A method for purifying HSV-1

Abstract

The present application relates to a virus purification method. The present application relates to a method for purification of herpes simplex virus (HSV-1). The present application also relates to HSV-1 purified according to the method. In particular, the method does not comprise 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 purification method of herpes simplex virus (HSV-1). The present application also relates to HSV-1 purified according to the method. In particular, the method does not comprise an ultrafiltration concentration step. BACKGROUND

[0002] Herpes simplex virus type 1 (HSV-1) is an enveloped double-stranded DNA virus with a molecular weight of about 152 kb, composed of more than 70 proteins, and the complete virion is 200 nm. At present, herpes simplex virus type 1 has been genetically modified to develop oncolytic herpes simplex virus type 1 (oHSV-1) for cancer treatment. Oncolytic herpes simplex virus type 1 is a virus inoculated into human osteosarcoma cells (U-2 OS), cultured, and then prepared into a drug product by harvesting and isolating and purifying. 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, it is necessary to maintain their activity during the isolation and purification process, which requires that the isolation and purification process be virus-stable, robust, reproducible, and suitable for commercial scale-up production. At present, there are relatively few literature reports on the isolation and purification method of herpes simplex virus. In these studies, in order to obtain herpes simplex virus with high purity, ultracentrifugation, density gradient centrifugation and other purification methods are usually used, however, such methods have the problems of low sample throughput, long operation time, and low virus purity and recovery rate. There are also reports that use ultrafiltration technology (such as flat membrane or hollow fiber) to concentrate viruses, and then use affinity chromatography or ion exchange chromatography for purification, but there are still problems such as complex process and easy virus inactivation.

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

[0005] The HSV-1 purification method provided by the present application has high virus titer yield and high impurity (e.g., host protein and / or host nucleic acid) removal rate. In addition, the HSV-1 purification method provided by the present application does not include an ultrafiltration concentration step, greatly simplifying the purification process and improving the purification efficiency. Specifically, the HSV-1 purification method provided by the present application can achieve high virus titer yield (e.g., at least 70%) and high impurity removal rate (e.g., at least 98%) through only a heparin affinity chromatography step; when the heparin affinity chromatography is combined with other chromatography steps (e.g., a multimodal chromatography and / or an ion exchange chromatography and a molecular sieve chromatography), it also has high virus titer yield (e.g., at least 50%) and high impurity removal rate (e.g., at least 99%). The HSV-1 purification method provided by the present application has low requirements for the initial sample to be purified (e.g., HSV-1 particles can be efficiently captured directly from the virus harvest liquid), the purification process is simple, can be used for large-scale preparation of HSV-1 complete particles, and the yield and purity meet the requirements of industrialization, 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 a herpes simplex virus (HSV-1), comprising:

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

[0008] (b) lysing the host cells 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 the 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 medium 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 complex mode medium chromatography.

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

[0019] In certain embodiments, the complex mode medium chromatography medium is Capto Core 700.

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

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

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

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

[0024] In certain embodiments, the molecular sieve chromatography medium can also be a molecular sieve chromatography medium of 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 of using heparin affinity chromatography for the purification of a portion of a virus, based on the diversity of different virus structures, different viruses can or can not bind to heparin or have different characteristics (e.g., affinity, specificity, etc.) of binding to heparin, and the method of the present application is particularly suitable for the purification of herpes simplex virus (HSV-1), and in particular, the oncolytic herpes simplex type I KOS virus (referred to as OVH-APD1 strain, i.e., rHSV-1-APD1).

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

[0028] In certain embodiments, the HSV-1 is an oncolytic Herpes Simplex Virus Type 1 KOS strain (OVH-APD1 strain, rHSV-1-APD1).

[0029] In certain embodiments, the oncolytic Herpes Simplex Virus Type 1 KOS strain is obtained by the method described in patent application No. PCT / CN2018 / 077518, i.e., knocking out the non-essential gene ICP0 and the neurovirulence factor gene ICP34.5, and using the human telomerase reverse transcriptase (hTERT) promoter with tumor specificity to regulate the expression of the immediate early protein ICP27 gene essential for viral replication, thereby obtaining the oncolytic Herpes Simplex Virus Type 1 KOS strain.

[0030] In certain embodiments, the host cell is selected from a mammal (e.g., human, murine) cell.

[0031] In certain embodiments, the host cell is selected from a human osteosarcoma cell (U-2 OS) cell, a Hela cell, a Vero adherent cell, a Hep-2 cell, a BHK cell, 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 (e.g., 0.02, 0.03, 0.04, 0.05, 0.06).

[0034] In certain embodiments, the inoculation is performed 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 ).

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

[0036] In certain embodiments, the method is performed by digesting the nucleic acid released by lysing the host cell with a nuclease.

[0037] In some 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 some embodiments, the treatment condition of the nuclease is treating at 33-37°C (e.g., 33°C, 34°C, 35°C, 36°C, 37°C) for 1-3 h or treating at 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 some embodiments, magnesium ions are also added when lysing the host cells.

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

[0041] In some embodiments, the lysis solution comprises NaCl and sucrose.

[0042] In some embodiments, the formulation of the lysis solution is 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 the pH value is 6.5-8.0 (e.g., pH=6.5, pH=7.0, pH=7.4, pH=7.5, pH=8.0).

[0043] In some embodiments, in step (c), the filtration employs single-stage or multi-stage filtration.

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

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

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

[0047] In some 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, in order of arrangement, a equilibration buffer, an elution buffer, and a regeneration buffer.

[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 residence time of the product in the chromatography medium is 3-10 min.

[0051] In certain embodiments, the buffer system of the buffer (e.g., the equilibration buffer, the elution buffer, the 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 buffer (e.g., the equilibration buffer, the elution buffer, the 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 value of 6-8.

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

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

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

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

[0058] In certain embodiments, the formulation of the regeneration buffer is 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 a equilibration buffer to equilibrate the chromatography column media and / or to flush the HSV-1.

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

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

[0063] In certain embodiments, the buffer (e.g., equilibration buffer, elution buffer) has a buffer system 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 buffer (e.g., equilibration buffer, elution buffer) is a phosphate buffer system (e.g., comprising H2PO4 - and HPO4 2- ).

[0065] In certain embodiments, the equilibration buffer used in the multimodal chromatography comprises NaH2PO4, Na2HPO4, NaCl, and sucrose, and has a pH of 6 to 8.

[0066] In certain embodiments, the formulation of the equilibration 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 has a pH of 7.

[0067] In certain embodiments, the equilibration buffer used in the ion exchange chromatography comprises NaH2PO4, Na2HPO4, NaCl, and sucrose, and has a pH of 6 to 8.

[0068] In certain embodiments, the formulation of the equilibration buffer used in the ion exchange chromatography is 3.9 mM NaH2PO4, 6.1 mM Na2HPO4, 0.5 M NaCl, and 1.7% (m / v) sucrose, and has a pH of 7.

[0069] In certain embodiments, the ion exchange chromatography uses an elution buffer comprising NaH2P04, Na2HP04, NaCl, and sucrose, and has 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 NaH2P04, 6.1 mM Na2HP04, 1.0 M NaCl, and 1.7% (m / v) sucrose, and has a pH of 7.

[0071] In certain embodiments, the composite mode chromatography column has a column height of 10 to 30 cm.

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

[0075] In certain embodiments, in step (f), the size exclusion chromatography uses a buffer to equilibrate the chromatography column media and / or to flush 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 equilibration buffer is a phosphate buffer system (e.g., comprising H2P04 - and HPO4 2- ). In certain embodiments, the buffer comprises NaH2P04, Na2HP04, histidine, NaCl, trehalose, and sorbitol.

[0078] In certain embodiments, the formulation of the buffer is 3.9 mM NaH2P04, 6.1 mM Na2HP04, 5 mM histidine, 50 mM NaCl, 4.2% (m / v) trehalose, and 0.8% (m / v) sorbitol.

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

[0080] In certain embodiments, the size exclusion chromatography has a linear flow rate of 30 to 120 cm / h.

[0081] In some embodiments, the method does not comprise a step of ultrafiltration concentration.

[0082] In some embodiments, the method does not comprise a step of ultrafiltration concentration between step (b) and step (d).

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

[0084] In some embodiments, the removal rate of impurities after step (d) 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 some embodiments, the impurities comprise host proteins and / or host nucleic acids.

[0086] In some embodiments, the titer yield of HSV-1 after step (d) 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 a HSV-1 virus obtained by the method of the first aspect.

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

[0089] In some embodiments, the HSV-1 is an oncolytic Herpes Simplex Virus Type 1 KOS strain (OVH-APD1 strain, rHSV-1-APD1).

[0090] In some embodiments, the oncolytic Herpes Simplex Virus Type 1 KOS strain is obtained by the method described in patent application No. PCT / CN2018 / 077518, i.e., knocking out the non-essential gene ICP0 and the neurovirulence factor gene ICP34.5, and using the human telomerase reverse transcriptase (hTERT) promoter with tumor specificity to regulate the expression of the immediate early protein ICP27 gene essential for virus replication, thereby obtaining the oncolytic Herpes Simplex Virus Type 1 KOS strain.

[0091] In some embodiments, the HSV-1 can be used for the development of tumor treatment drugs, diagnostic reagents or vaccines.

[0092] Term definition

[0093] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the viral, biochemical, immunological laboratory procedures used in the present application are those well known in the art. The definitions and explanations of the relevant terms are provided below for better understanding of the present application.

[0094] When the terms "for example," "for instance," "such as," "including," "containing," or "comprising" are used herein, these terms are not to be interpreted in an exclusionary sense, but rather in an illustrative sense.

[0095] The terms "a" and "an" and "the" and similar referents in the context of describing the application (especially in the context of the following claims) are to be interpreted in an inclusive rather than an exclusive sense unless otherwise noted.

[0096] As used herein, the term "chromatography" refers to a separation technique that employs 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 that contains the particular type of molecule to be separated and other molecules. The particular type of molecule 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 particular type of molecule to be separated can be separated from the other molecules due to different interactions of the different molecules in the mobile phase with the stationary phase or different residence times of the different molecules in the mobile phase in the stationary phase.

[0097] 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 support (or matrix) as a ligand is capable of interacting with molecules (e.g., HSV) in the 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 application, heparin affinity chromatography involves the addition of a sample containing HSV to a stationary phase that contains heparin (or heparin mimetic) as a ligand.

[0098] As used herein, the term "HSV" also known as herpes simplex virus, is a double- stranded DNA virus belonging to the Herpesviridae family. HSV is capable of causing a variety of diseases, including oral herpes and genital herpes. Based on antigenic differences, HSV is classified into two serotypes: HSV-1 and HSV-2. The affinity of HSV to heparin is mainly dependent on 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.

[0099] 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 is capable of effectively killing a variety of tumor cells, but has significantly reduced replication ability and killing ability 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.

[0100] As used herein, the term "ultrafiltration", also known as tangential flow filtration (TFF) and crossflow filtration, is a technique for purifying biomolecules. In the ultrafiltration process, the sample solution flows parallel to the membrane surface, part of the sample solution passes through the membrane (filtrate), and the rest of the feed solution (reflux) is circulated back to the sample container. Ultrafiltration technology generally includes hollow fiber and membrane bag methods. In certain embodiments herein, the ultrafiltration uses the membrane bag method.

[0101] As used herein, the term "virus harvest" is a liquid containing viral particles obtained from a host cell inoculated with a virus, and the steps of treatment include culturing the virus in the host cell, lysing the host cell, nuclease digestion, and filtration. After the above steps of treatment, the liquid containing viral particles obtained is the "virus harvest", and the obtained virus harvest can be used for further virus purification, titer determination, infectivity test, or other related biomedical research and applications. In certain embodiments herein, the virus harvest is further purified.

[0102] As used herein, the term "mixed mode chromatography", also known as "complex mode chromatography" or "multi-mode chromatography" or "hybrid mode chromatography", is a chromatographic separation technique that utilizes more than one chromatographic technique (e.g., gel filtration, ion exchange, hydrophobic, and affinity chromatography) acting alone or in concert with each other. In certain embodiments herein, the complex mode chromatography utilizes size exclusion chromatography and ion exchange chromatography.

[0103] As used herein, the term "ion exchange chromatography" (IEC) is a technique that utilizes electrostatic interactions between an ion exchanger and viral particles for viral purification. This technique is based on the surface charge characteristics of viral particles, which are separated from impurity components by selective adsorption and elution. In certain embodiments herein, the ion exchange chromatography is anion exchange chromatography.

[0104] As used herein, the term "regeneration" refers to the process in chromatography techniques, in which viral particles or other impurities bound to the chromatography media are eluted off, so that the chromatography media can be reused. The regeneration process ensures that the chromatography media can maintain activity and selectivity in multiple purification cycles. Regeneration usually involves the use of a regeneration buffer to disrupt the interactions between the viral particles and the chromatography media, so that the viral particles are dissociated from the media, and the activity and integrity of the viral particles are guaranteed. In certain embodiments herein, a regeneration buffer is used in the complex mode chromatography to achieve regeneration.

[0105] As used herein, the term "molecular sieve chromatography", also known as "gel filtration chromatography" or "exclusion chromatography", is a technique based on the separation of molecules in a sample by size differences. This technique generally selects a gel with a porous structure of different pore sizes as a stationary phase according to the size of the sample to be separated, and separates substances of different molecular sizes by its molecular sieve effect.

[0106] As used herein, the term "virus titer yield" refers to the retention ratio of the activity and quantity of viral particles from the virus harvest to the final harvested bulk of virus during the viral purification process. It is an index that comprehensively evaluates the activity retention and recovery efficiency of the virus during the purification process.

[0107] As used herein, the term "virus stabilizer" refers to a class of substances used to protect viral particles from physical and chemical damage. Virus stabilizers can be small molecule compounds (such as sugars and amino acids), polymers or proteins, which can maintain the structural integrity and biological activity of viral particles, and ensure the stability and functionality of viruses during the purification process, especially under high salt, pH change or other conditions that may cause virus inactivation. In certain embodiments herein, a virus stabilizer is used in the molecular sieve chromatography.

[0108] As used herein, the term "top wash" refers to the process of using a buffer to flush through the top of the chromatography column after the sample has passed through the chromatography media. Top wash can be used to remove sample components or other impurities that are not adsorbed in the chromatography media, thereby reducing the impact of these components on the purity of the subsequent collected purified target, or to elute the residual purified target in the chromatography media to improve the yield.

[0109] Advantages of the Invention

[0110] The HSV-1 purification method provided by the present application has high virus titer yield and high impurity (e.g., host protein and / or host nucleic acid) removal rate. In addition, the HSV-1 purification method provided by the present application does not include an ultrafiltration concentration step, greatly simplifying the purification process and improving the purification efficiency. Specifically, the HSV-1 purification method provided by the present application can achieve high virus titer yield (e.g., at least 70%) and high impurity removal rate (e.g., at least 98%) through only the heparin affinity chromatography step; when the heparin affinity chromatography is combined with other chromatography steps (e.g., multimodal chromatography and / or ion exchange chromatography and size exclusion chromatography), it also has high virus titer yield (e.g., at least 50%) and high impurity removal rate (e.g., at least 99%).

[0111] Embodiments of the present application will be described in detail below with reference to the accompanying drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present application, and are not a limitation on the scope of the present application. According to the following detailed description of the preferred embodiments and the accompanying drawings, various objects and advantageous aspects of the present application will become apparent to those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0112] Figure 1 The chromatogram of Capto Heparin chromatography medium used in the heparin affinity chromatography step in the preparation of highly purified HSV-1 virus based on heparin affinity chromatography and multimodal chromatography in Example 3 is shown. After Capto Heparin chromatography, the virus was concentrated, and more than 98% of the host proteins, host nucleic acids and other impurities were removed.

[0113] Figure 2 The chromatogram of Capto Core 700 chromatography medium used in the multimodal medium chromatography step in the preparation of highly purified HSV-1 virus based on heparin affinity chromatography and multimodal chromatography in Example 3 is shown. The host protein and host nucleic acid content is further reduced by more than 70% compared to the previous affinity chromatography step, and the virus titer yield is more than 90%.

[0114] Figure 3 The chromatogram of Sepharose 4 Fast Flow chromatography medium used in the size exclusion chromatography step in the preparation of highly purified HSV-1 virus based on heparin affinity chromatography and multimodal chromatography in Example 3 is shown. After Capto Core 700 chromatography, the sample was subjected to Sepharose 4 Fast Flow chromatography, and the solution was replaced with a virus stabilizer, and there was no loss of virus titer.

[0115] Figure 4A chromatogram of Bestarose Heparin chromatography medium used in the step of heparin affinity chromatography in the preparation of HSV-1 virus in Example 4 is shown. After the harvest fluid was subjected to Bestarose Heparin chromatography, the virus was concentrated and more than 98% of the host proteins, host nucleic acids and other impurities were removed.

[0116] Figure 5 A chromatogram of Capto DEAE chromatography medium used in the step of ion exchange chromatography in the preparation of HSV-1 virus in Example 4 is shown. After the sample was subjected to Capto DEAE chromatography after heparin affinity chromatography, the content of host proteins and host nucleic acids was further reduced by more than 70% compared to the previous step of affinity chromatography, and the yield of virus titer reached more than 80%.

[0117] Figure 6 A chromatogram of Sepharose 6 Fast Flow chromatography medium used in the step of molecular sieve chromatography in the preparation of HSV-1 virus in Example 4 is shown. After the sample was subjected to Sepharose 6 Fast Flow chromatography after Capto DEAE chromatography, the solution was replaced with a virus stabilizer, and there was no loss of virus titer.

[0118] Figure 7 A chromatogram of Capto Core 700 chromatography medium used in the step of complex mode chromatography in the preparation of HSV-1 virus in Example 5 is shown. After the concentrated harvest fluid was subjected to Capto Core 700 chromatography, more than 50% of the host proteins, host nucleic acids and other impurities were removed.

[0119] Figure 8 A chromatogram of Capto Q chromatography medium used in the step of ion exchange chromatography in the preparation of HSV-1 virus in Example 5 is shown. After the sample was subjected to Capto Q chromatography after Capto Core 700 chromatography, the removal rate of host proteins and host nucleic acids was further increased to more than 95%. DETAILED DESCRIPTION

[0120] The present application will now be described with reference to the following examples, which are intended to illustrate the application (but not to limit it).

[0121] Unless specifically indicated otherwise, the experiments and methods described in the examples were performed essentially according to conventional methods well known in the art and described in various references.

[0122] In addition, the specific conditions not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, which are conventional products that can be obtained by purchase. Those skilled in the art know that the examples describe the present application by way of example and are not intended to limit the scope of the present application claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

[0123] Example 1. Cultivation of viruses and obtaining of virus harvest

[0124] 1.1 Virus cultivation: U-2 OS suspension cells are cultured to 2E5 to 5E5 cells / cm 2 density, HSV-1 virus is inoculated at MOI = 0.04, and sampling is observed after inoculation of the virus, and the virus culture is harvested when the cytopathic rate reaches more than 90% (about 48-65 h after inoculation).

[0125] The HSV-1 virus strain used in this application is a human type I herpes simplex virus KOS strain (abbreviated as OVH-APD1 strain, i.e. rHSV-1-APD1) modified by reverse genetics, which is independently developed by the National Center for Infectious Disease Diagnosis Reagents and Vaccine Engineering Technology Research Center of Xiamen University, and is applied for invention patent protection, with the patent application number PCT / CN2018 / 077518.

[0126] 1.2 Cell lysis and enzyme digestion: add virus harvest additive to the harvested virus culture, the virus harvest additive contains 1000-2000 mM NaCl, contains 10%-20% m / v sucrose, pH value is 6.5-8.0, the addition amount of virus harvest additive is 1 / 9 (v:v). After adding, stir for 1-2 h for cell lysis to facilitate the release of intracellular viruses. At the same time of lysis, add nucleic acid enzyme to digest the host nucleic acid released into the virus harvest additive by cell lysis, and the nucleic acid enzyme can be purchased from Merck, Novozymes, Zhuchan or Seno. The effective concentration of nucleic acid enzyme is 20-100 U / ml, and the treatment condition of nucleic acid enzyme digestion is 33-37°C for 1-3 h or 18-26°C for 4-23 h. At the same time, 1-10 mM magnesium ions are additionally added to the lysis solution to improve the enzyme activity. The host nucleic acid content of the product after nucleic acid enzyme digestion under the above conditions is reduced to within 1% of the content before digestion.

[0127] The virus harvest addition liquid used in this example has a NaCl concentration of 2000 mM, a sucrose content of 15% (m / v), a pH of 7.4, and the amount of virus harvest addition liquid added is 1 / 9 (v:v); the nuclease is commercially available from Merck and is added to a final concentration of 40 U / ml; the 1 M magnesium ion stock solution is added at 1 / 200 (v:v) to a final concentration of 5 mM; and digestion is carried out at 18-26°C (room temperature) for 13 hours.

[0128] 1.3 Clarification filtration: the product after cell lysis and nuclease digestion is subjected to clarification filtration using a combination of a 5 μm PP filter and a 1+0.65 μm PES filter from GE Healthcare to remove a large amount of cell debris and particulate matter, while controlling the turbidity to be within 50 NTU, and the yield of virus titer before and after filtration is >80%. The virus harvest liquid is thus obtained.

[0129] Example 2. Formulation of solutions used in chromatography

[0130] 2.1 Heparin affinity chromatography

[0131] The heparin affinity chromatography uses an equilibration buffer, an elution buffer, and a regeneration buffer, and the formulations of the three solutions are as follows.

[0132] Equilibration buffer:

[0133] 3.9 mM NaH2PO4

[0134] 6.1 mM Na2HPO4

[0135] 0.3 M NaCl

[0136] 1.7% (m / v) sucrose

[0137] pH 7.0

[0138] Elution buffer:

[0139] 3.9 mM NaH2PO4

[0140] 6.1 mM Na2HPO4

[0141] 0.5 M NaCl

[0142] 1.7% (m / v) sucrose

[0143] pH 7.0

[0144] Regeneration buffer:

[0145] 3.9 mM NaH2PO4

[0146] 6.1 mM Na2HPO4

[0147] 1.0 M NaCl

[0148] 1.7% (m / v) sucrose

[0149] pH 7.0

[0150] 2.2 CEX

[0151] CEX uses an equilibration buffer with the following formulation.

[0152] Equilibration buffer:

[0153] 3.9 mM NaH2PO4

[0154] 6.1 mM Na2HPO4

[0155] 0.3 M NaCl

[0156] 1.7% (m / v) sucrose

[0157] pH 7.0

[0158] 2.3 Capto DEAE ion exchange chromatography

[0159] Capto DEAE ion exchange chromatography uses an equilibration buffer and an elution buffer, both of which have the following formulations.

[0160] Equilibration buffer:

[0161] 3.9 mM NaH2PO4

[0162] 6.1 mM Na2HPO4

[0163] 0.5 M NaCl

[0164] 1.7% (m / v) sucrose

[0165] pH 7.0

[0166] Elution buffer:

[0167] 3.9 mM NaH2PO4

[0168] 6.1 mM Na2HPO4

[0169] 1.0 M NaCl

[0170] 1.7% (m / v) sucrose

[0171] pH 7.0

[0172] 2.4 Size exclusion chromatography

[0173] The virus stabilizer solution used for the molecular sieve chromatography was formulated as follows.

[0174] Virus stabilizer:

[0175] 3.9 mM NaH2PO4

[0176] 6.1 mM Na2HPO4

[0177] 5 mM Histidine

[0178] 50 mM NaCl

[0179] 4.2% (m / v) Trehalose

[0180] 0.8% (m / v) Sorbitol

[0181] 2.5 Ultrafiltration concentration and replacement

[0182] The ultrafiltration concentration used 300K ultrafiltration replacement buffer, which was formulated as follows:

[0183] Ultrafiltration replacement buffer:

[0184] 3.9 mM NaH2PO4

[0185] 6.1 mM Na2HPO4

[0186] 0.3 M NaCl

[0187] 1.7% (m / v) Sucrose

[0188] pH 7.0

[0189] 2.6 Capto Q ion exchange chromatography

[0190] The Capto Q ion exchange chromatography used an equilibration buffer, a prewash buffer, and an elution buffer, which were formulated as follows.

[0191] Equilibration buffer:

[0192] 3.9 mM NaH2PO4

[0193] 6.1 mM Na2HPO4

[0194] 0.3 M NaCl

[0195] 1.7% (m / v) Sucrose

[0196] pH 7.0

[0197] Prewash buffer:

[0198] 3.9 mM NaH2PO4

[0199] 6.1 mM Na2HPO4

[0200] 0.5 M NaCl

[0201] 1.7%(m / v) Sucrose

[0202] pH 7.0

[0203] Elution buffer:

[0204] 3.9 mM NaH2PO4

[0205] 6.1 mM Na2HPO4

[0206] 1.0 M NaCl

[0207] 1.7%(m / v) Sucrose

[0208] pH 7.0

[0209] Example 3. HSV-1 virus polishing preparation based on heparin affinity chromatography in complex mode

[0210] According to the steps of virus culture, cell lysis and enzyme digestion and clarification filtration described in Example 1, the virus harvest was obtained to perform the following steps.

[0211] 3.1 Heparin affinity chromatography: The virus harvest after clarification filtration was first subjected to heparin affinity chromatography. The heparin affinity chromatography medium was Capto Heparin (Cytiva) chromatography medium, Heparin Sepharose FF (Cytiva) chromatography medium, Bestarose Heparin (BIA Separations) chromatography medium or Bestarose Heparin HR (BIA Separations chromatography medium), and Capto Heparin chromatography medium was selected for heparin affinity chromatography in this example. The heparin affinity chromatography used equilibrium buffer, elution buffer and regeneration buffer, and the formulations of the three solutions were as described in Example 2.1. The method of heparin affinity chromatography was to equilibrate the chromatography column with equilibrium buffer for 2-5 column volumes, then load the virus harvest onto the affinity chromatography column, and the loading amount was 15-25 column volumes, and the average residence time of the sample in the chromatography medium was controlled in the range of 3-10 min. After loading, the chromatography column was washed with equilibrium buffer for 2-5 column volumes to wash away the unbound impurities. Then the chromatography column was eluted with elution buffer for 1-3 column volumes, and the elution peak was collected, and the collected sample was the component containing the target virus (HSV-1).

[0212] As Figure 1As shown, more than 98% of host proteins and host nucleic acids can be removed by directly subjecting the obtained virus harvest to a single-step heparin affinity chromatography, and the virus titer yield under the above conditions reaches more than 70%.

[0213] 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 Core 700 (Cytiva), as long as the composite mode chromatography medium (outer molecular sieve, inner core anion exchange) has the same separation principle and specification to achieve the same impurity removal function. The composite mode chromatography medium in this embodiment is Capto Core 700. The composite mode chromatography uses an equilibration buffer, and the formula of the equilibration buffer solution is as described in embodiment 2.2. The chromatography steps are as follows: (I) equilibrating the chromatography medium with the equilibration buffer; (II) passing the HSV-1-containing sample after heparin affinity chromatography through the composite mode chromatography medium, wherein the impurities are combined with the chromatography medium and the HSV-1 virus flows through; (III) washing the composite chromatography medium with the equilibration buffer, and collecting the HSV-1 virus. The column height of the composite mode chromatography column is 10-30 cm, the sample loading amount is 0.2-2 column volumes, and the retention time is 2-5 min.

[0214] As shown in the formula, Figure 2 the host protein and host nucleic acid content of the sample after composite mode chromatography in this embodiment is further reduced by more than 70% compared to the previous step of affinity chromatography, and the virus titer yield reaches more than 90%.

[0215] 3.3 Molecular sieve chromatography: After the HSV-1 sample is subjected to composite mode chromatography, the HSV-1 sample is subjected to virus stabilizer replacement using molecular sieve chromatography. The molecular sieve chromatography medium is Sepharose 6 Fast Flow (Cytiva) or Sepharose 4 Fast Flow (Cytiva) chromatography medium or other domestic and foreign molecular sieve chromatography media with the same agarose matrix and cross-linking degree. The molecular sieve chromatography medium selected in this embodiment is Sepharose 4 Fast Flow. The molecular sieve chromatography includes the following steps: (I) equilibrating the chromatography medium with a virus stabilizer; (II) passing the HSV-1-containing sample through the molecular sieve chromatography medium, and the HSV-1 virus flows through; (III) washing the molecular sieve chromatography medium with the virus stabilizer, and collecting the HSV-1 virus. The column height of the chromatography column of the molecular sieve chromatography is 20-30 cm, the sample loading amount is 0.1-0.4 column volumes, and the linear flow rate of the molecular sieve chromatography is 30-120 cm / h, wherein the formula of the virus stabilizer is as described in embodiment 2.4.

[0216] As shown in the formula, Figure 3As shown, in this embodiment, after Capto Core 700 chromatography, the sample is replaced by molecular sieve chromatography to change the solution in the virus sample, replacing the solution in the virus sample with a virus stabilizer, which replaces the ultrafiltration solution replacement method. The virus activity is not lost in this step.

[0217] 3.4 Experimental Results: This embodiment purifies HSV-1 virus through heparin affinity chromatography, multimode chromatography, and molecular sieve chromatography. This reduces the need for ultrafiltration concentration and replacement processes. The final step uses mild molecular sieve chromatography instead of ultrafiltration replacement buffer, avoiding viral activity loss, significantly simplifying the purification process, making it suitable for linear scale-up, and improving purification efficiency. The overall removal rate of host proteins and host nucleic acids in the purified sample reached over 99%, and the total HSV-1 virus titer yield reached over 50%. The virus titer and impurity residues met pharmaceutical-grade standards.

[0218] Example 4. Preparation of purified HSV-1 virus by ion exchange chromatography based on heparin affinity chromatography

[0219] Following the steps of virus culture, cell lysis and enzyme digestion and clarification filtration described in Example 1, a virus harvest fluid was obtained and the following steps were performed.

[0220] 4.1 Heparin affinity chromatography: The obtained virus harvest fluid was subjected to heparin affinity chromatography. In this embodiment, Bestarose Heparin chromatography medium was used, and the experimental steps were as described in Example 3.1.

[0221] like Figure 4 As shown, directly passing the obtained virus harvest fluid through a single-step heparin affinity chromatography can remove more than 98% of the host protein and host nucleic acid, and under the above conditions, the virus titer yield reaches more than 70%.

[0222] 4.2 Ion exchange chromatography: The sample after heparin affinity chromatography is subjected to ion exchange chromatography, and 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 also achieve the same impurity removal function. The ion exchange chromatography medium in this embodiment is Capto DEAE. Ion exchange chromatography uses an equilibration buffer and an elution buffer, and the formulations of the two solutions are as described in Example 2.3. The chromatography steps are as follows: (I) equilibrate the chromatography medium with the equilibration buffer; (II) pass the sample containing HSV-1 after heparin affinity chromatography through the ion exchange medium, and the HSV-1 virus binds to the chromatography medium, and the impurities flow through; (III) elute the ion exchange chromatography medium with the elution buffer, and collect the elution peak, and the collected sample is the component containing the target virus (HSV-1 virus). The column height of the ion exchange chromatography column is 10-30 cm, the sample loading amount is 0.5-2 column volumes, and the retention time is 2-5 min.

[0223] As shown in Figure 5 , the host protein and host nucleic acid content of the sample after ion exchange chromatography in this embodiment is further reduced by more than 70% compared to the previous affinity chromatography, and the virus titer yield reaches more than 80%.

[0224] 4.3 Molecular sieve chromatography: After the HSV-1 sample is subjected to complex mode chromatography, the HSV-1 sample is subjected to virus stabilizer replacement using molecular sieve chromatography. The molecular sieve chromatography medium selected in this embodiment is Sepharose 6 Fast Flow, and the experimental steps are as described in Example 3.3, and the chromatogram is as shown in Figure 6 .

[0225] 4.4 Experimental results: In this embodiment, HSV-1 virus is purified through the steps of heparin affinity chromatography, ion exchange chromatography and molecular sieve chromatography. Among them, ion exchange chromatography and the complex mode chromatography used in Example 1 are both purification steps, and have the same purification effect. The total removal rate of host proteins and host nucleic acids in the purified sample is more than 99%, the total yield of HSV-1 virus titer is more than 50%, and the virus titer and impurity residue reach the pharmaceutical grade standard.

[0226] Example 5. HSV-1 virus purification preparation based on complex mode chromatography and ion exchange chromatography after ultrafiltration concentration

[0227] According to the steps of virus culture, cell lysis and enzyme digestion and clarification filtration described in Example 1, the virus harvest is obtained, and the following steps are performed.

[0228] 5.1 Ultrafiltration concentration: In this example, the virus harvest was concentrated 5-10 times by ultrafiltration using a 300K membrane. The ultrafiltration buffer used was as described in Example 2.5. The virus liquid composition after ultrafiltration concentration removed more than 50% of host proteins, host nucleic acids and other impurities, and the virus yield was about 40-50%.

[0229] 5.2 Composite mode chromatography: The composite mode medium used in this example was Capto Core 700 from Cytiva. The equilibration buffer used was the same as the ultrafiltration buffer in step 5.1 (see Example 2.5), with a pH of 6-9 and a conductivity of 20-30 mS / cm. When loading, the average retention time of the sample was 5-10 min, and the total loading amount was 2-6 column volumes. After loading, the peak (containing the target virus HSV-1) was collected by washing with the equilibration buffer.

[0230] As shown in Figure 7 , the virus liquid composition after ultrafiltration concentration was subjected to Capto Core 700 composite mode chromatography, which removed more than 50% of host proteins, host nucleic acids and other impurities, and the virus yield was more than 80%.

[0231] 5.3 Anion exchange chromatography: The sample after composite mode chromatography was subjected to ion exchange chromatography. The ion exchange chromatography medium used in this example was Capto Q (Cytiva). The ion exchange chromatography used equilibration buffer, pre-washing buffer and elution buffer, the formulations of which are described in Example 2.6. The chromatography steps were as follows: (I) equilibration of the chromatography medium with the equilibration buffer; (II) passing the sample containing HSV-1 after composite mode chromatography through the ion exchange medium, the HSV-1 virus was bound to the chromatography medium and the impurities flowed through; (III) pre-washing the chromatography medium with the pre-washing buffer, part of the impurities were washed out; (IV) eluting the ion exchange chromatography medium with the elution buffer, and collecting the elution peak, the collected sample was the component containing the target virus (HSV-1 virus). The height of the ion exchange chromatography column was 10-30 cm, the loading amount of the sample was 0.5-3 column volumes, and the retention time was 2-5 min. The virus sample after ion exchange chromatography was concentrated by 300K ultrafiltration to replace the virus stabilizer to obtain the virus stock solution. The Capto Q (Cytiva) chromatogram is shown in Figure 8 , the host protein and host nucleic acid content of the sample after ion exchange chromatography in this example was further reduced by more than 90% compared to the previous step of composite mode chromatography, and the virus titer yield was about 60%.

[0232] 5.4 Experimental results: Based on the two-step chromatography process (mixed mode chromatography and anion exchange chromatography) of this example, the overall removal of host proteins and host nucleic acids can reach more than 95%, and the virus titer yield is between 20% and 30%. Since HSV-1 virus is a large particle virus with a capsid structure, its activity is particularly sensitive to shear force. Compared with Examples 3 and 4, the two-step high-shear ultrafiltration process included in this example is the main reason for the loss of virus titer activity. The selectivity of anion exchange chromatography and mixed mode chromatography is also worse than that of heparin affinity chromatography, which is also the reason for the poor removal effect of host proteins and host nucleic acid impurities.

[0233] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details without departing from the spirit of the application disclosed in all aspects. The scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. A method for purifying herpes simplex virus (HSV-1), the method comprising: (a) inoculating HSV-1 to host cells to amplify HSV-1; (b) lysing the host cells 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; wherein the heparin affinity chromatography uses an equilibration buffer to equilibrate the chromatography media and / or to flush impurities, the equilibration buffer comprising NaH2PO4, Na2HPO4, NaCl and sucrose, and having a pH value of 6 to 8; the heparin affinity chromatography uses an elution buffer to elute HSV-1, the elution buffer comprising NaH2PO4, Na2HPO4, NaCl and sucrose, and having a pH value of 6 to 8; the heparin affinity chromatography uses a regeneration buffer to regenerate the chromatography column, the regeneration buffer comprising NaH2PO4, Na2HPO4, NaCl and sucrose, and having a pH value of 6 to 8; wherein the heparin affinity chromatography uses Capto Heparin chromatography media or Bestarose Heparin chromatography media.

2. The method of claim 1, further comprising: (e) subjecting the product obtained in step (d) to ion exchange chromatography and / or to multimodal chromatography.

3. The method of claim 2, further comprising: (f) subjecting the product obtained in step (e) to size exclusion chromatography.

4. The method of claim 1, having one or more technical features selected from the group consisting of: (1) subjecting the product obtained in step (d) to ion exchange chromatography or to multimodal chromatography; (2) the multimodal chromatography uses a media having an outer molecular sieve and a core anion exchange chromatography media; (3) the multimodal chromatography uses Capto Core 700; (4) the ion exchange chromatography uses Capto Q or Capto DEAE; (5) the size exclusion chromatography uses Sepharose 6 Fast Flow or Sepharose 4 Fast Flow.

5. The method of claim 1, in step (a), having one or more technical features selected from the group consisting of: (1) the HSV-1 is oncolytic herpes simplex virus type I; (2) the host cells are selected from mammalian cells; (3) the inoculation is performed at a MOI value of 0.02 to 0.

06. (4) the inoculation is performed at a cell density of 2E5 to 5E5 cells / cm 2 under the conditions.

6. The method of claim 5, wherein the HSV-1 is oncolytic herpes simplex virus type I KOS.

7. The method of claim 5, wherein the host cells are selected from human osteosarcoma cells (U-2 OS) cells, Hela cells, Vero adherent cells, Hep-2 cells, BHK cells, or any combination thereof.

8. The method of claim 1, in step (b), having one or more technical features selected from the group consisting of: (1) lysing the host cells by lysis solution; (2) nucleic acid released by lysing the host cell through nuclease digestion; (3) magnesium ions are also added when lysing the host cell.

9. The method of claim 8, wherein the nuclease concentration is 20-100 U / ml.

10. The method of claim 8, wherein the nuclease treatment condition is 33-37 °C for 1-3 h or 18-26 °C for 4-23 h.

11. The method of claim 1, wherein the filtration in step (c) is performed by single-stage or multi-stage filtration.

12. The method of claim 11, wherein the filtration is performed by a combination of 5 μm PP filter and 1+0.65 μm PES filter.

13. The method of claim 1, wherein the heparin affinity chromatography uses equilibration buffer, elution buffer and regeneration buffer in the order of arrangement.

14. The method of claim 1, wherein the product obtained in step (c) is loaded after using equilibration buffer and before using elution buffer, and the average residence time of the product in the chromatography medium is 3-10 min.

15. The method of claim 1, wherein the impurities include host proteins and / or host nucleic acids.

16. The method of claim 2, wherein the method has one or more technical features selected from the following in step (e): (1) the composite mode chromatography uses equilibration buffer to equilibrate the chromatography column medium and / or to flush HSV-1; (2) the ion exchange chromatography uses equilibration buffer to equilibrate the chromatography column medium; (2) the ion exchange chromatography uses elution buffer to elute HSV-1.

17. The method of claim 16, wherein the buffer system of the equilibration buffer and elution buffer is selected from phosphate buffer system, Tris buffer system, His buffer system, sodium acetate buffer system, or any combination thereof.

18. The method of claim 16, wherein the equilibration buffer used in the composite mode chromatography comprises NaH2PO4, Na2HPO4, NaCl and sucrose, and has a pH value of 6-8.

19. The method of claim 16, wherein the formulation of the equilibration buffer used in the composite mode chromatography is 3.9 mM NaH2PO4, 6.1 mM Na2HPO4, 0.3 M NaCl and 1.7% (m / v) sucrose, and has a pH value of 7.

20. The method of claim 16, wherein the equilibration buffer used in the ion exchange chromatography comprises NaH2PO4, Na2HPO4, NaCl and sucrose, and has a pH value of 6-8.

21. The method of claim 16, wherein the formulation of the equilibration buffer used in the ion exchange chromatography is 3.9 mM NaH2PO4, 6.1 mM Na2HPO4, 0.5 M NaCl and 1.7% (m / v) sucrose, and has a pH value of 7.

22. The method of claim 16, wherein the elution buffer used in the ion exchange chromatography comprises NaH2PO4, Na2HPO4, NaCl and sucrose, and has a pH value of 6-8.

23. The method of claim 16, wherein the ion exchange chromatography uses an elution buffer formulation of 3.9 mM NaH2PO4, 6.1 mM Na2HPO4, 1.0 M NaCl, and 1.7% (m / v) sucrose, and a pH of 7.

24. The method of claim 3, wherein in step (f), the size exclusion chromatography uses a buffer to equilibrate the chromatography column media and / or to rinse the HSV-1.

25. The method of claim 24, wherein the buffer has a buffer system selected from the group consisting of a phosphate buffer system, a Tris buffer system, a His buffer system, a sodium acetate buffer system, or any combination thereof.

26. The method of claim 24, wherein the buffer comprises NaH2PO4, Na2HPO4, histidine, NaCl, trehalose, and sorbitol.

27. The method of claim 1, wherein the method does not comprise a step of ultrafiltration concentration.

28. The method of claim 1, wherein the method does not comprise a step of ultrafiltration concentration between step (b) and step (d).

29. The method of claim 1, wherein the method does not comprise a step of ultrafiltration concentration after step (d).

30. The method of claim 1, wherein the removal of impurities is at least 80% after step (d); wherein, wherein the impurities comprise host proteins and / or host nucleic acids.

31. The method of claim 1, wherein the HSV-1 titer yield after step (d) is at least 50%.

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