HSV-1 purification method

Abstract

The invention relates to a virus purification method. The invention relates to a method for purifying herpes simplex virus (HSV-1). The invention also relates to the HSV-1 purified according to the method. In particular, the method does not include an ultrafiltration concentration step. In particular, the method has a high virus titer yield and a high impurity removal rate.

Description

Technical Field

[0001] The present application relates to a virus purification method. The present application relates to a method for purifying herpes simplex virus (HSV-1). The present application also relates to HSV-1 purified according to the method. In particular, the method does not include an ultrafiltration concentration step. Background Art

[0002] Herpes simplex virus type 1 (HSV-1) is an enveloped double-stranded DNA virus with a molecular weight of approximately 152 kb, composed of more than 70 proteins, and a complete virion of 200 nm. Currently, herpes simplex virus type 1 has been able to be genetically modified and developed into oncolytic herpes simplex virus type 1 (oncolytic herpes simplex virus type 1, abbreviated as oHSV-1) for cancer treatment. Oncolytic herpes simplex virus type 1 is a virus that is inoculated into human osteosarcoma cells (U-2 OS), cultured, harvested, separated and purified to prepare drugs. The purification process needs to effectively remove related impurities, including host proteins, host nucleic acids, and process additives such as nucleases.

[0003] However, due to the particularity of oncolytic viruses, their activity needs to be maintained during the separation and purification process, which requires the separation and purification process to be friendly to virus stability, robust, reproducible, and suitable for commercial scale-up production. At present, there are relatively few literature reports on the separation and purification methods of herpes simplex virus. In these studies, in order to obtain herpes simplex virus with higher purity, purification methods such as ultracentrifugation and density gradient centrifugation are usually used. However, such methods process small amounts of samples, low flux, long operation time, and low virus purity and recovery rates. There are also reports of ultrafiltration technology (such as flat membrane or hollow fiber) to concentrate the virus, followed by purification methods such as affinity chromatography or ion exchange chromatography, but there are still problems such as complex processes and easy inactivation of the virus.

[0004] Therefore, it is necessary to develop a purification method for HSV-1 virus to solve the problem of large-scale, efficient and robust purification and preparation of HSV-1. On the one hand, it can achieve high HSV-1 virus titer yield and low impurity residues, and on the other hand, it has a more streamlined and efficient process suitable for large-scale commercial production. Summary of the invention

[0005] The HSV-1 purification method provided in the present application has a high virus titer yield and a high impurity (e.g., host protein and / or host nucleic acid) removal rate. In addition, the HSV-1 purification method provided in the present application does not include an ultrafiltration concentration step, which greatly simplifies the purification process and improves the purification efficiency. Specifically, the HSV-1 purification method provided in the present application can obtain a high virus titer yield (e.g., at least 70%) and a high impurity removal rate (e.g., at least 98%) only through a heparin affinity chromatography step; when heparin affinity chromatography is combined with other chromatography steps (e.g., composite mode chromatography and / or ion exchange chromatography and molecular sieve chromatography), it also has a high virus titer yield (e.g., at least 50%) and a high impurity removal rate (e.g., at least 99%). The HSV-1 purification method provided in the present application has low requirements for the initial sample to be purified (for example, HSV-1 particles can be efficiently captured directly from the virus harvest fluid), the purification process is simple, and can be used for large-scale preparation of HSV-1 complete particles. The yield and purity meet industrial requirements and can be used for the development of tumor treatment drugs, diagnostic reagents or vaccines.

[0006] In a first aspect, the present application provides a method for purifying herpes simplex virus (HSV-1), the method comprising:

[0007] (a) inoculating HSV-1 into host cells to amplify HSV-1;

[0008] (b) lysing the host cell to release the amplified HSV-1;

[0009] (c) filtering the product obtained in step (b) to remove impurities;

[0010] (d) subjecting the product obtained in step (c) to heparin affinity chromatography.

[0011] In certain embodiments, the medium for heparin affinity chromatography is selected from Capto Heparin chromatography medium, Heparin Sepharose FF chromatography medium, Bestarose Heparin chromatography medium, Bestarose Heparin HR chromatography medium, or any combination thereof.

[0012] In certain embodiments, the method further comprises:

[0013] (e) subjecting the product obtained in step (d) to ion exchange chromatography and / or multimodal chromatography.

[0014] In certain embodiments, the method further comprises:

[0015] (f) subjecting the product obtained in step (e) to molecular sieve chromatography.

[0016] In certain embodiments, the heparin affinity chromatography medium is Capto Heparin chromatography medium or Bestarose Heparin chromatography medium.

[0017] In certain embodiments, the method comprises subjecting the product obtained in step (d) to ion exchange chromatography or multimodal media chromatography.

[0018] In certain embodiments, the multimodal medium chromatography medium has an outer layer of molecular sieve and an inner layer of anion exchange chromatography medium.

[0019] In certain embodiments, the medium for multimodal medium chromatography is Capto Core700.

[0020] In certain embodiments, the medium for ion exchange chromatography is Capto Q or Capto DEAE.

[0021] In certain embodiments, the medium for ion exchange chromatography is Capto DEAE.

[0022] In certain embodiments, the medium for molecular sieve chromatography is Sepharose 6 Fast Flow or Sepharose 4 Fast Flow.

[0023] In certain embodiments, the ion exchange chromatography medium may also be an ion exchange chromatography medium having the same ion exchange type and matrix as Capto Q or Capto DEAE.

[0024] In certain embodiments, the molecular sieve chromatography medium may also be a molecular sieve chromatography medium having the same agarose matrix and cross-linking degree as Sepharose 6 Fast Flow or Sepharose 4 Fast Flow.

[0025] In certain embodiments, the chromatography medium Capto Q or Capto DEAE includes but is not limited to Capto Q or Capto DEAE chromatography medium purchased from Cytiva.

[0026] Although there are cases in the art where heparin affinity chromatography has been used to purify some viruses, based on the diversity of different viral structures, different viruses vary greatly in whether they can bind to heparin or in the properties of binding to heparin (e.g., affinity, specificity, etc.). The method of the present application is particularly suitable for the purification of herpes simplex virus (HSV-1), in particular, the purification of oncolytic herpes simplex KOS virus type I (abbreviated as OVH-APD1 strain, i.e., rHSV-1-APD1).

[0027] Therefore, in certain embodiments, the HSV-1 is an oncolytic Herpes Simplex Virus Type 1 (oHSV-1 for short).

[0028] In certain embodiments, the HSV-1 is an oncolytic herpes simplex KOS virus type 1 (abbreviated as OVH-APD1 strain, ie, rHSV-1-APD1).

[0029] In certain embodiments, the oncolytic herpes simplex KOS virus type I is obtained by the method described in patent application number PCT / CN2018 / 077518, i.e., the non-essential gene ICP0 and the neurovirulence factor gene ICP34.5 are knocked out, and the expression of the immediate early protein ICP27 gene essential for viral replication is regulated by the tumor-specific human telomerase reverse transcriptase (hTERT) promoter, thereby obtaining the oncolytic herpes simplex KOS virus type I.

[0030] In certain embodiments, the host cell is selected from mammalian (eg, human, mouse) cells.

[0031] In certain embodiments, the host cell is selected from human osteosarcoma cells (U-2 OS) cells, Hela cells, Vero adherent cells, Hep-2 cells, BHK cells, or any combination thereof.

[0032] In certain embodiments, the host cell is a human osteosarcoma cell (U-2 OS) cell.

[0033] In certain embodiments, the inoculation is performed at an MOI value of 0.02 to 0.06 (eg, 0.02, 0.03, 0.04, 0.05, 0.06).

[0034] In certain embodiments, the seeding is carried out at a cell density of 2E5 to 5E5 cells / cm 2 (e.g., 2E5 cells / cm 2 、3E5 cells / cm 2 、4E5 cells / cm 2 , 5E5 cells / cm 2 ) conditions.

[0035] In certain embodiments, in step (b), the method comprises lysing the host cell by a lysis solution.

[0036] In certain embodiments, the methods involve nuclease digestion of nucleic acids released by lysis of the host cells.

[0037] In certain embodiments, the nuclease concentration is 20-100 U / ml (e.g., 20-40 U / ml, 40-60 U / ml, 60-80 U / ml, 80-100 U / ml).

[0038] In certain embodiments, the nuclease treatment conditions are 33-37°C (e.g., 33°C, 34°C, 35°C, 36°C, 37°C) for 1-3 h or 18-26°C (e.g., 18°C, 20°C, 22°C, 24°C, 26°C) for 4-23 h (e.g., 4 h, 8 h, 12 h, 13 h, 16 h, 20 h, 23 h).

[0039] In certain embodiments, magnesium ions are also added when lysing the host cells.

[0040] In certain embodiments, the concentration of the magnesium ions is 1-10 mM (eg, 1 mM, 2 mM, 4 mM, 5 mM, 6 mM, 8 mM, 10 mM).

[0041] In certain embodiments, the lysate comprises NaCl and sucrose.

[0042] In certain embodiments, the lysate has a formula of 1000-2000 mM (e.g., 1000 mM, 1200 mM, 1400 mM, 1600 mM, 1800 mM, 2000 mM) of NaCl and 10%-20% m / v (e.g., 10% m / v, 12% m / v, 14% m / v, 15% m / v, 16% m / v, 18% m / v, 20% m / v) of sucrose, and a pH of 6.5-8.0 (e.g., pH=6.5, pH=7.0, pH=7.4, pH=7.5, pH=8.0).

[0043] In certain embodiments, in step (c), the filtration is performed by single-stage or multi-stage filtration.

[0044] In certain embodiments, the filtration uses a combination of a 5 μm PP material filter and a 1+0.65 μm PES material filter.

[0045] In certain embodiments, the impurities include host proteins, host nucleic acids, and process additives, such as nucleases.

[0046] In certain embodiments, in step (d), the heparin affinity chromatography uses an equilibration buffer to equilibrate the chromatography column medium and / or flush out impurities.

[0047] In certain embodiments, the heparin affinity chromatography uses an elution buffer to elute HSV-1.

[0048] In certain embodiments, the heparin affinity chromatography uses a regeneration buffer to regenerate the chromatography column.

[0049] In certain embodiments, the heparin affinity chromatography uses an equilibration buffer, an elution buffer, and a regeneration buffer in that order.

[0050] In certain embodiments, the product obtained in step (c) is loaded after using the equilibration buffer and before using the elution buffer, and the average retention time of the product in the chromatography medium is 3 to 10 min.

[0051] In certain embodiments, the buffer system of the buffer (eg, equilibration buffer, elution buffer, regeneration buffer) is selected from a phosphate buffer system, a Tris buffer system, a His buffer system, a sodium acetate buffer system, or any combination thereof.

[0052] In certain embodiments, the buffer system of the equilibration solution (e.g., equilibration buffer, elution buffer, regeneration buffer) is a phosphate buffer system (e.g., comprising H2PO4 - and HPO4 2- ).

[0053] In certain embodiments, the equilibration buffer comprises NaH2PO4, Na2HPO4, NaCl, and sucrose, and has a pH of 6 to 8.

[0054] In certain embodiments, the elution buffer comprises NaH2PO4, Na2HPO4, NaCl, and sucrose, and has a pH of 6 to 8.

[0055] In certain embodiments, the regeneration buffer comprises NaH2PO4, Na2HPO4, NaCl, and sucrose, and has a pH of 6 to 8.

[0056] In certain embodiments, the equilibration buffer has a formula of 3.9 mM NaH2PO4, 6.1 mM Na2HPO4, 0.3 M NaCl, and 1.7% (m / v) sucrose, and has a pH of 7.

[0057] In certain embodiments, the elution buffer has a formulation of 3.9 mM NaH2PO4, 6.1 mM Na2HPO4, 0.5 M NaCl, and 1.7% (m / v) sucrose, and has a pH of 7.

[0058] In certain embodiments, the regeneration buffer has a formula of 3.9 mM NaH2PO4, 6.1 mM Na2HPO4, 1.0 M NaCl, and 1.7% (m / v) sucrose, and has a pH of 7.

[0059] In certain embodiments, the impurities include host proteins and / or host nucleic acids.

[0060] In certain embodiments, in step (e), the multimodal chromatography uses an equilibration buffer to equilibrate the chromatography column medium and / or to wash HSV-1.

[0061] In certain embodiments, the ion exchange chromatography uses an equilibration buffer to equilibrate the chromatography column medium.

[0062] In certain embodiments, the ion exchange chromatography uses an elution buffer to elute HSV-1.

[0063] In certain embodiments, the buffer system of the buffer (eg, equilibration buffer, elution buffer) is selected from a phosphate buffer system, a Tris buffer system, a His buffer system, a sodium acetate buffer system, or any combination thereof.

[0064] In certain embodiments, the buffer system of the equilibration solution (e.g., equilibration buffer, elution buffer) is a phosphate buffer system (e.g., comprising H2PO4 - and HPO4 2- ).

[0065] In certain embodiments, the multimodal chromatography uses an equilibration buffer comprising NaH2PO4, Na2HPO4, NaCl, and sucrose, and a pH value of 6 to 8.

[0066] In certain embodiments, the equilibration buffer used in the multimodal chromatography has a formulation of 3.9 mM NaH2PO4, 6.1 mM Na2HPO4, 0.3 M NaCl and 1.7% (m / v) sucrose, and a pH of 7.

[0067] In certain embodiments, the ion exchange chromatography uses an equilibration buffer comprising NaH2PO4, Na2HPO4, NaCl, and sucrose, and a pH of 6 to 8.

[0068] In certain embodiments, the ion exchange chromatography uses an equilibration buffer having a formula of 3.9 mM NaH2PO4, 6.1 mM Na2HPO4, 0.5 M NaCl and 1.7% (m / v) sucrose, and a pH value of 7.

[0069] In certain embodiments, the ion exchange chromatography uses an elution buffer comprising NaH2PO4, Na2HPO4, NaCl and sucrose, and having a pH of 6 to 8.

[0070] In certain embodiments, the ion exchange chromatography uses an elution buffer having a formulation of 3.9 mM NaH2PO4, 6.1 mM Na2HPO4, 1.0 M NaCl and 1.7% (m / v) sucrose, and a pH value of 7.

[0071] In certain embodiments, the column height of the multimodal chromatography column is 10 to 30 cm.

[0072] In certain embodiments, the average residence time of the product in the multimodal chromatography medium is 2 to 5 min.

[0073] In certain embodiments, the ion exchange chromatography column has a column height of 10 to 30 cm.

[0074] In certain embodiments, the average residence time of the product in the ion exchange chromatography medium is 2 to 5 min.

[0075] In certain embodiments, in step (f), the molecular sieve chromatography uses a buffer to equilibrate the chromatography column medium and / or to wash the HSV-1.

[0076] In certain embodiments, the buffer system of the buffer is selected from a phosphate buffer system, a Tris buffer system, a His buffer system, a sodium acetate buffer system, or any combination thereof.

[0077] In certain embodiments, the buffer system of the balancing solution is a phosphate buffer system (e.g., comprising H2PO4 - and HPO4 2- ). In certain embodiments, the buffer comprises NaH2PO4, Na2HPO4, histidine, NaCl, trehalose, and sorbitol.

[0078] In certain embodiments, the buffer has a formula of 3.9 mM NaH2PO4, 6.1 mM Na2HPO4, 5 mM histidine, 50 mM NaCl, 4.2% (m / v) trehalose, and 0.8% (m / v) sorbitol.

[0079] In certain embodiments, the molecular sieve chromatography column has a column height of 20 to 30 cm.

[0080] In certain embodiments, the linear flow rate of the molecular sieve chromatography is 30-120 cm / h.

[0081] In certain embodiments, the method does not include the step of concentrating by ultrafiltration.

[0082] In certain embodiments, the method does not include an ultrafiltration concentration step between step (b) and step (d).

[0083] In certain embodiments, the method does not include a step of ultrafiltration concentration after step (d).

[0084] In certain embodiments, after step (d), the impurity removal rate can reach at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%.

[0085] In certain embodiments, the impurities include host proteins and / or host nucleic acids.

[0086] In certain embodiments, after step (d), the titer yield of HSV-1 can reach at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%.

[0087] In a second aspect, the present application provides an HSV-1 virus obtained by the method described in the first aspect.

[0088] In certain embodiments, the HSV-1 is oncolytic Herpes Simplex Virus Type 1 (oHSV-1).

[0089] In certain embodiments, the HSV-1 is an oncolytic herpes simplex KOS virus type 1 (abbreviated as OVH-APD1 strain, ie, rHSV-1-APD1).

[0090] In certain embodiments, the oncolytic herpes simplex KOS virus type I is obtained by the method described in patent application number PCT / CN2018 / 077518, i.e., the non-essential gene ICP0 and the neurovirulence factor gene ICP34.5 are knocked out, and the expression of the immediate early protein ICP27 gene essential for viral replication is regulated by the tumor-specific human telomerase reverse transcriptase (hTERT) promoter, thereby obtaining the oncolytic herpes simplex KOS virus type I.

[0091] In certain embodiments, the HSV-1 can be used in the development of tumor therapeutic drugs, diagnostic reagents or vaccines.

[0093] Definition of terms

[0094] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the virology, biochemistry, and immunology laboratory operation steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0095] When the terms "for example," "such as," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0096] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0097] As used herein, the term "chromatography" refers to a separation technique that uses a mobile phase and a stationary phase to separate one type of molecule (e.g., HSV-1) from other molecules (e.g., impurities) in a sample. The liquid mobile phase is a mixture containing the specific type of molecules to be separated and other molecules. The specific type of molecules to be separated and other molecules are transported across or through the stationary phase (e.g., a solid matrix) by the mobile phase, and the specific type of molecules to be separated in the mobile phase can be separated from other molecules due to different interactions between different molecules in the mobile phase and the stationary phase or different retention times of different molecules in the mobile phase in the stationary phase.

[0098] As used herein, the term "heparin affinity chromatography" refers to a chromatography method that relies on the specific binding of a target molecule to a heparin molecule (or heparin mimetic), wherein the heparin molecule (or heparin mimetic) coupled to a solid phase carrier (or matrix) as a ligand can interact with a molecule (e.g., HSV) in a mobile phase (sample), i.e., the heparin molecule (or heparin mimetic) has a specific affinity for the molecule to be purified. As understood in the context of the present invention, heparin affinity chromatography involves adding a sample containing HSV to a stationary phase comprising heparin (or heparin mimetic) as a ligand.

[0099] As used herein, the term "HSV", also known as herpes simplex virus, is a double-stranded DNA virus belonging to the herpesviridae family. HSV can cause a variety of diseases, including oral herpes and genital herpes. According to antigenic differences, HSV is divided into two serotypes: HSV-1 and HSV-2. The affinity of HSV to heparin mainly depends on the viral surface glycoproteins gC and gB, which are present in both HSV-1 and HSV-2. In certain embodiments, the HSV used is HSV-1, also known as herpes simplex virus type 1.

[0100] In particular, "oncolytic herpes simplex virus type 1", also known as "oHSV-1", refers to a genetically engineered recombinant HSV virus that has a high level of replication ability in tumor cells and can effectively kill a variety of tumor cells, but has significantly reduced replication and killing abilities in normal cells, and oHSV-1 has the same physicochemical properties (e.g., purification properties) as HSV. In certain embodiments, the HSV used is oHSV-1.

[0101] As used herein, the term "ultrafiltration" is also known as tangential flow filtration (TFF) and cross-flow filtration, which is a technique for purifying biomolecules. During ultrafiltration, the feed liquid flows parallel to the membrane surface, part of the sample liquid passes through the membrane (filtrate), and the remaining feed liquid (reflux liquid) circulates back to the feed container. Ultrafiltration technology generally includes hollow fiber and membrane package methods. In certain embodiments herein, the ultrafiltration uses a membrane package method.

[0102] As used herein, the term "virus harvest liquid" is a liquid containing virus particles harvested from host cells inoculated with viruses, and the processing steps include culturing viruses in host cells, lysing host cells, nuclease digestion and filtration. After the above steps, the liquid containing virus particles is the "virus harvest liquid", and the obtained virus harvest liquid can be used for further virus purification, titer determination, infectivity test or other related biomedical research and application. In certain embodiments herein, the virus harvest liquid is further purified.

[0103] As used herein, the term "compound mode chromatography", also known as "compound mode medium chromatography" or "multi-mode chromatography" or "mixed mode chromatography", is a chromatographic separation technique that utilizes more than one chromatographic technique (e.g., gel filtration, ion exchange, hydrophobic and affinity chromatography) to act alone or in synergy with each other. In certain embodiments herein, the compound mode chromatography utilizes molecular sieve chromatography and ion exchange chromatography.

[0104] As used herein, the term "ion exchange chromatography" (IEC) is a technique for purifying viruses by utilizing electrostatic interactions between ion exchangers and virus particles. This technique is based on the surface charge characteristics of virus particles, and selectively adsorbs and elutes to separate virus particles from impurity components. In certain embodiments herein, the ion exchange chromatography is anion exchange chromatography.

[0105] As used herein, the term "regeneration" refers to the process of washing away viral particles or other impurities bound to a chromatographic medium in a chromatographic technique so that the chromatographic medium can be reused. The regeneration process ensures that the chromatographic medium can maintain activity and selectivity over multiple purification cycles. Regeneration typically involves the use of a regeneration buffer to disrupt the interaction between the viral particles and the chromatographic medium, thereby dissociating the viral particles from the medium and ensuring the activity and integrity of the viral particles. In certain embodiments herein, a regeneration buffer is used in a multimodal chromatography to achieve regeneration.

[0106] As used herein, the term "molecular sieve chromatography", also known as "gel filtration chromatography" or "exclusion chromatography", is a technique for separation based on the size differences of molecules in a sample. This technique generally selects a porous gel with different pore sizes as the stationary phase according to the size of the sample to be separated, and separates substances of different molecular sizes through its molecular sieve effect.

[0107] As used herein, the term "virus titer yield" refers to the retention ratio of the activity and quantity of virus particles from the virus harvest solution to the final harvested virus stock solution during the virus purification process. It is an indicator for comprehensively evaluating the activity retention and recovery efficiency of the virus during the purification process.

[0108] As used herein, the term "virus stabilizer" refers to a class of substances used to protect virus particles from physical and chemical damage. Virus stabilizers can be small molecule compounds (such as sugars and amino acids), polymers or proteins that can maintain the structural integrity and biological activity of virus particles, ensuring that the virus is kept stable and functional during the purification process, especially downstream of high salt, pH changes or other conditions that may cause virus inactivation. In certain embodiments herein, virus stabilizers are used in molecular sieve chromatography.

[0109] As used herein, the term "wash" refers to the process of washing a sample through the top of a chromatography column with a buffer solution after the sample passes through the chromatography medium. The wash can be used to remove sample components or other impurities that are not adsorbed in the chromatography medium, thereby reducing the impact of these components on the purity of the purified target collected subsequently, and can also be used to elute the purified target remaining in the chromatography medium to improve the yield.

[0110] Advantageous Effects of the Invention

[0111] The HSV-1 purification method provided in the present application has a high virus titer yield and a high impurity (e.g., host protein and / or host nucleic acid) removal rate. In addition, the HSV-1 purification method provided in the present application does not include an ultrafiltration concentration step, which greatly simplifies the purification process and improves the purification efficiency. Specifically, the HSV-1 purification method provided in the present application can obtain a high virus titer yield (e.g., at least 70%) and a high impurity removal rate (e.g., at least 98%) only through a heparin affinity chromatography step; when heparin affinity chromatography is combined with other chromatography steps (e.g., composite mode chromatography and / or ion exchange chromatography and molecular sieve chromatography), it also has a high virus titer yield (e.g., at least 50%) and a high impurity removal rate (e.g., at least 99%).

[0112] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. According to the following detailed description of the accompanying drawings and preferred embodiments, various objects and advantages of the present invention will become practicable to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0113] Figure 1 The chromatogram of the Capto Heparin chromatography medium used in the heparin affinity chromatography step in the purified preparation of HSV-1 virus based on the composite mode chromatography of heparin affinity chromatography in Example 3 is shown. After the harvest fluid is chromatographed by Capto Heparin, the virus is concentrated and more than 98% of the host protein, host nucleic acid and other impurities are removed.

[0114] Figure 2 The chromatogram of the Capto Core700 chromatography medium used in the composite mode medium chromatography step in the purified preparation of HSV-1 virus based on heparin affinity chromatography in Example 3 is shown. The host protein and host nucleic acid content are further reduced by more than 70% compared with the previous affinity chromatography, and the virus titer yield reaches more than 90%.

[0115] Figure 3 The chromatogram of the Sepharose 4 Fast Flow chromatography medium after the molecular sieve chromatography step in the purification preparation of HSV-1 virus based on the composite mode chromatography of heparin affinity chromatography in Example 3 is shown. After the sample after Capto Core 700 chromatography was chromatographed by Sepharose 4 Fast Flow, its solution was replaced with a virus stabilizer, and there was no loss of virus titer.

[0116] Figure 4The chromatogram of the Bestarose Heparin chromatography medium used in the heparin affinity chromatography step in the purified preparation of HSV-1 virus based on ion exchange chromatography of heparin affinity chromatography in Example 4 is shown. After the harvest fluid is chromatographed by Bestarose Heparin, the virus is concentrated and more than 98% of host proteins, host nucleic acids and other impurities are removed.

[0117] Figure 5 The chromatogram of the Capto DEAE chromatography medium used in the ion exchange chromatography step in the purified preparation of HSV-1 virus based on heparin affinity chromatography and ion exchange chromatography in Example 4 is shown. After the sample after heparin affinity chromatography was chromatographed by Capto DEAE, the host protein and host nucleic acid content of the sample after heparin affinity chromatography was further reduced by more than 70% compared with the previous affinity chromatography, and the virus titer yield reached more than 80%.

[0118] Figure 6 The chromatogram of the Sepharose 6 Fast Flow chromatography medium after the molecular sieve chromatography step in the purification preparation of HSV-1 virus based on ion exchange chromatography of heparin affinity chromatography in Example 4 is shown. After the Capto DEAE chromatography sample was chromatographed on Sepharose 6 Fast Flow, its solution was replaced with a virus stabilizer without loss of virus titer.

[0119] Figure 7 The chromatogram of the Capto Core 700 chromatography medium used in the multimodal chromatography step in the purification preparation of HSV-1 virus based on multimodal chromatography and ion exchange chromatography after ultrafiltration concentration in Example 5 is shown. After the concentrated harvest liquid is chromatographed on Capto Core 700, more than 50% of host proteins, host nucleic acids and other impurities are removed.

[0120] Figure 8 The chromatogram of the Capto Q chromatography medium after the ion exchange chromatography step in the purification preparation of HSV-1 virus based on the composite mode chromatography and ion exchange chromatography after ultrafiltration concentration in Example 5 is shown. After the sample after Capto Core 700 chromatography was subjected to Capto Q chromatography, the removal rate of host proteins and host nucleic acids was further increased to more than 95%. DETAILED DESCRIPTION

[0121] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0122] Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references.

[0123] In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

[0124] Example 1. Virus culture and virus harvest fluid acquisition

[0125] 1.1 Virus culture: U-2 OS suspension cells were cultured to 2E5 to 5E5 cells / cm 2 When the density was 500 μg / mL, HSV-1 virus was inoculated at MOI = 0.04. Samples were taken for observation after virus inoculation, and the virus culture was harvested when the cytopathic rate reached more than 90% (about 48-65 h after inoculation).

[0126] The HSV-1 virus strain used in this application is the human herpes simplex virus type 1 KOS strain (abbreviated as OVH-APD1 strain, i.e. rHSV-1-APD1) modified by reverse genetics. This strain was independently developed by the National Infectious Disease Diagnostic Reagents and Vaccine Engineering Technology Research Center of Xiamen University and applied for invention patent protection. The patent application number is PCT / CN2018 / 077518.

[0127] 1.2 Cell lysis and enzyme digestion: Add virus harvesting supplement to the harvested virus culture. The virus harvesting supplement contains 1000-2000 mM NaCl, 10%-20% m / v sucrose, and the pH value is 6.5-8.0. The amount of virus harvesting supplement is 1 / 9 (v:v). Stir for 1-2 hours after addition to perform cell lysis to promote the full release of intracellular viruses. While lysing, add nuclease to digest the host nucleic acid released into the virus harvesting supplement by cell lysis. The nuclease can be purchased from Merck, Novozyme, Zhudian or Sino. The effective concentration of nuclease is 20-100 U / ml. The treatment conditions for nuclease digestion are digestion at 33℃-37℃ for 1-3 hours or digestion at 18-26℃ for 4-23 hours. At the same time, add 1-10 mM magnesium ions to the lysate to increase enzyme activity. After nuclease digestion under the above conditions, the host nucleic acid content of the products after detection enzyme digestion was reduced to less than 1% of the content before digestion.

[0128] The virus harvest additive used in this example has a NaCl concentration of 2000 mM, a sucrose content of 15% (m / v), a pH of 7.4, and an amount of the virus harvest additive of 1 / 9 (v:v); nuclease was purchased from Merck and added at a final concentration of 40 U / ml; 1 M magnesium ion stock solution was added at 1 / 200 (v:v) to a final concentration of 5 mM; and the digestion was allowed to proceed at 18-26°C (room temperature) for 13 hours.

[0129] 1.3 Clarification and filtration: The products after cell lysis and nuclease digestion are clarified and filtered using a combination of Cobetter's 5 μm PP filter and 1+0.65 μm PES filter to remove a large amount of cell debris and particulate matter, while controlling the turbidity within 50 NTU, and the virus titer yield before and after filtration is >80%. Thus, the virus harvest solution is obtained.

[0130] Example 2. Formula of solution used for chromatography

[0131] 2.1 Heparin affinity chromatography

[0132] Heparin affinity chromatography uses equilibration buffer, elution buffer, and regeneration buffer. The formulas of the three solutions are as follows.

[0133] Equilibration Buffer:

[0134] 3.9 mM NaH2PO4

[0135] 6.1 mM Na2HPO4

[0136] 0.3 M NaCl

[0137] 1.7% (m / v) sucrose

[0138] pH 7.0

[0139] Elution Buffer:

[0140] 3.9 mM NaH2PO4

[0141] 6.1 mM Na2HPO4

[0142] 0.5 M NaCl

[0143] 1.7% (m / v) sucrose

[0144] pH 7.0

[0145] Regeneration Buffer:

[0146] 3.9 mM NaH2PO4

[0147] 6.1 mM Na2HPO4

[0148] 1.0 M NaCl

[0149] 1.7% (m / v) sucrose

[0150] pH 7.0

[0151] 2.2 Multimodal chromatography

[0152] The equilibration buffer is used for multimodal chromatography. The formula of the equilibration buffer is as follows.

[0153] Equilibration Buffer:

[0154] 3.9 mM NaH2PO4

[0155] 6.1 mM Na2HPO4

[0156] 0.3 M NaCl

[0157] 1.7% (m / v) sucrose

[0158] pH 7.0

[0159] 2.3 Capto DEAE ion exchange chromatography

[0160] Capto DEAE ion exchange chromatography uses equilibration buffer and elution buffer. The formulas of the two solutions are as follows.

[0161] Equilibration Buffer:

[0162] 3.9 mM NaH2PO4

[0163] 6.1 mM Na2HPO4

[0164] 0.5 M NaCl

[0165] 1.7% (m / v) sucrose

[0166] pH 7.0

[0167] Elution Buffer:

[0168] 3.9 mM NaH2PO4

[0169] 6.1 mM Na2HPO4

[0170] 1.0 M NaCl

[0171] 1.7% (m / v) sucrose

[0172] pH 7.0

[0173] 2.4 Molecular sieve chromatography

[0174] Molecular sieve chromatography uses a virus stabilizer, and the formula of the virus stabilizer solution is as follows.

[0175] Virus Stabilizer:

[0176] 3.9 mM NaH2PO4

[0177] 6.1 mM Na2HPO4

[0178] 5 mM Histidine

[0179] 50 mM NaCl

[0180] 4.2% (m / v) Trehalose

[0181] 0.8%(m / v) Sorbitol

[0182] 2.5 Ultrafiltration concentration replacement

[0183] 300K ultrafiltration concentration uses ultrafiltration exchange buffer, the formula is as follows:

[0184] Ultrafiltration exchange buffer:

[0185] 3.9 mM NaH2PO4

[0186] 6.1 mM Na2HPO4

[0187] 0.3 M NaCl

[0188] 1.7% (m / v) sucrose

[0189] pH 7.0

[0190] 2.6 Capto Q ion exchange chromatography

[0191] Capto Q ion exchange chromatography uses equilibration buffer, pre-wash buffer and elution buffer. The formulas of the three solutions are as follows.

[0192] Equilibration Buffer:

[0193] 3.9 mM NaH2PO4

[0194] 6.1 mM Na2HPO4

[0195] 0.3 M NaCl

[0196] 1.7% (m / v) sucrose

[0197] pH 7.0

[0198] Prewash Buffer:

[0199] 3.9 mM NaH2PO4

[0200] 6.1 mM Na2HPO4

[0201] 0.5 M NaCl

[0202] 1.7% (m / v) sucrose

[0203] pH 7.0

[0204] Elution Buffer:

[0205] 3.9 mM NaH2PO4

[0206] 6.1 mM Na2HPO4

[0207] 1.0 M NaCl

[0208] 1.7% (m / v) sucrose

[0209] pH 7.0

[0210] Example 3. Preparation of HSV-1 virus purification by multi-mode chromatography based on heparin affinity chromatography

[0211] According to the steps of virus culture, cell lysis and enzyme digestion and clarification filtration described in Example 1, the virus harvest solution is obtained and the following steps are performed.

[0212] 3.1 Heparin affinity chromatography: The clarified and filtered virus harvested liquid is first subjected to heparin affinity chromatography. The heparin affinity chromatography medium is Capto Heparin (Cytiva) chromatography medium, Heparin Sepharose FF (Cytiva) chromatography medium, Bestarose Heparin (Bogeron) chromatography medium or Bestarose Heparin HR (Bogeron chromatography medium). In this embodiment, Capto Heparin chromatography is used for heparin affinity chromatography. Heparin affinity chromatography uses equilibrium buffer, elution buffer, and regeneration buffer. The formulas of the three solutions are as described in Example 2.1. The method of heparin affinity chromatography is to balance the chromatography column with equilibrium buffer for 2 to 5 column volumes, and then load the virus harvested liquid onto the affinity chromatography column. The loading volume is 15 to 25 column volumes, and the average retention time of the sample in the chromatography medium is controlled in the range of 3 to 10 min. After loading, the chromatography column is rinsed with equilibrium buffer for 2 to 5 column volumes to wash away unbound impurities. Then, the chromatography column is eluted with elution buffer for 1 to 3 column volumes, and the elution peak is collected. The collected sample is the component containing the target virus (HSV-1).

[0213] like Figure 1As shown, directly subjecting the obtained virus harvest to a single-step heparin affinity chromatography can remove more than 98% of the host proteins and host nucleic acids, and under the above conditions, the virus titer yield reaches more than 70%.

[0214] 3.2 Composite mode chromatography: The sample after heparin affinity chromatography is subjected to composite mode chromatography. The composite mode chromatography medium used includes but is not limited to Capto Core700 (Cytiva). As long as it is a composite mode chromatography medium (outer layer molecular sieve, inner core anion exchange) with the same separation principle and specifications, the same impurity removal function can be achieved. Capto Core700 is selected as the composite mode chromatography medium in this embodiment. The composite mode chromatography uses an equilibrium buffer, and the formula of the equilibrium buffer solution is as described in Example 2.2. The chromatography steps are as follows: (I) Equilibrium buffer is used to equilibrate the chromatography medium; (II) The sample after heparin affinity chromatography containing HSV-1 is passed through the composite mode chromatography medium, wherein the impurities bind to the chromatography medium and the HSV-1 virus flows through; (III) The composite chromatography medium is top washed with an equilibrium buffer to collect the HSV-1 virus. The column height of the composite mode chromatography column is 10 to 30 cm, the sample loading amount is 0.2 to 2 column volumes, and the retention time is 2 to 5 min.

[0215] like Figure 2 As shown, the host protein and host nucleic acid content of the sample after the composite mode chromatography in this embodiment is further reduced by more than 70% compared with the previous affinity chromatography, and the virus titer yield reaches more than 90%.

[0216] 3.3 Molecular sieve chromatography: After the HSV-1 sample is subjected to composite mode chromatography, the virus stabilizer is replaced by molecular sieve chromatography. The medium of the molecular sieve chromatography is Sepharose 6 Fast Flow (Cytiva) or Sepharose4 Fast Flow (Cytiva) chromatography medium or other molecular sieve chromatography medium with the same agarose matrix and cross-linking degree at home and abroad. The molecular sieve chromatography medium selected in this embodiment is Sepharose 4 Fast Flow. The molecular sieve chromatography includes the following steps: (I) balancing the chromatography medium with a virus stabilizer; (II) passing the sample containing HSV-1 through the molecular sieve chromatography medium, and the HSV-1 virus flows through; (III) top-washing the molecular sieve chromatography medium with a virus stabilizer to collect the HSV-1 virus. The column height of the molecular sieve chromatography column is 20 to 30 cm, the sample loading amount is 0.1 to 0.4 column volumes, the linear flow rate of the molecular sieve chromatography is 30 to 120 cm / h, and the virus stabilizer formula is as described in Example 2.4.

[0217] like Figure 3As shown, in this embodiment, the sample after Capto Core 700 chromatography is subjected to molecular sieve chromatography for liquid exchange, and the solution in the virus sample is replaced with a virus stabilizer, replacing the ultrafiltration liquid exchange method, and the virus activity is not lost in this step.

[0218] 3.4 Experimental results: In this embodiment, HSV-1 virus is purified by the steps of heparin affinity chromatography, composite mode chromatography and molecular sieve chromatography. The process of ultrafiltration concentration and displacement is reduced, and the ultrafiltration displacement buffer is replaced by mild molecular sieve chromatography in the last step, which avoids the loss of virus activity, significantly simplifies the purification process, is suitable for linear amplification, and improves the purification efficiency. The overall removal rate of host protein and host nucleic acid in the purified sample reaches more than 99%, the total recovery rate of HSV-1 virus titer reaches more than 50%, and the virus titer and impurity residues reach the pharmaceutical grade standard.

[0219] Example 4. Preparation of HSV-1 virus purification by ion exchange chromatography based on heparin affinity chromatography

[0220] According to the steps of virus culture, cell lysis and enzyme digestion and clarification filtration described in Example 1, a virus harvest solution was obtained and the following steps were performed.

[0221] 4.1 Heparin affinity chromatography: The obtained virus harvest solution was subjected to heparin affinity chromatography. The heparin affinity chromatography of this example selected Bestarose Heparin chromatography medium, and the experimental steps were as described in Example 3.1.

[0222] like Figure 4 As shown, directly subjecting the obtained virus harvest to a single-step heparin affinity chromatography can remove more than 98% of host proteins and host nucleic acids, and the virus titer yield under the above conditions reaches more than 70%.

[0223] 4.2 Ion exchange chromatography: The sample after heparin affinity chromatography is subjected to ion exchange chromatography. The ion exchange chromatography medium used includes but is not limited to Capto Q (Cytiva) or Capto DEAE (Cytiva), or an anion exchange chromatography medium with the same separation principle and specifications can achieve the same impurity removal function. The ion exchange chromatography medium in this embodiment uses Capto DEAE. Ion exchange chromatography uses an equilibrium buffer and an elution buffer, and the formulas of the two solutions are as described in Example 2.3. The chromatography steps are as follows: (I) Equilibrium chromatography medium with an equilibrium buffer; (II) Pass the sample after heparin affinity chromatography containing HSV-1 through the ion exchange medium, HSV-1 virus binds to the chromatography medium, and impurities flow through; (III) Elute the ion exchange chromatography medium with an elution buffer, and collect the elution peak. The collected sample is the component containing the target virus (HSV-1 virus). The column height of the ion exchange chromatography column is 10 to 30 cm, the sample loading amount is 0.5 to 2 column volumes, and the retention time is 2 to 5 min.

[0224] like Figure 5 As shown, the host protein and host nucleic acid content of the sample after ion exchange chromatography in this example is further reduced by more than 70% compared with the previous affinity chromatography, and the virus titer yield reaches more than 80%.

[0225] 4.3 Molecular sieve chromatography: After the HSV-1 sample was subjected to composite mode chromatography, the virus stabilizer was replaced by molecular sieve chromatography. The molecular sieve chromatography medium used in this example was Sepharose 6 Fast Flow. The experimental steps were as described in Example 3.3. The chromatogram was as shown in FIG. Figure 6 shown.

[0226] 4.4 Experimental results: In this example, HSV-1 virus was purified by heparin affinity chromatography, ion exchange chromatography and molecular sieve chromatography. Among them, ion exchange chromatography and the composite mode chromatography used in Example 1 are both purification steps, which have the same purification effect. After purification, the overall removal rate of host protein and host nucleic acid in the sample reached more than 99%, the total recovery rate of HSV-1 virus titer reached more than 50%, and the virus titer and impurity residues reached pharmaceutical grade standards.

[0227] Example 5. Preparation of HSV-1 virus purification based on composite mode chromatography and ion exchange chromatography after ultrafiltration concentration

[0228] According to the steps of virus culture, cell lysis and enzyme digestion and clarification filtration described in Example 1, a virus harvest solution was obtained and the following steps were performed.

[0229] 5.1 Ultrafiltration and concentration: In this embodiment, the virus harvest solution is concentrated 5 to 10 times by ultrafiltration using a 300K membrane package, and the ultrafiltration replacement buffer used is as described in Example 2.5. The virus liquid phase composition after ultrafiltration replacement is then ultrafiltered and concentrated by a composite mode chromatography to remove more than 50% of host proteins, host nucleic acids and other impurities, and the virus yield is about 40% to 50%.

[0230] 5.2 Multimode chromatography: The multimode medium used in this example is Cytiva's Capto Core700, and its equilibrium solution is the same as the ultrafiltration replacement buffer in step 5.1 (see Example 2.5), pH 6-9, and conductivity 20-30mS / cm. When loading, the average retention time of the sample is 5-10 min, and the total loading volume is 2-6 column volumes. After the loading is completed, the flow-through peak (containing the target virus HSV-1 virus) is collected by top washing with the equilibrium solution.

[0231] like Figure 7 As shown, the ultrafiltration and concentration of the virus liquid phase composition was subjected to Capto Core 700 composite mode chromatography, which removed more than 50% of the host protein, host nucleic acid and other impurities, and the virus yield could reach more than 80%.

[0232] 5.3 Anion exchange chromatography: The sample after the composite mode chromatography is subjected to ion exchange chromatography. The ion exchange chromatography medium in this embodiment is Capto Q (Cytiva). The ion exchange chromatography uses an equilibrium buffer, a pre-wash buffer and an elution buffer, and the formulas of the three solutions are as described in Example 2.6. The chromatography steps are as follows: (I) Equilibrium the chromatography medium with an equilibrium buffer; (II) The sample after the composite mode chromatography containing HSV-1 is passed through the ion exchange medium, the HSV-1 virus binds to the chromatography medium, and impurities flow through; (III) The chromatography medium is pre-washed with a pre-wash buffer, and some impurities are washed away; (IV) The ion exchange chromatography medium is eluted with an elution buffer, and the elution peak is collected. The collected sample is the component containing the target virus (HSV-1 virus). The column height of the ion exchange chromatography column is 10 to 30 cm, the sample loading amount is 0.5 to 3 column volumes, and the retention time is 2 to 5 min. The virus sample after ion exchange chromatography was concentrated by 300K ultrafiltration to replace the virus stabilizer to obtain the virus stock solution. CaptoQ (Cytiva) chromatography map is as follows Figure 8 As shown, the host protein and host nucleic acid content of the sample after ion exchange chromatography in this embodiment is further reduced by more than 90% compared with the previous composite mode chromatography, and the virus titer yield is about 60%.

[0233] 5.4 Experimental results: Based on the two-step chromatography process (composite mode chromatography and anion exchange chromatography) of this embodiment, the overall removal effect of host protein and host nucleic acid can reach more than 95%, and the virus titer yield is between 20% and 30%. Since the HSV-1 virus is a large particle virus with an envelope structure, its activity is particularly sensitive to shear force. Compared with Examples 3 and 4, the two-step high shear force ultrafiltration process included in this embodiment is the main reason for the loss of virus titer activity. The selectivity of anion exchange chromatography and composite mode chromatography is also worse than that of heparin affinity chromatography, which is also the reason for the poor removal effect of host protein and host nucleic acid impurities.

[0234] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. A method for purifying herpes simplex virus (HSV-1), the method comprising: (a) inoculating HSV-1 into host cells to amplify HSV-1; (b) lysing the host cell to release the amplified HSV-1; (c) filtering the product obtained in step (b) to remove impurities; (d) subjecting the product obtained in step (c) to heparin affinity chromatography; Preferably, the medium for heparin affinity chromatography is selected from Capto Heparin chromatography medium, Heparin SepharoseFF chromatography medium, Bestarose Heparin chromatography medium, Bestarose Heparin HR chromatography medium, or any combination thereof; Optionally, the method further comprises: (e) subjecting the product obtained in step (d) to ion exchange chromatography and / or multimodal chromatography; Optionally, the method further comprises: (f) subjecting the product obtained in step (e) to molecular sieve chromatography.

2. The method according to claim 1, wherein the method has one or more technical features selected from the following: (1) The heparin affinity chromatography medium is Capto Heparin chromatography medium or Bestarose Heparin chromatography medium; (2) subjecting the product obtained in step (d) to ion exchange chromatography or multimode chromatography; (3) The composite mode chromatography medium has an outer layer of molecular sieve and a core anion exchange chromatography medium; (4) The medium of the multimodal chromatography is Capto Core 700; (5) The medium of the ion exchange chromatography is Capto Q or Capto DEAE; (6) The medium of the molecular sieve chromatography is Sepharose 6 Fast Flow or Sepharose 4 Fast Flow.

3. The method according to any one of claims 1 or 2, wherein in step (a), the method has one or more technical features selected from the following: (1) The HSV-1 is an oncolytic herpes simplex virus type 1 (oHSV-1 for short); Preferably, the HSV-1 is an oncolytic herpes simplex KOS virus type I (referred to as OVH-APD1 strain, i.e., rHSV-1-APD1); (2) The host cell is selected from mammalian (e.g., human, mouse) cells; Preferably, the host cell is selected from human osteosarcoma cells (U-2 OS) cells, Hela cells, Vero adherent cells, Hep-2 cells, BHK cells, or any combination thereof; Preferably, the host cell is a human osteosarcoma cell (U-2 OS) cell; (3) the inoculation is performed at an MOI value of 0.02 to 0.06 (e.g., 0.02, 0.03, 0.04, 0.05, 0.06); (4) The inoculation is carried out at a cell density of 2E5 to 5E5 cells / cm 2 (e.g., 2E5 cells / cm 2 、3E5 cells / cm 2 、4E5 cells / cm 2 , 5E5 cells / cm 2 ) conditions.

4. The method according to any one of claims 1 to 3, wherein in step (b), the method has one or more technical features selected from the following: (1) lysing the host cells using a lysis solution; (2) digesting nucleic acids released by lysis of the host cells by nucleases; Preferably, the nuclease concentration is 20-100 U / ml (e.g., 20-40 U / ml, 40-60 U / ml, 60-80 U / ml, 80-100 U / ml); Preferably, the treatment conditions of the nuclease are 33-37°C (e.g., 33°C, 34°C, 35°C, 36°C, 37°C) for 1-3 h or 18-26°C (e.g., 18°C, 20°C, 22°C, 24°C, 26°C) for 4-23 h (e.g., 4 h, 8 h, 12 h, 13 h, 16 h, 20 h, 23 h); (3) adding magnesium ions when lysing the host cells; Preferably, the concentration of the magnesium ions is 1 to 10 mM (e.g., 1 mM, 2 mM, 4 mM, 5 mM, 6 mM, 8 mM, 10 mM); Preferably, the lysate comprises NaCl and sucrose; Preferably, the lysate has a formula of 1000-2000 mM (e.g., 1000 mM, 1200 mM, 1400 mM, 1600 mM, 1800 mM, 2000 mM) of NaCl and 10%-20% m / v (e.g., 10% m / v, 12% m / v, 14% m / v, 15% m / v, 16% m / v, 18% m / v, 20% m / v) of sucrose, and a pH of 6.5-8.0 (e.g., pH=6.5, pH=7.0, pH=7.4, pH=7.5, pH=8.0).

5. The method according to any one of claims 1 to 4, wherein in step (c), the filtration is carried out in a single-stage or multi-stage filtration manner; Preferably, the filtration uses a combination of a 5 μm PP material filter and a 1+0.65 μm PES material filter.

6. The method according to any one of claims 1 to 5, wherein in step (d), the method has one or more technical features selected from the following: (1) The heparin affinity chromatography uses an equilibrium buffer to balance the chromatography column medium and / or flush impurities; (2) the heparin affinity chromatography uses an elution buffer to elute HSV-1; (3) The heparin affinity chromatography uses a regeneration buffer to regenerate the chromatography column; Preferably, the heparin affinity chromatography uses an equilibration buffer, an elution buffer and a regeneration buffer in the order in which they are arranged; Preferably, the product obtained in step (c) is loaded after using the equilibration buffer and before using the elution buffer, and the average retention time of the product in the chromatography medium is 3 to 10 min; Preferably, the buffer system of the buffer (e.g., equilibration buffer, elution buffer, regeneration buffer) is selected from a phosphate buffer system, a Tris buffer system, a His buffer system, a sodium acetate buffer system, or any combination thereof; Preferably, the buffer system of the balancing solution (e.g., balancing buffer, elution buffer, regeneration buffer) is a phosphate buffer system (e.g., containing H2PO4 - and HPO4 2- ); Preferably, the equilibration buffer comprises NaH2PO4, Na2HPO4, NaCl and sucrose, and has a pH value of 6 to 8; Preferably, the elution buffer comprises NaH2PO4, Na2HPO4, NaCl and sucrose, and has a pH of 6 to 8; Preferably, the regeneration buffer comprises NaH2PO4, Na2HPO4, NaCl and sucrose, and has a pH of 6 to 8; Preferably, the equilibration buffer has a formula of 3.9 mM NaH2PO4, 6.1 mM Na2HPO4, 0.3 M NaCl and 1.7% (m / v) sucrose, and a pH value of 7; Preferably, the elution buffer has a formula of 3.9 mM NaH2PO4, 6.1 mM Na2HPO4, 0.5 M NaCl and 1.7% (m / v) sucrose, and a pH of 7; Preferably, the formula of the regeneration buffer is 3.9 mM NaH2PO4, 6.1 mM Na2HPO4, 1.0 M NaCl and 1.7% (m / v) sucrose, and the pH value is 7; Preferably, the impurities include host proteins and / or host nucleic acids.

7. The method according to any one of claims 1 to 6, wherein in step (e), the method has one or more technical features selected from the following: (1) The multimodal chromatography uses an equilibrium buffer to equilibrate the chromatography column medium and / or flush HSV-1; (2) The ion exchange chromatography uses an equilibrium buffer to equilibrate the chromatography column medium; (2) the ion exchange chromatography uses an elution buffer to elute HSV-1; Preferably, the buffer system of the buffer (e.g., equilibration buffer, elution buffer) is selected from a phosphate buffer system, a Tris buffer system, a His buffer system, a sodium acetate buffer system, or any combination thereof; Preferably, the buffer system of the balancing solution (e.g., balancing buffer, elution buffer) is a phosphate buffer system (e.g., containing H2PO4 - and HPO4 2- ); Preferably, the multimodal chromatography uses an equilibrium buffer comprising NaH2PO4, Na2HPO4, NaCl and sucrose, and has a pH value of 6 to 8; Preferably, the formula of the equilibrium buffer used in the multimodal chromatography is 3.9 mM NaH2PO4, 6.1 mM Na2HPO4, 0.3 M NaCl and 1.7% (m / v) sucrose, and the pH value is 7; Preferably, the ion exchange chromatography uses an equilibrium buffer comprising NaH2PO4, Na2HPO4, NaCl and sucrose, and has a pH value of 6 to 8; Preferably, the ion exchange chromatography uses an equilibrium buffer having a formula of 3.9 mM NaH2PO4, 6.1 mM Na2HPO4, 0.5 M NaCl and 1.7% (m / v) sucrose, and a pH value of 7; Preferably, the ion exchange chromatography uses an elution buffer comprising NaH2PO4, Na2HPO4, NaCl and sucrose, and the pH value is 6 to 8; Preferably, the ion exchange chromatography uses an elution buffer having a formula of 3.9 mM NaH2PO4, 6.1 mM Na2HPO4, 1.0 M NaCl and 1.7% (m / v) sucrose, and a pH value of 7.

8. The method according to any one of claims 1 to 7, wherein in step (f), the molecular sieve chromatography uses a buffer to balance the chromatography column medium and / or flush the HSV-1; Preferably, the buffer system of the buffer solution is selected from a phosphate buffer system, a Tris buffer system, a His buffer system, a sodium acetate buffer system, or any combination thereof; Preferably, the buffer system of the balancing solution is a phosphate buffer system (e.g., comprising H2PO4 - and HPO4 2- ); Preferably, the buffer comprises NaH2PO4, Na2HPO4, histidine, NaCl, trehalose and sorbitol; Preferably, the formula of the buffer is 3.9 mM NaH2PO4, 6.1 mM Na2HPO4, 5 mM histidine, 50 mM NaCl, 4.2% (m / v) trehalose and 0.8% (m / v) sorbitol.

9. The method according to any one of claims 1 to 8, which does not include the step of ultrafiltration concentration; Preferably, the method does not include an ultrafiltration concentration step between step (b) and step (d); Preferably, the method does not include an ultrafiltration concentration step after step (d).

10. The method according to any one of claims 1 to 9, wherein after step (d), the impurity removal rate can reach at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%; Preferably, the impurities include host proteins and / or host nucleic acids.

11. The method of any one of claims 1-10, wherein after step (d), the titer yield of HSV-1 can reach at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%.

12. The HSV-1 virus obtained by the method according to any one of claims 1 to 11; Preferably, the HSV-1 is oncolytic herpes simplex virus type 1 (oncolytic Herpes Simplex Virus Type 1, referred to as oHSV-1); Preferably, the HSV-1 is an oncolytic herpes simplex KOS virus type 1 (abbreviated as OVH-APD1 strain, i.e. rHSV-1-APD1).

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