debris and the like may be removed and used as a sample. Note that many reagents and kits for preparing virus-containing samples from virus-producing cells are commercially available, and the sample may be prepared using these reagents and kits.

[0044] Step A of placing the virus particle mixture, Liquid L, and Liquid H1 in the zonal rotor in this order, and step C of fractionating and recovering the contents of the zonal rotor, can be performed using an automated fluid processing system. Examples of automated processing systems include those disclosed in WO2005 / 058452 and WO2010 / 144037, which are incorporated herein by reference.

[0045] Although there are differences from the automatic fluid processing system according to the present invention, the automatic fluid processing system shown in FIG. 2 of WO2005 / 058452 is particularly useful as a reference.

[0046] An automated fluid treatment system according to the present invention includes a zonal rotor (not shown in Figure 2 of WO 2005 / 058452) having an inlet end and an outlet end.

[0047] Similar to Figure 2 of WO 2005 / 058452, the automated fluid processing system of the present invention includes reservoirs (solution storage containers) that can contain a virus particle mixture, a low-density liquid L, and a high-density liquid H1, respectively, and are connectable to a computer-controlled multi-path inlet valve 13. The inlet valve 13 can be controlled by a computer 14 so that flow from one of the reservoirs is routed to the inlet end of a zonal rotor via a four-port, four-position selector valve. The outlet end of the selector valve can be connected to the zonal rotor, a detector, and the computer-controlled multi-path outlet valve.

[0048] The zonal outlets can be connected to the fluid inlets of detectors, such as UV detector 19A and / or conductivity detector 19B (instruments that measure ultraviolet absorbance or conductivity), so that the fluid exiting the zonal rotor passes through the detectors. UV detector 19A generates an output electrical signal whose intensity varies depending on the amount of UV light absorbed by the fluid passing through the detector, and this signal is transmitted to computer 16. Conductivity detector 19B generates an output electrical signal whose intensity varies depending on the conductivity of the fluid passing through the detector, and this signal is transmitted to computer 16. Fluid exiting the detector(s) flows to a computer-controlled multipath outlet valve 21. Outlet valve 21 can be connected to a waste reservoir 25, a fraction collector 27, and a plurality of fraction storage reservoirs F3-FN. Outlet valve 21 can be controlled by computer 16 to selectively route fluid exiting the detectors to any of fraction storage reservoirs F1-FN, waste reservoir 25, and fraction collector 27. Fraction collector 27 is movable and has a fixed outlet tube 33. Fraction collector 27 can be moved under the control of computer 16 so that 33 is positioned above a fraction collection reservoir, for example above a well 35 in a microtiter plate 37. To drive fluid flow through the system, the system includes a pump 39, the pump output and direction being under the control of computer 14. Preferably, the pump, valves, column, loop, detector, and fraction collector are all mounted in / on a housing 43 (shown in dashed lines) to form a single unit. Such a unit is commercially available from Cytiva as Äktapure 150™.

[0049] The computer 14 includes a memory 45 containing software for controlling the operation of the pumps, valves and fraction collectors and for processing the output signals from the detectors 19 A, 19 B. Suitable software programs are commercially available from Cytiva, Inc.

[0050] Steps A and C are performed by the automated fluid processing system described above. In step A, the virus particle mixture, low-density liquid L, and high-density liquid H1 are filled through the detector flow path before being filled into the zonal rotor. The path through which these three solutions pass can be set to either fill the zonal rotor directly or pass through the detector flow path before being filled into the zonal rotor. However, when the zonal rotor is filled directly, air inside the zonal rotor gets mixed into the downstream detector flow path, causing detector malfunction and resulting in false detection of the fractionation start point. By passing through the detector flow path before filling the zonal rotor, false detection does not occur and accurate fractionation is possible.

[0051] In step C, the solution centrifuged in the zonal rotor flows through a channel in the detector and is fractionated and collected. By using the above-mentioned automated fluid processing system, fraction collection can be performed automatically, and whole virus particles can be efficiently collected.

[0052] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0053] <1> Methods <1-1> Preparation of AAV Vectors <Cell Culture Medium> Thermo Fisher's suspension HEK293 cell line, Viral Production Cells 2.0 (VPCs 2.0), was used for cell line 1 and cell line 2. The medium used was Fujifilm Irvine Scientific's Balan-CD-HEK293 medium. The cells were cultured in shake flasks (manufactured by Corning) or bioreactors. Shake flasks were cultured in an incubator at 37°C with 120 rpm agitation and 8% CO ２ The cells were cultured in a humidified atmosphere at 0.5 × 10 ６ The cells were seeded at 100 cells / mL, and the cell density was approximately 2 × 10 ６ When the cell density reached 100 cells / mL, gene transfer was carried out by adding a nucleic acid complex.

[0054] <Nucleic Acid> Three plasmid DNAs were used to prepare adeno-associated virus vectors (rAAV): (1) a plasmid containing a gene of interest flanked by ITRs, (2) a packaging plasmid containing rep and cap genes, and (3) an adenovirus helper plasmid containing adenovirus E2, E4, and VA RNA genes. All plasmids used were AAVpro Packaging Plasmid (AAV5,6650, Takara Bio). For (1), EGFP (Enhanced Green Fluorescent Protein) was inserted downstream of the CMV promoter as the gene of interest.

[0055] <Cationic Polymer> Linear polyethyleneimine (PEI) "PEI pro" molecular weight 25 kDa (manufactured by Polyplus) was used as a gene transfer reagent.

[0056] <Transfection> The total amount of plasmid DNA used for transfection was 0.5 μg per 1 million cells. PEI / DNA complexes were prepared using a 2:1 ratio of PEI and plasmid DNA and incubated at room temperature for 15 minutes. The plasmid DNA / PEI complexes were then added to the cell culture medium for transfection. AAV vectors were recovered 72 hours after transfection.

[0057] For recovery, 2 mM MgCl ２ AAV vector was incubated at 1.25 × 10 －５　 The cells were incubated with U / cells Benzonase (Merck) at 37°C for 2 hours to obtain a cell lysate. The cell lysate was then filtered through a Merck depth filter DOSP to remove cell debris. After the entire volume of the cell lysate was loaded, the AAV vectors remaining in the filter unit were recovered by pushing with water.

[0058] <1-2> Purification of intact AAV vector by cesium chloride equilibrium density gradient ultracentrifugation using a zonal rotor. CsCl (FUJIFILM Wako) was added to the prepared AAV vector (5% CsCl). A cesium chloride density gradient was formed by injecting the following solutions into a zonal rotor (P35ZT, Eppendorf Himac Technologies) from the outer edge in the order of (1), (2), (3), and (4), followed by centrifugation at 3,000 rpm.

[0059] (1) 200 mL of HN buffer (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.4, 0.15 M NaCl), (2) 1 L of 5 w / v% CsCl (in pure water) containing AAV vector, (3) 300 mL of 25-27% CsCl (in pure water), (4) 200-300 mL of 40% CsCl (in pure water).

[0060] After forming the density gradient, the zonal rotor was centrifuged at 35,000 rpm for 4 hours (himac CP 80NX, Eppendorf Himac Technologies) to separate the intact AAV vector from the intermediate and empty vector. After ultracentrifugation, 42-45% CsCl (HN buffer) was slowly injected into the rotor from the outside while the zonal rotor was centrifuged at 3,000 rpm, pushing the solution inside the rotor (cesium chloride density gradient solution containing AAV vectors) out of the rotor. The pushed-out solution was fractionated using a fraction collector, and fractions from the intersection of UV260 nm and UV280 nm until the conductivity reached 355 mS / cm were collected. Each collected fraction was buffer-exchanged with PBS using a centrifugal ultrafiltration filter unit (Amicon Ultra, molecular weight cutoff: 50 kDa).

[0061] <1-3> Measurement of UV 260 nm and UV 280 nm The absorbance of each fraction was measured at UV 260 nm and UV 280 nm. A UV meter was installed midway between the outlet of the zonal rotor and the fraction collector, and the absorbance of UV 260 nm and UV 280 nm was continuously measured while the solution extruded from the rotor was being collected.

[0062] <1-4> Measurement of Electrical Conductivity The electrical conductivity of each fraction was measured (Monitor pH / C-900, manufactured by Cytiva). A conductivity meter was installed midway between the outlet of the zonal rotor and the fraction collector, and the electrical conductivity of the solution extruded from the rotor was continuously measured.

[0063] <1-5> Evaluation of whole particles (including full-length genomes) and hollow particles by anion exchange high-performance chromatography (AEX-HPLC) The purity of the AAV vector was determined by injecting 15 μL of sample into an anion exchange column (Thermo Fisher, ProPac SAX-10, 10 μm, 4 × 50 mm) equilibrated with 20 mM ammonium bicarbonate, pH 9.2 (hereinafter referred to as mobile phase A), and separating and quantifying whole and hollow particles by continuously changing the mixing ratio of mobile phase A to 30 mM acetic acid, 15 mM formic acid, pH 2.7 (hereinafter referred to as mobile phase B). The flow rate was 1.75 mL / min, detection was by fluorescence (Ex: 280 nm, Em: 348 nm), and the column temperature was 30°C. Of the elution peaks in the AEX-HPLC analysis, the first peak was defined as Full and the second peak as Empty, and the evaluation of the complete particles (referred to as evaluation of the Full rate (%)) was carried out based on the peak area.

[0064] <1-6> Measurement of genome titer. The genome titer of each fraction obtained after ultracentrifugation using a zonal rotor was measured. The fractions were heat-treated at 95°C for 15 minutes using a thermal cycler to inactivate benzonase and dissolve the capsid. Subsequently, concentration quantification was performed by ddPCR using the AAV ITR region as the target, using a method similar to that described in Hum Gene Ther Methods. 2019 Aug;30(4):127-136.

[0065] <2> Results: Separation of complete AAV vectors (containing the full-length genome including the gene of interest) and hollow particles (not containing the gene of interest) by cesium chloride density gradient ultracentrifugation using a zonal rotor. The conductivity, AAV concentration by ddPCR, and full rate measurements for each fraction in two experiments (Experiment 1 and Experiment 2) are shown in Tables 1 and 2. The relationships between each parameter in Experiment 1 are shown in Figures 1 to 4, and the relationships between each parameter in Experiment 2 are shown in Figures 5 to 8.

[0066] To establish a method for separating large quantities of intact AAV vector particles from hollow particles in a short time, we used a zonal rotor to form a density gradient with two concentrations of cesium chloride solutions and attempted to separate intact particles using an equilibrium density gradient. Because the longer the contact time with cesium chloride, the lower the efficiency of AAV vector infection in cells, it was necessary to shorten the centrifugation time as much as possible.

[0067] Two concentrations of cesium chloride (25-27 wt% and 40 wt%) were injected into the rotor to form a density gradient, and ultracentrifugation was performed. This enabled the processing of large volumes of AAV vector (900-1,000 mL). A peak was detected by ddPCR and AEX-HPLC (intact AAV vector) in the fraction between 300 and 355 mS / cm. This result indicates that intact particles were collected in the fraction between 300 and 355 mS / cm.

[0068]

[0069]

[0070] The above results demonstrate that a density gradient formed using two concentrations of cesium chloride solution can separate whole virus particles and hollow particles in a short time (4-5 hours), and that a large amount of mixed virus particle sample can be processed. In this example, fraction collection was terminated when the conductivity of the eluate reached 355 mS / cm or less, enabling efficient collection and production of whole virus particles. The present invention provides a method for efficiently preparing highly pure viruses (particles), such as viral vectors. Therefore, this method is expected to be useful in medical fields such as gene therapy.

Claims

1. A method for producing whole virus particles, comprising: step A: rotating a zonal rotor at a low speed; and arranging, in this order from the rotor axis toward the outside, a virus particle mixture containing hollow virus particles and whole virus particles, liquid L having a lower density than the whole virus particles, and liquid H1 having a higher density than liquid L; step B: operating the zonal rotor in ultracentrifugal mode after step A to separate the hollow virus particles from the whole virus particles; and step C: fractionating and recovering the contents of the zonal rotor after step B, in which fraction recovery is started when A260 / A280, the ratio of the ultraviolet absorbance at 260 nm to the ultraviolet absorbance at 280 nm of the eluate, reaches an inflection point or when the electrical conductivity of the eluate becomes 300 ms / cm or higher, and ends when the electrical conductivity of the eluate becomes 370 ms / cm or lower.

2. The method for producing whole virus particles according to claim 1, wherein in step C, the collection of fractions is terminated when the conductivity of the eluate is 360 ms / cm or less.

3. A method for producing whole virus particles as described in claim 1, wherein in step C, the collection of fractions is terminated when the conductivity of the eluate is 355 mS / cm or less.

4. A method for producing whole virus particles according to any one of claims 1 to 3, wherein the virus particles are adeno-associated virus particles.

5. The method for producing whole virus particles according to any one of claims 1 to 3, wherein the virus particle mixture is obtained by culturing cells capable of producing virus particles to obtain a virus particle-containing culture solution, and filtering the virus particle-containing culture solution.

6. The method for producing whole virus particles according to claim 5, wherein the filtration is carried out using a depth filter having a nominal pore size of 0.1 μm or more.

7. A method for producing whole virus particles according to any one of claims 1 to 3, wherein in step C, the conductivity of the eluate is monitored after the start of fraction collection.

8. A method for producing whole virus particles described in any one of claims 1 to 3, wherein in step C, the volume of the conductivity measurement section is 1 mL or less and the measurement interval is 10 times / minute or more.

9. A method for producing whole virus particles according to claim 1 or 2, wherein the liquid L and the liquid H1 contain cesium chloride.

10. The density of liquid L is 1.21 g / cm ３ ~1.38g / cm ３ and the density of liquid H1 is 1.39 g / cm ３ The method for producing whole virus particles according to claim 4, which is as described above.

11. A method for producing whole virus particles as described in claim 1, using an automatic fluid processing system comprising a reservoir containing a virus particle mixture, a low-density liquid L or a high-density liquid H1 connected to a zonal rotor by a flow path, and a detector, wherein step A of placing the virus particle mixture, the low-density liquid L and the high-density liquid H1 into the zonal rotor is performed after the mixture has passed from the reservoir through the flow path of the detector.

12. A method for recovering whole virus particles from a virus particle mixture containing hollow virus particles and whole virus particles, comprising: step A: rotating a zonal rotor at a low speed; and arranging, in this order from the rotor axis toward the outside, a virus particle mixture containing hollow virus particles and whole virus particles, liquid L having a lower density than the whole virus particles, and liquid H1 having a higher density than liquid L; after step A, step B: operating the zonal rotor in ultracentrifugal mode to separate the hollow virus particles from the whole virus particles; and after step B, step C: eluting and fractionating the contents of the zonal rotor to recover fractions, wherein fraction recovery is started when A260 / A280, the ratio of the ultraviolet absorbance at 260 nm to the ultraviolet absorbance at 280 nm of the eluate, reaches an inflection point or when the conductivity of the eluate becomes 300 ms / cm or higher, and ends when the conductivity of the eluate becomes 370 ms / cm or lower.

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